CN116083367A - Method for preparing induced hepatic parenchymal cells and its application - Google Patents

Abstract

The invention provides a method for preparing induced hepatic parenchymal cells and its application. The method of the present invention can effectively avoid the influence of HNF1A, HNF4A and FOXA3 virus quality on the purity and functional stability of hepatic parenchymal cells derived from transdifferentiation, and ensure the functional stability of terminal hepatic parenchymal cells. The present invention also provides hepatic parenchymal cells prepared by the method.

Description

Method for preparing induced hepatic parenchymal cells and its application

technical field

The invention belongs to the field of cell biology, and more specifically, the invention relates to a method for preparing induced hepatic parenchymal cells and its application.

Background technique

The liver is the largest secretory gland in the human body, and it has both endocrine and exocrine functions. The endocrine function is mainly to secrete various hormones such as insulin-like growth factor, angiotensinogen, and thrombopoietin. The corresponding exocrine function is the synthesis of bile. Hepatocytes use cholesterol as raw material to synthesize primary bile acids through multiple steps, and the key enzyme is cholesterol 7α-hydroxylase. Converting cholesterol into bile acids is the main way the liver clears cholesterol. In addition, the important role of the liver in the human body is also manifested in: (1) glycogen storage, the liver stores excess glucose in the blood in the form of liver glycogen, providing energy for the body when a large amount of energy is consumed; (2) drug metabolism and detoxification function, the drug metabolism and detoxification enzymes in the liver are divided into ① drug metabolism one-phase enzymes, the main family of which is the CYP450 superfamily, which participates in the oxidation of exogenous and endogenous organic substances; ② drug metabolism two-phase enzymes, whose Mainly catalyze enzymes that use the first-phase enzyme reaction product as the substrate for binding reactions; ③Drug metabolism three-phase enzymes, whose main function is to transfer toxic substances in liver cells out of the liver; (3) Regulate the synthesis and transport of cholesterol ; (4) urea metabolism; (5) secretion of serum protein, the main serum protein secreted by the juvenile liver is AFP, the main serum protein in the adult liver is albumin (ALB), and the liver also secretes serum trypsin inhibitor AAT ( Si-Tayeb et al., 2010).

Because the liver has such an important physiological function, liver diseases such as hepatitis, cirrhosis, and acute and chronic liver failure have brought serious economic burdens to families and society. At present, the most effective treatment for patients with severe liver disease is liver transplantation. However, there is a shortage of liver donors. Therefore, finding novel therapeutic methods is an urgent problem to be solved. Recently, bioartificial liver therapy has brought good news to patients with liver diseases. At present, the seed cells used in bioartificial liver devices are some alternative hepatic parenchymal cells, mainly including immortalized primary human hepatic parenchymal cells, human liver cancer cell lines, porcine hepatocytes, and hepatic parenchymal cells derived from human fibroblast transdifferentiation and human induced pluripotent stem cell-derived hepatic parenchymal cells.

However, in clinical practice, the function of immortalized hepatocytes is limited, and the karyotype is often unstable; human cancer cell lines have lost many important functions of liver cells, and there is a greater risk of carcinogenesis; pig hepatocytes face the risk of animal disease transmission ; There is a risk of teratoma formation in hepatic parenchymal cells derived from pluripotent stem cells; the production stability of the three-factor virus in the human fibroblast transdifferentiation method seriously affects the purity and functional stability of the final hepatic parenchymal cells, and the source of transdifferentiation The expansion process of hepatocytes is often accompanied by the loss of hepatocyte function. Therefore, there is an urgent need in this field to develop new, safe and effective alternative hepatic parenchymal cells as seed cells for bioartificial livers.

Contents of the invention

The purpose of the present invention is to provide a method for preparing induced hepatic parenchymal cells and its application.

In the first aspect of the present invention, there is provided a method for preparing induced hepatic parenchymal cells, the method comprising:

(1) Using fibroblasts as initial cells (donor cells) to prepare immortalized cells;

(2) In the cells of (1), the following elements are introduced: antisense Tet transcriptional activator (rtTA);

(3) In the cells of (2), introduce the following elements in sequence (upstream to downstream, or 5' to 3') in series: tetracycline regulatory element (TRE), FOXA3, HNF1A, HNF4A;

(4) In the cells of (3), introduce the following elements in sequence (upstream to downstream, or 5' to 3') in series: tetracycline regulatory element (TRE), HNF4A, so as to obtain inducible hepatic parenchymal cells.

In one or more embodiments, the method further includes the step of: treating the cells of (4) with doxycycline (Dox) or 4-epidoxycycline (4-ED), inducing the cell Express FOXA3, HNF1A, HNF4A; preferably, induce for 0.5-10 days (preferably induce for 1-8 days, more preferably induce for 1.5-6 days, such as 2, 3, 4 or 5 days).

In one or more embodiments, the method further includes the step of subculturing the hepatic parenchymal cells obtained in (4) to obtain subcultured cells.

In one or more embodiments, the described elements are operatively connected.

In one or more embodiments, the testosterone metabolism ability of the hepatic parenchymal cells prepared by the method is significantly improved.

In one or more embodiments, the hepatocytes comprise cell culture or isolated/purified cells.

In one or more embodiments, the immortalized cells are cells in which senescent or apoptotic pathways have been disrupted (or inhibited).

In one or more embodiments, the fibroblasts are ex vivo cells, or cell cultures.

In one or more embodiments, the induced hepatocytes are isolated cells or cell cultures.

In one or more embodiments, said FOXA3, HNF1A, HNF4A includes variants thereof.

In one or more embodiments, the HNF4A or its variant is: (a1) a polypeptide of the amino acid sequence shown in SEQ ID NO: 1; or (b1) substituted in the amino acid sequence defined in (a1), Deletion or addition of one or several (such as 1-30, preferably 1-20, preferably 1-10, more preferably 1-5, such as 1, 2, 3, 4 or 5) A polypeptide derived from (a1) that is amino acid and has the polypeptide function of (a1); or (c1) and (a1) defined protein sequence have 90% (preferably 95%; more preferably 98%; more preferably 99% ) A polypeptide derived from (a1) having the above homology and having the function of the polypeptide of (a1).

In one or more embodiments, the HNF1A or its variant is: (a2) a polypeptide of the amino acid sequence shown in SEQ ID NO: 2; or (b2) substituted in the amino acid sequence defined in (a2), Deletion or addition of one or several (such as 1-30, preferably 1-20, preferably 1-10, more preferably 1-5, such as 1, 2, 3, 4 or 5) A polypeptide derived from (a2) that is amino acid and has the polypeptide function of (a2); or (c2) has 90% (preferably 95%; more preferably 98%; more preferably 99%) of the protein sequence defined by (a2) ) A polypeptide derived from (a2) having the above homology and having the function of the polypeptide of (a2).

In one or more embodiments, the FOXA3 or its variant is: (a6) a polypeptide of the amino acid sequence shown in SEQ ID NO: 6; or (b6) substituted in the amino acid sequence defined in (a6), Deletion or addition of one or several (such as 1-30, preferably 1-20, preferably 1-10, more preferably 1-5, such as 1, 2, 3, 4 or 5) A polypeptide derived from (a6) that is amino acid and has the polypeptide function of (a6); or (c6) and (a6) have 90% (preferably 95%; more preferably 98%; more preferably 99%) of the protein sequence defined by (a6) ) A polypeptide derived from (a6) having the above homology and having the function of the polypeptide of (a6).

In one or more embodiments, in (1), the fibroblasts are umbilical cord fibroblasts; or, the fibroblasts are mammalian-derived fibroblasts.

In one or more embodiments, in (1), immortalized cells are prepared by introducing SV40 large T antigen (Large T), or immortalized cells are prepared by introducing human telomerase reverse transcriptase (hTERT).

In one or more embodiments, the fibroblasts are, for example, from (but not limited to) primates, artiodactyls, or rodents.

In one or more embodiments, the fibroblasts are, for example, from (but not limited to) humans, monkeys, mice, rabbits, dogs, pigs, cows, sheep, horses and the like. Preferably, the fibroblasts are from human.

In one or more embodiments, the fibroblasts are fibroblasts of human origin.

In one or more embodiments, in (3), between the FOXA3 and HNF1A, between the HNF1A and HNF4A are connected by 2A; preferably, the FOXA3 and HNF1A are connected by E2A; relatively Preferably, the HNF1A and HNF4A are connected by T2A.

In one or more embodiments, viruses are used to introduce the elements, SV40 large T antigen or human telomerase reverse transcriptase.

In one or more embodiments, the virus includes: lentivirus, adenovirus, and adeno-associated virus.

In one or more embodiments, the element is constructed in a viral vector, packaged into a virus, and infected into a cell; more preferably, the virus is a lentivirus.

In one or more embodiments, more preferably, at the time of infection: (2), 10-120, preferably 10-110 (such as 20, 30, 40, 50, 60, 70, 80, 90, 100 wait).

In one or more embodiments, in (3), the MOI is 8-100, preferably 10-60 (such as 12, 15, 20, 30, 40, 50, 60, etc.).

In one or more embodiments, in (4), the MOI is 5-100 (such as 8, 15, 20, 30, 40, 50, 70, etc.), preferably 6-60, more preferably 8-12 .

In one or more embodiments, the dosage of doxycycline is 0.1-2ug/mL; preferably 0.5-1.5ug/mL.

In one or more embodiments, the doxycycline also includes its functional analogs, derivatives, and isomers.

In one or more embodiments, after the cells are induced to express FOXA3, HNF1A, and HNF4A, the cells have typical characteristics of hepatic parenchymal cells; preferably, the hepatic parenchymal cells have properties selected from the following:

1) Have the ability to metabolize testosterone;

2) Express/secrete one or more proteins selected from the group consisting of albumin (ALB), α1-antitrypsin (AAT), Ttr, CK8, CK18, E-cadherin, Gjp1, Cldn2, tdo2, transferrin, pah , fxr, vitaminectin;

3) Formation of epithelioid cell clones;

4) accumulation of liver glycogen;

5) Transport acetylated low-density lipoprotein;

6) Expression of one or more genes or proteins selected from the group consisting of: glucose metabolism, drug metabolism, fatty acid metabolism, cholesterol metabolism, bile acid metabolism-related genes or proteins, genes or proteins regulating hemostasis and fibrinolysis, class I And class II heterologous metabolism genes, secreted protein genes.

In one or more embodiments, the testosterone metabolic capacity of hepatocytes prepared by the method is higher than 75%, preferably higher than 80%, more preferably higher than 85%, further preferably higher than 90%, 95%, 98%, 99%; optimal testosterone metabolism 100%.

In one or more embodiments, the hepatic parenchymal cells prepared by the method highly express and secrete albumin (ALB).

In one or more embodiments, the amount of ALB secreted by the hepatic parenchymal cells prepared by the method is higher than 1200ng/day/million cells, preferably higher than 1400ng/day/million cells, more preferably higher than 1600ng /day/million cells, more preferably higher than 1800ng/day/million cells, further preferably higher than 2000, 2200, 2500, ng/day/million cells.

In one or more embodiments, the hepatic parenchymal cells prepared by the method highly express and secrete α1-antitrypsin (AAT).

In one or more embodiments, the amount of AAT secreted by the hepatic parenchymal cells prepared by the method is higher than 1500ng/day/million cells, preferably higher than 1800ng/day/million cells, more preferably higher than 2000ng /day/million cells, more preferably higher than 2500ng/day/million cells, further more preferably higher than 3000, 3500, 4000ng/day/million cells.

In another aspect of the present invention, there is provided a construct for preparing induced hepatic parenchymal cells, comprising:

Construct 1 comprising a polynucleotide encoding an antisense Tet transcriptional activator;

Construct 2, which contains polynucleotides encoding (upstream to downstream, or 5' to 3') the following elements in tandem: tetracycline regulatory element (TRE), FOXA3, HNF1A, HNF4A;

Construct 3, containing polynucleotides encoding (upstream to downstream, or 5' to 3') the following elements in tandem: tetracycline regulatory element (TRE), HNF4A.

In one or more embodiments, the construct further includes: construct 4, which contains a polynucleotide encoding SV40 large T antigen or human telomerase reverse transcriptase.

In one or more embodiments, the construct contains a promoter driving expression of the polynucleotide, and an appropriate terminator, operably linked to the functional elements.

In one or more embodiments, the promoter comprises an inducible promoter of TRE 3G.

In one or more embodiments, the construct is an expression vector, preferably a viral vector; more preferably, the virus is a lentivirus.

In another aspect of the present invention, there is provided an induced hepatic parenchymal cell transfected with the construct.

In one or more embodiments, the induced hepatic parenchymal cells are prepared by any of the methods described above.

In another aspect of the present invention, the use of the induced hepatocytes is provided for: (a) preparing a composition for promoting liver regeneration or treating (including alleviating) liver damage; (b) as an in vitro model, performing Research on liver-related diseases or drugs; preferably, the research on liver-related diseases or drugs includes: research on drug transport, drug metabolism, liver formation, liver regeneration, or liver toxicity testing, screening of hepatotoxic compounds, screening of regulatory Liver cell functional compound; preferably, used to prepare a hepatitis virus infection model, and the hepatitis virus infection model is used to screen anti-hepatitis virus drugs; or, (c) prepare albumin (ALB) and/or α1-anti- Trypsin (AAT).

In one or more embodiments, the liver injury includes (but not limited to): end-stage liver disease, liver cirrhosis, alcoholic liver disease, diabetes, obesity, acute liver failure, hepatitis, liver fibrosis, liver cancer, liver metabolic disease or liver damage from liver failure.

In one or more embodiments, the hepatitis virus includes hepatitis B virus, hepatitis C virus and the like.

In another aspect of the present invention, a composition is provided, which contains the induced hepatic parenchymal cells; and a pharmaceutically acceptable carrier.

In one or more embodiments, the pharmaceutical composition is used for promoting liver regeneration; or treating (including alleviating) diseases related to liver loss.

In another aspect of the present invention, a kit or kit is provided, which contains the construct, the induced hepatic parenchymal cells or the composition.

In one or more embodiments, the kit or kit further contains doxycycline (Dox) or 4-epidoxycycline (4-ED).

In one or more embodiments, the kit or kit further includes: a cell culture medium.

In one or more embodiments, the kit or kit further includes: instructions for preparing the induced hepatic parenchymal cells, or instructions for using the induced hepatic parenchymal cells.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Arrangement of FOXA3, HNF1A and HNF4A transcription factors and 2A combination screening for inducible hiHep preparation;

A. q-PCR detection of the gene expression of HNF1A and HNF4A in the tandem of five kinds of FOXA3, HNF1A and HNF4A; PT, ET, EP, TE in the figure refer to P2AT2A, E2AT2A, E2AP2A, T2AE2A respectively;

B. Western blot detection of the protein expression of HNF1A and HNF4A in the series of five FOXA3, HNF1A and HNF4A.

Figure 2. Selection of UCF cell donors; Flow cytometry detection of the large T cell positive rate of UCF cells from different donors after Large T infection.

Figure 3. Immortalization of UCF cells;

A. Cell morphology of 0916-UCF cells infected by Large T (UCFT) and hTERT viruses;

B. Flow cytometry detection of Large T cell purity of UCFT cells;

C. Immunofluorescence detection of the Large T protein expression of UCFT cells;

D. Statistics of cell doubling time of 0916-UCF cells infected by Large T and hTERT virus (time unit of doubling time: hour); P5～P10 respectively represent the number of passages.

Figure 4. Investigation of rtTA virus infection titer in Large T-rtTA-UCF cell preparation; q-PCR detection of rtTA gene expression level under different rtTA virus infection titers, 0122 is another donor source of Large T-rtTA -UCF cells.

Figure 5. Investigation of TRE-A3-1A-4A virus infection titer in Large T-rtTA-TRE-A3-1A-4A-UCF cell preparation;

A. Exploration strategy for optimal infectious titer of TRE-A3-1A-4A virus;

B. Immunofluorescence detection of HNF1A cell positive rate under different MOI infection conditions;

C. Statistical results of the positive rate of HNF1A cells in immunofluorescence detection.

Figure 6. Functional detection of Large T-rtTA-TRE-A3-1A-4A-UCF cells after Dox induction;

A. Immunofluorescence detection of the expression of FOXA3, HNF4A, HNF1A, ALB and AAT in Large T-rtTA-TRE-A3-1A-4A cells after Dox induction;

B. ELISA detection of the secretion levels of ALB and AAT in Large T-rtTA-TRE-A3-1A-4A cells after Dox induction;

C. Detection of testosterone metabolism in Large T-rtTA-TRE-A3-1A-4A cells after Dox induction.

Figure 7. Investigation of TRE4A virus infection titer in the preparation of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells;

A. Exploring the optimal infectious titer of TRE4A virus;

B. Immunofluorescence detection of HNF4A cell positive rate under different MOI infection conditions;

C. Statistical results of the positive rate of HNF4A cells in immunofluorescence detection.

Figure 8. Functional detection of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells after Dox induction;

A. Immunofluorescence detection of the expression of FOXA3, HNF4A, HNF1A, ALB and AAT in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells after Dox induction;

B. ELISA detection of the secretion levels of ALB and AAT in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells after Dox induction;

C. Detection of testosterone metabolism in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells after Dox induction.

Figure 9. Single clone selection and positive cell line identification of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells;

A.Large T-rtTA-TRE-A3-1A-4A-TRE4A cell monoclonal selection strategy;

B. HNF1A immunofluorescence staining identification of Large T-rtTA-TRE-A3-1A-4A-TRE4A cell clones.

Figure 10. Functional detection of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells after Dox induction;

A. Immunofluorescence detection of the expression of FOXA3, HNF4A, HNF1A, ALB, AAT and FABP1 in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells (#85 clone) after Dox induction;

B. ELISA detection of the secretion levels of ALB and AAT in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells (#85 clone) after Dox induction;

C. Detection of testosterone metabolism after Dox induction in Large T-rtTA-TRE-A3-1A-4A-TRE4A cells (#85 clone).

Figure 11. Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells (#85 clone) rescued mice with acute liver failure after Dox induction;

A. Cell morphology after microencapsulation of #85-D-hiHep;

B. The therapeutic effect of #85-D-hiHep microencapsulation on concanavalin-induced acute liver failure mice.

Detailed ways

The reprogramming process of somatic cells often involves the integration of multiple transcription factors into the genome of somatic cells through lentivirus infection to realize the process of cell transdifferentiation. However, this virus-dependent multi-transcription factor-independent infection of somatic cells often leads to inappropriate viral copy numbers in the genome, which may cause insertional mutations or reactivation of silent viral transcripts in cells, limiting the ability of transdifferentiated source cells. Safety of use. How to achieve proper expression of exogenous genes in cells, how well the expressed exogenous proteins cooperate and interact with each other and effectively compatible with endogenous proteins, improve the efficiency of cell transdifferentiation, and obtain better quality target cells are the key sex factor.

In the art, a method for preparing hepatic parenchymal cells by introducing transcription factors FOXA3, HNF1A and HNF4A into fibroblasts by using somatic cell reprogramming technology has been established. The present inventors devoted themselves to the optimization of the established method and the improvement of the quality of the product cells on this basis, and provided an optimized method for preparing induced hepatic parenchymal cells (induced hiHep, D-hiHep). The method of the present invention can effectively avoid the influence of HNF1A, HNF4A and FOXA3 virus quality on the purity and functional stability of hepatic parenchymal cells derived from transdifferentiation, and ensure the functional stability of terminal hepatic parenchymal cells.

the term

As used herein, unless otherwise stated, the "cells" or "cells for overexpressing transcription factors" are fibroblasts, which can form hepatic parenchymal cells after being transferred with the transcription factors. Preferably, said cells are mammalian somatic cells. Preferably, said cells are fibroblasts derived from umbilical cord.

As used herein, the "mammal" is an animal of the class Mammalia of the subphylum Vertebrata of the phylum Chordata. The mammals include but not limited to primates (including humans), artiodactyls, carnivores, rodents and the like. Mammals in the present invention include humans and non-human mammals. The non-human mammals are, for example, mice, rats, rabbits, dogs, rabbits, apes, pigs, cows, sheep, horses, etc. Preferably, said fibroblasts are from human.

As used herein, the "hepatic parenchymal cell" refers to the ability to form epithelial cell clones, express liver genes (such as: ALBUMIN, AAT, etc.), proliferate in the liver, and have the ability to rebuild liver function.

As used herein, the terms "introducing", "importing", "expressing", "overexpressing" or "infecting" are used interchangeably.

As used herein, the "overexpression" means that the content (such as expression) of the transcription factor in the cell exceeds the level of the initial cell (the cell that has not been transferred with the exogenous gene); if compared with the initial cell, its content 20% higher, preferably 50% higher; more preferably more than 100% higher, such as 200%, 300%...500% higher or higher. A situation of "overexpression" is that the coding gene of an exogenous transcription factor is transferred into the cell and expressed.

As used herein, the "low expression" refers to the content (such as expression) of a certain gene in the cell is significantly lower than its normal level (such as compared with wild-type cells, its expression is 20% lower, preferably lower 50%; more preferably lower than 100%, such as lower than 200%, 300%...500% or lower), said low expression also includes the case of no expression. Low expression can be achieved by techniques well known in the art, such as gene knockout, gene silencing, and protein inhibition.

As used herein, the "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the "promoter" or "promoter region (domain)" refers to a nucleic acid sequence, which usually exists upstream (5' end) of the coding sequence of the gene of interest and can guide the transcription of the nucleic acid sequence into mRNA . Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it has been inserted.

As used herein, an "endogenous" gene or protein refers to a gene or protein that is naturally or intrinsically contained in the genome of an animal.

As used herein, the words "comprising", "having" or "comprising" include "comprising", "consisting essentially of", "consisting essentially of", and "consisting of";" "Mainly consist of", "essentially consist of" and "consist of" belong to the sub-concepts of "contain", "have" or "include".

transcription factor

The present inventors used three transcription factors, FOXA3, HNF1A and HNF4A, to introduce them into fibroblasts. After the three transcription factors are introduced into cells and overexpressed, non-hepatic cells can be formed into hepatic parenchymal cells. The obtained cells express liver genes, can proliferate in the liver, and can restore liver function.

The HNF4A can have the amino acid sequence shown in SEQ ID NO: 1, or its amino acid sequence can be found in GenBank accession number NM_000457.

The HNF1A may have the amino acid sequence shown in SEQ ID NO: 2, or its amino acid sequence may refer to GenBank accession number NM_000545.

The FOXA3 can have the amino acid sequence shown in SEQ ID NO: 3, or its amino acid sequence can be found in GenBank accession number NM_004497.

Based on the above three transcription factors, the inventor proposed an optimized method for preparing hepatic parenchymal cells by establishing effective constructs and optimizing expression conditions, thereby effectively improving the efficiency of forming hepatic parenchymal cells from non-hepatic cells.

In the present invention, the term "transcription factor" refers to the above-mentioned polypeptide (protein). The term also includes variant forms that have the same function as the polypeptides of the above sequences. These variations include (but are not limited to): one or more (such as 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, Insertion and/or substitution, and addition or deletion of one or several (eg, within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, addition and/or deletion of one or several amino acids at the C-terminus and/or N-terminus will generally not change the function of the protein. The term also includes active fragments and active derivatives of the aforementioned polypeptides. Such variant forms have the proviso that they retain or substantially retain the function of the full-length polypeptide.

The variant forms of the polypeptide include: conservative variants, homologous sequences, allelic variants, natural mutants, induced mutants and the like. The present invention also provides other polypeptides, such as fusion proteins comprising said polypeptides or fragments thereof. In addition to substantially full-length polypeptides, the present invention also includes soluble fragments of said polypeptides as long as they retain full-length said transcription factor function.

The invention also includes analogs of said transcription factors. The difference between these analogues and the natural transcription factor may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

The gene encoding the transcription factor may be a polynucleotide encoding the transcription factor, or a polynucleotide further comprising additional coding and/or non-coding sequences.

The present invention also relates to hybridization with the above-mentioned transcription factor coding gene and there is at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% between the two sequences , more preferably at least 98% identical variants. The present invention particularly relates to polynucleotides hybridizable under stringent conditions to the transcription factor polynucleotides of the present invention.

Under the suggestion of the present invention, the variants or derivatives of the transcription factor or its coding gene can be obtained and applied by those skilled in the art. The gene sequence encoding the transcription factor can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences.

Preparation method of hepatic parenchymal cells

Since different types of cells in each individual have the same genome, it is theoretically possible that various types of cells have the potential to form hepatic parenchymal cells. However, in the present invention, fibroblasts are preferably used; in a more preferred embodiment, umbilical cord-derived fibroblasts are used.

The production stability of the FOXA3, HNF1A, and HNF4A three-factor virus in the fibroblast transdifferentiation method seriously affects the purity and functional stability of the final hepatocytes, and the hepatocytes from transdifferentiation are accompanied by hepatocytes during the expansion process For the technical problem of function loss, optimization and improvement have been carried out in the present invention. Although the preparation of hepatic parenchymal cells by the co-expression of the three factors has been studied before, the present invention proposes for the first time to rationally and effectively regulate the expression of the three factors, optimize their expression and activity state in the cells, and obtain better performance and purity. And stable hepatic parenchymal cells.

For the introduction and expression of the above three factors into fibroblasts, in the strategy provided by the present invention, on the one hand, they are jointly constructed in series in the same cis-transcription element and introduced into the cells; on the other hand, HNF4A is further introduced into the cells (also That is, the introduction of "three factors" + "HNF4A") into the cells. The inventors unexpectedly found that these factors, as exogenous genes, adopt such an expression strategy, which can make the expressed proteins cooperate and function well, and can also cooperate well with the endogenous proteins of the cells, so that the obtained liver parenchyma The cells have a more efficient testosterone metabolism ability, and a stronger ALB, AAT secretion ability

When the above-mentioned three factors are co-constructed in the same cis-transcription element, as a preferred mode of the present invention, the 2A protein is used for mutual tandem construction. In a more preferred manner, the FOXA3 and HNF1A are connected by E2A, and the HNF1A and HNF4A are connected by T2A. This connection method is conducive to the effective and proper expression of the fusion protein in cells, and has significant advantages compared with other connection methods.

In order to make the transformation of fibroblasts into hepatic parenchymal cells "inducible", the tetracycline-induced gene expression system (Tet-on) is preferably used in the present invention to control the expression of the three factors and the expression of HNF4A. Introduce and express antisense Tet transcriptional activator (rtTA) in the cells, and set tetracycline responsive element (TRE) upstream of the three-factor tandem gene/HNF4A. In the absence of doxycycline (Doxycycline, Dox), rtTA cannot bind to TRE, and the downstream gene expression of TRE-3G is turned off; when doxycycline is present, the conformation of rtTA changes, and rtTA binds to TRE, and the downstream gene expression of TRE-3G is blocked. open. The Dox-induced Tet-On system enables transient transcriptional regulation of genes.

In the present invention, the Tet-on system is preferably used to make the transformation of fibroblasts into hepatic parenchymal cells "inducible". However, the present invention does not exclude that other systems that can achieve similar inductive effects can also be applied to this technical solution, which is included in the overall solution of the present invention.

Methods for overexpressing foreign genes (transcription factors in the present invention) in cells are well known to those skilled in the art. A polynucleotide sequence encoding a transcription factor can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to virus (such as lentivirus, adenovirus, retrovirus), bacterial plasmid, phage, yeast plasmid or other vectors well known in the art. Various plasmids and vectors can be used as long as they are replicable and stable in the host. A vector comprising a polynucleotide sequence encoding a transcription factor and an appropriate promoter or control sequence can be used to transform a cell so that it can express the transcription factor.

The method for culturing cells can refer to techniques known in the art, and various media are available, such as some conventional media that are conducive to cell growth, preferably those that are conducive to fibroblast culture or hepatocyte culture. . In addition, some growth factors can be added to these media to promote cell growth and division. In addition, the medium can also be appropriately adjusted depending on the type of starting cells and the culture conditions. Those skilled in the art know which media are suitable for the culture of hepatocytes.

Methods for enriching (or separating and purifying) cells are also well known to those skilled in the art, for example, enrichment can be performed based on the epithelial-like morphological characteristics of hepatic parenchymal cells; or based on special proteins expressed by hepatic parenchymal cells (such as albumin, etc.) or Molecular markers for selective collection (eg, using specific antibodies or ligands). As an optional embodiment, flow cytometry technology can be used to separate and purify target cells through molecular markers on the surface of hepatic parenchymal cells. In the technical solution of the present invention, high-purity hepatic parenchymal cells can be obtained, which facilitates cell enrichment (or separation and purification), effectively simplifies the operation process, and reduces costs.

Since adult cells cannot proliferate in vitro, it is likely that adult cells obtained by reprogramming cannot proliferate in vitro. Cell senescence is the main reason for inhibiting cell proliferation. When mammalian adult cells are isolated and cultured in vitro, they remain in a state of replicative senescence after several rounds of replication. These cells are irreversibly arrested in the G1 phase of the cell cycle and are no longer sensitive to growth factor stimulation. Therefore, in the technical solution of the present invention, the fibroblasts are immortalized to destroy their aging or apoptosis pathways. Preferably, immortalized cells are prepared by introducing SV40 large T antigen (Large T), or prepared by introducing human telomerase reverse transcriptase (hTERT).

p53 and Rb are genes related to cell senescence signaling pathways, the existence of which can make cells unable to proliferate under in vitro culture conditions, resulting in the loss of proliferative ability of cells transformed into hepatic parenchymal cells. LT can inhibit the activity of p53, Rb, therefore, alternatively, make the cells underexpress p53, Rb (or not express) or overexpress LT. Methods for preparing cells with low expression of p53, Rb (or no expression) or high expression of LT are well known to those skilled in the art, including but not limited to gene silencing (such as making the gene not expressed at the mRNA level by siRNA) or gene knockout In addition to (such as gene editing targeting specific gene loci for knockout, gene targeting at the genome level so that the gene is not expressed), protein level inhibition (such as through antibodies or ligands that specifically bind to proteins) other genes in cells The method of low expression or no expression of genes (such as p53, Rb) and high expression of LT is well known in the art and can be applied in this application.

In a specific embodiment of the present invention, a tet-on promoter system is used to control gene expression. The rtTA plasmid system equipped with tet-on inducible three factors and trans-acting elements can be transfected into cells to realize the packaging of lentivirus for infection. After the immortalized human fibroblasts were infected with rtTA and the inducible three-factor virus, the single cell clones were further selected to realize the establishment of the inducible three-factor Large T/hTERT-rtTA-fibroblasts. The cells can be successfully transdifferentiated into hepatic parenchymal cells (D-hiHep) after being induced by Doxycyclin, and the obtained hepatic parenchymal cells have functions of hepatic parenchymal cells such as ALB and AAT secretion, testosterone metabolism, and the ability to rescue animals with acute liver failure.

The preparation method of the present invention can fundamentally avoid the influence of HNF1A, HNF4A and FOXA3 virus quality on the purity and functional stability of hepatic parenchymal cells derived from transdifferentiation, ensure the functional stability of terminal hepatic parenchymal cells, and simplify the preparation of transdifferentiated hepatic parenchymal cells. The hepatic parenchymal cell production process saves the cost of cell production and increases the throughput of cell production.

The preparation method of the present invention reduces the number of cell expansion generations and simplifies the operation procedure. Specifically, in the present invention, there is no need to amplify the terminal hiHep cells, and direct induction preparation can be realized, reducing the passage of hiHep cells. As a result, the cell expansion efficiency of the overall scheme is very high.

Application of hepatic parenchymal cells

In view of the novel method for preparing hepatic parenchymal cells created in the present invention, a unique expression construct is introduced into the cells, and elements for inducing expression are introduced, the hepatic parenchymal cells of the present invention retain the typical functions of hepatic parenchymal cells Under the precursor of the hepatic parenchymal cells of the prior art, there are aspects that are different from those of the hepatic parenchymal cells in the prior art. Therefore, the present invention also provides novel constructs for preparing induced hepatic parenchymal cells, and novel induced hepatic parenchymal cells.

The hepatic parenchymal cells prepared by the invention have various applications. Including but not limited to: for the preparation of compositions (pharmaceutical compositions) for promoting liver regeneration; for the preparation of liver damage (including but not limited to: end-stage liver disease, cirrhosis, alcoholic liver, diabetes, obesity, acute liver failure, Hepatitis, liver fibrosis, liver cancer, liver metabolic disease or liver damage caused by liver failure) composition (pharmaceutical composition); used as an in vitro model for research on liver-related diseases or drug efficacy, such as for researching drugs Transport, drug metabolism, liver formation, liver regeneration, for hepatotoxicity testing, for albumin production, etc. The hepatic parenchymal cells of the present invention can also be used in the study of liver toxicity.

The induced hepatic parenchymal cells of the present invention will have a wide range of application prospects: providing a good model cell and screening platform for liver drug screening, screening of hepatotoxic compounds, screening of compounds that regulate liver cell function, etc.; providing a steady stream for the treatment of liver diseases Continuously mature functional liver cells; provide good seed cells for artificial liver devices for treating patients with acute liver failure.

Compositions and kits

The present invention also provides a composition, which contains: an effective amount of the hepatic parenchymal cells (such as 1×10 ⁴ -1×10 ¹² ; preferably 1×10 ⁵ -1×10 ¹⁰ each); and a pharmaceutically acceptable carrier. It contains an effective amount of the liver parenchymal cells and a pharmaceutically acceptable carrier. The composition has no visible toxicity and side effects on animals.

The "effective amount" refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in compositions may contain liquids, such as water, saline, buffers. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carrier may also contain cell transfection reagents.

The present invention also provides a method for promoting liver regeneration, the method comprising: administering an effective amount of the hepatic parenchymal cells of the present invention to a subject in need of treatment.

When the composition is used for administration, usually 1×10 ² -1×10 ¹⁰ cells/kg body weight, preferably 1×10 ³ -1×10 ⁸ cells/kg body weight is suitable, which is also It depends on the clinician's diagnosis and the severity of the patient's symptoms.

Based on the new discovery of the present invention, a kit is also provided, which contains the raw materials or reagents for preparing the hepatic parenchymal cells of the present invention.

As an embodiment of the present invention, a kit/medicine kit for inducing differentiation of hepatic parenchymal cells is provided, the kit/medicine kit contains the construct described in the present invention, the inducible type described in the present invention Liver parenchymal cells or the composition of the present invention.

More preferably, the kit also contains one or more selected from the following: cells for overexpressing transcription factors; trypsin; cell culture medium and the like.

More preferably, the kit also contains instructions for use.

The main excellent effects of the in vitro preparation system of induced hepatic parenchymal cells of the present invention are:

(1) The expression strategy of combining the three factors in series and further expressing HNF4A was adopted. On the one hand, it effectively saved the cost of viruses prepared by hepatic parenchymal cells, and on the other hand, it ensured that the exogenous protein was relatively optimized in the cells. Express to obtain ideal hepatic parenchymal cells;

(2) Overcoming the difficulty of differences in the stability of hepatocytes caused by differences in virus stability;

(3) It provides an ideal source of liver cells, and provides practical research tools for related applications including high-throughput research on in vitro drug metabolism, artificial liver devices, in vitro remodeling of liver organs, and terminal cell or organ-based therapy.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, or according to the manufacturer's suggestion conditions of.

Materials and Methods

1. In vitro isolation and expansion of fibroblasts

The human umbilical cord was taken out from the PBS, cut into small sections of 3-5 cm, the three blood vessels in the umbilical cord were separated with scissors and forceps, and the tissue was washed 3 times with PBS containing 2% double antibody (penic-streptomycin). Transfer the tissue to a clean and sterile 10cm petri dish, cut the tissue into pieces with scissors, transfer the chopped tissue into a 50mL centrifuge tube, and put every 3-5cm small piece of tissue into a 50mL centrifuge tube. Add 20 mL of PBS solution containing 4 mg/mL collagenase IV and 2% double antibody to the tube, seal it, and incubate in a 37°C water bath for 3 h. Add 10 mL of trypsin digestion solution to the tube, seal and transfer to a 37°C water bath for incubation for 10 min. Add 20mL HFM medium to the tube to stop the digestion, centrifuge at 600×g for 10min, carefully discard the supernatant, resuspend the cells in HFM medium and pass through a 20μm cell sieve, centrifuge at 600×g for 5min, discard the supernatant, and then use HFM medium again Resuspend and inoculate three 10cm culture dishes per tube of cells. Transfer the culture dish to a CO ₂ incubator, culture at 37°C and 5% CO ₂ , record it as passage 0 cells, change the medium 24 hours after inoculation, and change the medium every other day in the next culture, and passage after the cells are full nourish.

2. Establishment of Large T, hTERT, rtTA, TRE-A3-1A-4A virus vectors and virus packaging

Establishment of Large T virus vector: Insert the coding sequence of SV40LargeT (positions 2683-4572 and 4919-5164 in the sequence of GenBank accession number AY271817) into the backbone vector pHIV (the promoter is EF1A).

Construction of hTERT virus vector: the coding sequence of human telomerase reverse transcriptase (hTERT) (position 965-1972 in the sequence of GenBank accession number MN996841) was inserted into the backbone vector pHIV (the promoter is EF1A).

Establishment of rtTA viral vector: Insert the coding sequence of rtTA into the backbone vector pHIV (the promoter is EF1A). Among them, rtTA includes antisense TetR (rTetR) and a transcription activation region at the C-terminus of herpes simplex virus (HSV) VP16 protein, which are fused (GenBank accession number MN996841, 965-1972 in the sequence).

Establishment of TRE-4A-T2A-A3-E2A-1A viral vector: See GenBank accession number MN996842 for the coding sequence of the TRE3G promoter at positions 237-613 in the sequence, FOXA3 (abbreviated as A3) coding sequence see GenBank accession number NM_004497, HNF1A (abbreviated as 1A) See GenBank accession number NM_000545 for the coding sequence, and GenBank accession number NM_000457 for the coding sequence of HNF4A (4A for short). Insert T2A between 4A and A3 (sequentially: GagggcaggggaagTCTGCTGCGCGGTGTGGGGGAATCCCCCCCCT (SEQ ID NO: 4)), between A3 and 1A, E2A (sequence is: CagtgtgtaatgctCTCTC between A3 and 1A TTGAAAATTGGGGGGATGTGAGAGAGACCCCCCCCCCC (SEQ ID NO: 5)). The tandem sequence was inserted into the pHIV vector, from 5' to 3' in order TRE3G, 4A, T2A, A3, E2A, 1A.

Establishment of TRE4A virus vector: HNF4A (abbreviated as 4A) coding sequence, see GenBank accession number NM_000457, was inserted into the backbone vector pHIV (promoted by TRE3G).

TRE-A3-P2A-1A-T2A-4A: the establishment method is the same as that of the TRE-4A-T2A-A3-E2A-1A viral vector, and the type of the connecting element is adjusted, wherein the P2A sequence is: gccacaaacttctctctgctaaagcaagcaggtgatgttgaagaaaaccccgggcct (SEQ ID NO: 6), and Adjust the order of components.

TRE-4A-P2A-A3-T2A-1A: The establishment method is the same as that of the TRE-4A-T2A-A3-E2A-1A virus vector, and the type and sequence of the regulatory elements are adjusted.

TRE-A3-E2A-1A-T2A-4A: The establishment method is the same as that of the TRE-4A-T2A-A3-E2A-1A virus vector, and the type and sequence of the regulatory elements are adjusted.

TRE-A3-E2A-1A-P2A-4A: The establishment method is the same as that of the TRE-4A-T2A-A3-E2A-1A viral vector, and the type and sequence of the regulatory elements are adjusted.

293FT cells were transfected with viral packaging plasmid one day after passage. The virus packaging plasmid preparation system (10cm petri dish) is as follows:

Firstly, the plasmid was thoroughly mixed with water, then 2.5M CaCl ₂ solution was added, and mixed well; the mixed plasmid CaCl ₂ solution was fully mixed with 2X Hepes solution (using an electric shaker for mixing). The homogeneously mixed solution of plasmid, calcium chloride and Hepes was allowed to stand at room temperature for 15 minutes, and was evenly dropped into the pre-plated 293FT cells. After 5 hours, replace the 293FT cells with fresh medium, 6mL/dish. Fluorescent observation and photographing were performed 48hrs after plasmid transfection to determine the packaging effect of the virus. Collect the cell supernatant, centrifuge the supernatant at 3000 rpm to remove cell debris, and filter the supernatant after centrifugation with a 0.45 μm filter membrane. If the virus needs to be further concentrated, ultracentrifugation can be used for concentration, otherwise the virus can be directly subpackaged and stored at -80°C.

3. Large T flow cytometry detection

Large T lentivirus: obtained from Jinan Yiming Cell Engineering Co., Ltd.

For flow staining of UCF cells infected with Large T virus, the cells were fixed with 4% paraformaldehyde, and the staining buffer was PBS solution containing 10% FBS. The proportion of positive cells was analyzed by flow cytometry (CalliaburBD).

4. Immunofluorescence of cells

Dox-induced Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were fixed with 4% PFA for one hour, then permeabilized with 0.3% Triton-100 for 5 minutes, washed three times with PBS at room temperature , 3% BSA at room temperature to block for two hours. The primary antibody was overnight at 4°C, washed three times with PBS, the secondary antibody was kept at room temperature for two hours, washed three times with PBS, and stained with DAPI for nuclei. Leica TCS SP5 laser confocal microscope was used to analyze the results of stained cells.

5. RNA extraction and real-time quantitative PCR

The cells were collected by centrifugation, and the resulting cells were treated with cell lysate. RNA extraction was performed according to the instructions of the RNA extraction kit. The prepared RNA was further completed cDNA synthesis using a reverse transcriptase kit. Real-time quantitative PCR was completed using ABI 7900fast (Invitrogen) under the instruction of SYBR Green fluorescent dye. Gene expression was calibrated using the internal reference gene GAPDH.

6. High performance liquid chromatography mass spectrometry drug metabolism detection

To detect the testosterone metabolism ability of Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells induced by Dox, the cells were treated with CYP3A4 substrate testosterone (100 μM) for 6 hours. The final detection analysis of the reaction supernatant was performed by high performance liquid chromatography mass spectrometry.

7. Selection of Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cell clones

After the Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were digested with 0.25% trypsin, single cells were selected under a microscope with a mouth pipette (Sigma, A5177-5EA) into 96 cells equipped with HFM. inside the orifice.

8. Detection of serum albumin secretion

After Dox-induced Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were cultured for 48 hours, the cell culture supernatant was collected. The serum albumin secretion detection kit was used to detect albumin secretion in the collected samples.

9. AAT secretion detection

After Dox-induced Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were cultured for 48 hours, the cell culture supernatant was collected. The collected samples were tested for albumin secretion using the AAT detection kit.

10. Microencapsulation of D-hiHep

After Dox-induced Large T/hTERT-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were digested with 0.25% trypsin and collected, an appropriate amount of sodium alginate was added at a ratio of 1X10 ⁷ cells/mL sodium alginate Mix with the cells, drop the sodium alginate cell solution into 100mM CaCl ₂ with a syringe pump, and let it stand for 20 minutes to fully calcify. Discard the CaCl ₂ solution, transfer the cytogel beads to a 50mL centrifuge tube, and fully wash 3 times with normal saline. Then add 5 times the volume of polylysine (PLL) solution, and react at room temperature for 5-8 minutes to complete the film-forming reaction. It is best to fully wash 3 times with normal saline, and prepare for the transplantation experiment.

11. Abdominal transplantation of microencapsulated D-hiHep in ConA mice

C57BI6/J mice were injected with 20 mg/kg concanavalin A (Sigma) via tail vein. Microencapsulated D-hiHep was transplanted into the abdominal cavity of mice with acute liver failure at 1X10 ⁷ /mouse. Blood samples from surviving mice were collected every 24 hours, and liver tissue samples were collected.

12. Transcription factor amino acid sequence

HNF4A (SEQ ID NO: 1):

MRLSKTLVDM DMADYSAALD PAYTTLEFEN VQVLTMGNDT SPSEGTNLNA PNSLGVSALC 60

AICGDRATGK HYGASSCDGC KGFFRRSVRK NHMYSCRFSR QCVVDKDKRN QCRYCRLKKC 120

FRAGMKKEAV QNERDRIST RSSYEDSSLP SINALLQAEV LSRQITSPVS GINGDIRAKK 180

IASIAADVCES MKEQLLVLVE WAKYIPAFCE LPLDDQVALL RHAGEHLLL GATKRSMVFK 240

DVLLLGNDYI VPRHCPELAE MSRVSIRILD ELVLPFQELQ IDDNEYAYLK AIIFFDPDAK 300

GLSDPGKIKR LRSQVQVSLE DYINDRQYDS RGRFGELLLL LPTLQSITWQ MIEQIQFIKL 360

FGMAKIDNLL QEMLLGGSPS DAPHAHHPLH PHLMQEHMGT NVIVANTMPT HLSNGQMCEW 420

PRPRGQAATP ETPQPSPPGG SGSEPYKLLP GAVATIVKPL SAIPQPTITK QEVI 474

HNF1A (SEQ ID NO:2):

MVSKLSQLQT ELLAALLESG LSKEALIQAL GEPGPYLLAG EGPLDKGESC GGGRGELAEL 60

PNGLGETRGS EDETDDDGED FTPPILKELE NLSPEEAAHQ KAVVETLLQE DPWRVAKMVK 120

SYLQQHNIPQ REVVDTTGLN QSHLSQHLNK GTPMKTQKRA ALYTWYVRKQ REVAQQFTHA 180

GQGGLIEEPT GDELPTKKGR RNRFKWGPAS QQILFQAYER QKNPSKEERE TLVEECNRAE 240

CIQRGVSPSQ AQGLGSNLVT EVRVYNWFAN RRKEEAFRHK LAMDTYSGPP PPGPGPGPALP 300

AHSSPGLPPP ALSPSKVHGV RYGQPATSET AEVPSSSGGP LVTVSTPLHQ VSPTGLEPSH 360

SLLSTEAKLV SAAGGLPPPV STLTALHSLE QTSPGLNQQP QNLIMASLPG VMTIGPGEPA 420

SLGPTFTNTG ASTLVIGLAS TQAQSVPVIN SMGSSLTTLQ PVQFSQPLHP SYQQPLMPV 480

QSHVTQSPFM ATMAQLQSPH ALYSHKPEVA QYTHTGLLPQ TMLITDTTNL SALASLTPTK 540

QVFTSDTEAS SESGLHTPAS QATTLHVPSQ DPAGIQHLQP AHRLSSPTV SSSSLVLYQS 600

SDSSNGQSHL LPSNHSVIET FISTQMASSS Q 631

FOXA3 (SEQ ID NO: 3):

MLGSVKMEAH DLAEWSYYPE AGEVYSPVTP VPTMAPLNSY MTLNPLSSPY PPGGLPASPL 60

PSGPLAPPAP AAPLGPTFPG LGVSGGSSSS GYGAPPGGLV HGKEMPKGYR RPLAHAKPPY 120

SYISLITMAI QQAPGKMLTL SEIYQWIMDL FPYYRENQQR WQNSIRHSLS FNDCFVKVAR 180

SPDKPGKGSY WALHPSSGNM FENGCYLRRQ KRFKLEEKVK KGGSGAATTT RNGTGSAAST 240

TTPAATVTSP PQPPPPAPEP EAQGGEDVGA LDCGSPASST PYFTGLELPG ELKLDAPYNF 300

NHPFSINNLM SEQTPAPPKL DVGFGGYGAE GGEPGVYYQG LYSRSLLNAS 350

Example 1. Arrangement of FOXA3, HNF1A and HNF4A transcription factors for preparation of inducible hiHep (D-hiHep) and 2A combination screening

Previous studies have shown that human fibroblasts immortalized by Large T virus can successfully transform into liver parenchyma with liver function under the action of important liver transcription factors FOXA3 (abbreviated as A3), HNF4A (abbreviated as 4A) and HNF1A (abbreviated as 1A). cell. However, in the transdifferentiation method of human fibroblasts, the three-factor virus needs to be packaged separately, and the stability of virus production seriously affects the purity and functional stability of the final hepatocytes, and the hepatocytes derived from transdifferentiation are often accompanied by hepatocytes during the expansion process. Loss of parenchymal cell function.

In order to simplify the virus preparation process, the inventors serially connected the important liver transcription factors FOXA3, HNF1A and HNF4A in order to simplify the expression. After analysis and comparison, the connection method of 2A protein was selected, and they were constructed in the same cis-transcription element, and the expression of the gene was controlled by the tet-on promoter system. 2A peptides include E2A, P2A and T2A, etc. However, the research results of the inventors show that different gene tandem sequences and connection through different 2A peptide segments will affect gene transcription and expression.

The inventor constructed the following five series of FOXA3, HNF1A and HNF4A:

TRE-4A-T2A-A3-E2A-1A;

TRE-A3-P2A-1A-T2A-4A;

TRE-4A-P2A-A3-T2A-1A;

TRE-A3-E2A-1A-T2A-4A;

TRE-A3-E2A-1A-P2A-4A;

Plasmids carrying the above fusion peptides were established and transformed into 293FT cells to verify the transcription and expression levels of FOXA3, HNF1A and HNF4A in different tandem combinations. The cells transfected with HNF1A and HNF4A plasmids alone were used as positive controls. q-PCR results showed that the gene expression levels of HNF4A and HNF1A in TRE-A3-E2A-1A-T2A-4A, TRE-A3-P2A-1A-T2A-4A and TRE-4A-P2A-A3-T2A-1A plasmids relatively high (Fig. 1A).

Further, Western Blot was performed to detect protein expression. The results showed that the expression levels of HNF4A and HNF1A in the TRE-A3-E2A-1A-T2A-4A tandem pattern were higher (Fig. 1B).

According to the above, the tandem method of TRE-A3-E2A-1A-T2A-4A is preferably used as the preparation method of the inducible hiHep.

Example 2, UCF cell donor

The present inventors used Large T virus to infect donor UCF cells (0402, 0419, 0829 and 0916, respectively obtained from different cell donors) (MOI=10), and detected the positive rate of Large T cells after infection.

According to the results of flow cytometry analysis, the UCF cells of the 0916 donor had good sensitivity to the Large T virus (Figure 2), and the UCF cells of the 0916 donor (0916-UCF) were subsequently used as the seed bank for the preparation of inducible hiHep cells. However, it should be understood that donors that can be infected by Large T virus are not limited to 0916-UCF.

Embodiment 3, the preparation of immortalized UCF cell

The immortalization of UCF cells is crucial to the in vitro expansion of inducible hiHep cells. Here, the inventors used Large T antigen virus and telomerase virus hTERT to infect 0916-UCF cells in vitro respectively. After infection, the cell purity was calculated. and cell doubling time to determine the expansion ability of immortalized cells.

The results showed that the cell morphology of 0916-UCF cells was significantly reduced after being infected with Large T virus (0916UCFT)/hTERT virus (0916hTERT) (Figure 3A).

After the 0916-UCF cells were infected with Large T virus (MOI=20), the purity was detected by flow cytometry, and the results showed that the purity was as high as 90% (Fig. 3B).

The results of immunofluorescence staining further proved that Large T-UCF cells showed high purity (Fig. 3C).

The doubling time showed that the proliferation ability of UCF cells could be significantly improved after being infected by Large T virus (Fig. 3D). The doubling time of hTERT cells after continuous amplification of telomerase virus was counted ( FIG. 3D ).

In conclusion, UCF cells can proliferate continuously in vitro after being infected by Large T virus or telomerase virus hTERT.

Embodiment 4, the preparation of Large T-rtTA-UCF cells

After the purity of the immortalized Large T-UCF cells was tested by flow cytometry, the inventors further introduced rtTA to prepare Large T-rtTA-UCF (also known as 0916-UCFT-rtTA) cells.

Investigate the appropriate MOI for rtTA virus infection, and set five MOI conditions of 10, 20, 50, 100, and 150, respectively. The results of q-PCR detection showed that with the increase of MOI, the expression level of rtTA gene gradually increased, but after increasing to a certain extent, that is, after MOI=100, the expression level did not increase even if the MOI value was increased (Figure 4).

Therefore, take MOI=100 as the infectious titer of rtTA virus.

Example 5, Preparation of Large T-rtTA-TRE-A3-1A-4A-UCF cells

Using Large T-rtTA-UCF cells to further investigate the appropriate MOI of TRE-A3-E2A-1A-P2A-4A virus infection, the MOI was set to 1, 10, 20, 50, and 100 respectively. After virus infection, the cells contained 1ug/mL Doxycycline was induced in HFM medium for 3 days, and the positive rate of HNF1A cells was counted (Figure 5A).

The immunofluorescence detection and statistical results of HNF1A showed that with the increase of MOI, the positive rate of HNF1A cells gradually increased and tended to be stable (Fig. 5B-C). value, the expression level no longer increased significantly, and even decreased when it was too high. Therefore, MOI=20 is selected as the suitable infectious titer of TRE-A3-E2A-1A-P2A-4A virus.

After Large T-rtTA-UCF cells were infected with TRE-A3-1A-4A virus, Doxycycline was used to induce and culture Large T-rtTA-TRE-A3-E2A-1A-P2A-4A-UCF to further verify its FOXA3, HNF4A, HNF1A, ALB and AAT expression and testosterone metabolism, immunofluorescence results showed that Large T-rtTA-TRE-A3-E2A-1A-P2A-4A-UCF induced by HMM medium containing 1ug/mL Doxycycline had a certain amount of FOXA3 , HNF4A, HNF1A, ALB and AAT expression (Fig. 6A).

However, in further research and analysis, the inventors found that the protein secretion of ALB and AAT were only 600ng/24hrs/million cells and 300ng/24hrs/million cells respectively (Figure 6B). Further testosterone metabolism results showed that the testosterone metabolism capacity of the cells was only about 30% ( FIG. 6C ). It can be seen that these indicators still have room for further improvement.

In summary, Large T-rtTA-UCF cells infected with TRE-A3-E2A-1A-P2A-4A can express FOXA3, HNF4A, HNF1A, ALB and AAT after being induced by Doxycycline. On the other hand, there is still a relatively weak ability to metabolize testosterone, and there is room for further improvement.

Example 6, Preparation of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells

In order to solve the shortcomings of the large T-rtTA-TRE-A3-1A-4A-UCF cells with low HNF4A expression level and weak testosterone metabolism ability, the inventors further explored an improved solution.

The inventors conducted various experimental analyzes and researches to explore a feasible method for improving the metabolism of testosterone in cells and at the same time increasing the protein secretion of ALB and AAT. In the process of repeated experiments, the inventors unexpectedly found that it is beneficial to further introduce the TRE-4A expression vector in the Large T-rtTA-TRE-A3-1A-4A-UCF cells of Example 5, which may be due to the presence of these factors in The cells are expressed according to a certain ratio, and this introduction is conducive to a more coordinated expression.

From more alternative strategies, the inventors optimized and selected to re-introduce the TRE4A expression vector independently into the Large T-rtTA-TRE-A3-1A-4A-UCF cells, that is, to further infect the TRE4A virus and obtain the Large T- rtTA-TRE-A3-1A-4A-TRE4A-UCF cells.

The appropriate MOI for TRE4A infection was investigated. The MOI was set to 1, 10, 20, 50, and 100 respectively. After virus infection, the cells were induced by Doxycycline for 3 days and the positive rate of HNF1A cells was counted ( FIG. 7A ). The immunofluorescence detection and statistical results of HNF4A showed that with the increase of MOI, the positive rate of HNF4A cells gradually increased, but after increasing to a certain extent, that is, after MOI=10, the expression level decreased when the MOI value was increased (Fig. 7B and Figure 7C). Therefore, MOI=10 is the suitable infectious titer of TRE4A virus.

After the Large T-rtTA-TRE-A3-1A-4A-UCF cells were infected with the appropriate TRE4A virus, the HMM medium Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF containing 1ug/mL Doxycycline was used to further verify its The expression of FOXA3, HNF4A, HNF1A, ALB and AAT and the metabolic capacity of testosterone.

Immunofluorescence results showed that after being induced by Doxycycline, Large T-rtTA-TRE-A3-1A-4A-TRE4A UCF presented a more ideal expression ratio of FOXA3, HNF4A, and HNF1A, which promoted the expression of ALB and AAT significantly lift (Fig. 8A).

According to the average level of overall cell protein secretion, the protein secretion of ALB and AAT are 1800ng/24hrs/million cells and 2100ng/24hrs/million cells respectively (Figure 8B), which have very high secretion.

Further testosterone metabolism results showed that the testosterone metabolism capacity of the cells was increased to 80% ( FIG. 8C ).

In summary, after the Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells are further introduced into TRE-4A, induced by Doxycycline, it can promote the effective expression and effect of exogenous genes in the cells, or help The improvement of the activity of exogenous proteins, or the promotion of better cooperation between exogenous proteins, thereby significantly improving the ability of cells to express ALB and AAT, and significantly improving the ability to metabolize testosterone.

Example 7, Monoclonal selection of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells and identification of positive cell lines

In this embodiment, the purity of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells for the preparation of inducible hiHep (D-hiHep) was further improved. -4A-TRE4A-UCF cells were selected for monoclonal selection, Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were selected by the Mouth pipette into a 96-well plate, and the cells were passaged and positive clones were performed after the cells were full Strain identification (Figure 9A). The results of immunofluorescence identification showed that the positive clones were #6, #8, #14 and #85 (Fig. 9B).

In summary, Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells can be selected through monoclonal selection to obtain cell clones that can efficiently produce D-hiHep.

Example 8. In vivo and in vitro functional identification of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF monoclonal cell line

In vitro and in vivo functional identification of the positive clone of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells.

Immunofluorescence staining results showed that the positive clones of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells were induced by Doxycycline; the positive clones (#85 in the figure) had higher levels of FOXA3, HNF4A, HNF1A, Expression of ALB, AAT and FABP (Figure 10A); ALB and AAT secretion protein ELISA detection results showed that the positive clones had strong ALB and AAT secretion ability (Figure 10B); testosterone metabolism results showed that the positive clones had up to 100% Testosterone metabolism capacity of the testosterone (Fig. 10C).

Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cell #85 clone was expanded in vitro and induced into D-hiHep cells by Doxycycline, and D-hiHep cells were microencapsulated by sodium alginate in vitro , D-hiHep cells had ideal morphology after microencapsulation ( FIG. 11A ).

The Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cell clone #85 was transplanted into concanavalin A-induced acute liver failure mice at a cell number of 1X10 ⁷ , and acute liver failure after transplantation The survival rate of the mice was about 60%, which was significantly improved compared with the control group (25%) ( FIG. 11B ).

In summary, the positive clones of Large T-rtTA-TRE-A3-1A-4A-TRE4A-UCF cells can be efficiently induced into hiHep cells in vitro, and D-hiHep cells can secrete ALB and AAT in vitro, and have a strong Testosterone metabolism ability, transplanted into the abdominal cavity of mice with acute liver failure can effectively improve the survival rate of diseased mice.

In addition, through observation and routine experiments, the hepatic parenchymal cells obtained above also have the following properties: forming epithelial-like cell clones; accumulating liver glycogen; transporting acetylated low-density lipoprotein.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

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Claims (10)

1. A method of preparing an induced hepatocyte, the method comprising:

(1) Preparing immortalized cells by taking fibroblasts as initial cells;

(2) In the cell of (1), the following elements are introduced: antisense Tet transcriptional activator;

(3) In the cells of (2), the following series of elements are introduced: tetracycline regulatory elements, FOXA3, HNF1A, HNF4A;

(4) In the cells of (3), the following series of elements are introduced: tetracycline regulatory elements, HNF4A, thereby obtaining inducible hepatic parenchymal cells.

2. The method of claim 1, wherein the method further comprises the step of: treating the cells of (4) with doxycycline or 4-epi doxycycline to induce the cells to express FOXA3, HNF1A, HNF4A; preferably, the induction is carried out for 0.5 to 10 days; or (b)

The method further comprises the steps of: and (3) subculturing the hepatic parenchymal cells obtained in the step (4) to obtain the subcultured cells.

3. The method of claim 1, wherein in (1), the fibroblast is an umbilical cord fibroblast; or, the fibroblast is a mammalian-derived fibroblast; and/or

(1) In (3) immortalized cells are prepared by introducing SV40 large T antigen or by introducing human telomerase reverse transcriptase.

4. The method of claim 1, wherein in (3), the FOXA3 and HNF1A are connected by 2A between HNF1A and HNF 4A; preferably, the FOXA3 and HNF1A are connected by E2A; preferably, the HNF1A and HNF4A are connected by T2A.

5. The method of claim 1 or 3, wherein the element, SV40 large T antigen or human telomerase reverse transcriptase is introduced using a virus; preferably, the virus comprises: lentiviruses, adenoviruses, adeno-associated viruses; preferably, the element is constructed in a viral vector, packaged into a virus, infected cell; more preferably, the virus is a lentivirus; more preferably, at the time of infection:

(2) The MOI is 10 to 120, preferably 10 to 110;

(3) The MOI is 8-100, preferably 10-60; or (b)

(4) The MOI is 5 to 100, preferably 6 to 60, more preferably 8 to 12.

6. A construct for preparing an induced hepatic parenchymal cell, comprising:

construct 1 comprising a polynucleotide encoding an antisense Tet transcriptional activator;

construct 2 comprising polynucleotides encoding the following sets of elements in tandem: tetracycline regulatory elements, FOXA3, HNF1A, HNF4A;

construct 3 comprising polynucleotides encoding the following sets of elements in tandem: a tetracycline regulatory element, HNF4A;

construct 4, which contains a polynucleotide encoding an SV40 large T antigen or a human telomerase reverse transcriptase, is also preferably included.

7. An induced liver parenchymal cell transfected with the construct of claim 6; preferably, the induced liver parenchymal cells are prepared by the method of any one of claims 1 to 5.

8. Use of the induced liver parenchymal cells of claim 7 for:

(a) Preparing a composition for promoting liver regeneration or treating liver injury;

(b) As an in vitro model, a study of liver-related diseases or drugs was performed; preferably, the study of liver-related diseases or drugs comprises: study drug delivery, drug metabolism, liver formation, liver regeneration, or hepatotoxicity testing, screening for hepatotoxic compounds, screening for compounds that modulate hepatocyte function; preferably, the method is used for preparing a hepatitis virus infection model, wherein the hepatitis virus infection model is used for screening anti-hepatitis virus medicines;

(c) Albumin and/or alpha 1-antitrypsin are prepared.

9. A composition comprising the induced hepatocyte of claim 7; and a pharmaceutically acceptable carrier.

10. A kit or kit comprising:

the construct of claim 6; or (b)

The induced liver parenchyma cell of claim 7; or (b)

The composition of claim 9;

preferably, doxycycline or 4-epi doxycycline is also contained therein.

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