CN108070567A - A kind of urine derived cell strain of immortalization and its construction method - Google Patents

Abstract

The invention discloses an immortalized urine-derived cell line and a construction method thereof, belonging to the field of biotechnology. The immortalized urine-derived cell line is obtained by transfecting the recombinant plasmid carrying the immortalized gene or DNA fragment into the urine-derived cell. The applicant transfected the SV40LT recombinant plasmid into urine-derived cells to construct an immortalized urine-derived cell line. And through comparison, a safer and more reliable gene editing method was selected to obtain immortalized urine-derived cell lines. When the immortalized urine-derived cell line was cultured to the 35th generation, the cell morphology was very young, and there was still no obvious aging phenomenon; the cells almost showed logarithmic growth, which was the growth rate of the primary urine-derived cell cultured to the 4th generation. 3 times. The identification analysis showed that all indicators of the immortalized urine-derived cell line were normal. After being frozen for 1 month, the cell adhesion rate is as high as 95%, and it can grow and pass passage stably for a long time.

Description

A kind of immortalized urine-derived cell line and its construction method

technical field

The invention belongs to the field of biotechnology, and in particular relates to an immortalized urine-derived cell line and a construction method thereof.

Background technique

A large number of existing studies have shown that due to the influence of eating habits and social environment, contemporary people have a variety of diseases in the urinary tract system (including bladder and kidney). Acute renal failure, hypertensive nephropathy, etc., and bladder tumors caused by exposure to benzidine and other chemical substances due to occupational exposure and other factors, and so on. Researchers who conduct research on these diseases related to the urinary tract system usually obtain research objects through biopsy sampling. This invasive behavior will cause related trauma and local tissue bleeding to the donor bladder or kidney. , infection, or donor discomfort, which often lead to patient resistance. The cells collected from urine are also derived from the squamous epithelium of the renal pelvis, ureter, bladder, and anterior urethra, which can be used as an alternative in vitro model system for research on diseases related to the urinary tract system. In addition, urine-derived cells are collected and cultured from urine, and the operation method is relatively simple and non-invasive. Therefore, urine-derived cells are good in vitro models for diseases of the urinary tract system. Researchers can use urine-derived cell models to study related urinary tract system diseases. Therefore, collecting urine-derived cells from different donors is very important for the construction of different urine-derived cell models and related research on urinary tract system diseases. Urine-derived cells are a kind of primary cells, which are obtained by collecting and culturing donor urine. However, the success of collecting and culturing urine-derived cells from donor urine is restricted by many factors. Factors such as volume of urine sample, pH value, osmotic pressure, age of urine donor, oxalic acid content in urine, amylase activity in urine, and time of urine retention in the bladder, etc., if one of them fails to meet The optimal state will affect the results of urine-derived cell collection and culture. R. Belik, W et al. have reported that only 0-6300 urine-derived cells can be collected from 1L of urine, and the amount of urine-derived cells collected in most samples is less than 1000, and in urine samples Only 37% of all urine samples contained urine-derived cells that could be collected and survived. In addition, even if there is an appropriate number of urine-derived cells in the sample, there are many factors that restrict the obtaining of urine-derived cells during the culture process. Unlike other conventional primary cells, urine-derived cells are not derived from tissues or organs. Because of their small base, they need to undergo multiple in vitro divisions and proliferations to obtain the amount of cells we need. However, in the case of a small amount of cells in the early culture of cells, the urine-derived cells will be in a longer incubation period, and it is difficult to enter the logarithmic growth phase, so the cells are prone to apoptosis and death. Moreover, as a primary cell, the urine-derived cell itself has a very limited number of passages in vitro. Generally, the cells gradually age at the 6th to 8th passage and cannot be used for further passage. Research on diseases, drug screening and diagnosis of urinary tract system. Therefore, in addition to research at the level of primary urine-derived cells, the construction of immortalized urine-derived cell lines can greatly solve the problem that primary urine-derived cells cannot be cultured for a long time in vitro, and can go further at the cellular level. Study diseases related to urinary tract system.

At present, viruses are mostly used as gene transfer carriers to introduce foreign genes into cells. Although the viral vectors have been modified many times and the toxic genes in them have been eliminated, there are still potential side effects when expressed in vivo, such as mutations. wild-type virus. In addition, repeated freezing and thawing of the virus will lead to a decrease in the virus titer, so each time the virus is used for infection, the virus packaging needs to be repackaged, resulting in the difference between batches of cells in the cell bank, and the virus operation needs to be performed under conditions of a higher safety level conduct. In the past ten years, the emergence of artificial nucleases using genetic engineering has achieved more efficient and more precise gene editing, greatly improved the efficiency of non-homologous end repair and homologous recombination, and brought new possibilities to the genetic modification of species. opportunity. Gene editing technologies include CRISPR-Cas9, TALEN, ZFN, CRISPR-Cpf1, other gene editing methods that cause DNA breaks and insert foreign DNA, DNA break-free homologous recombination, site-specific recombination and other gene editing methods. Editing technology has the characteristics of low cost, simple design, easy operation, and short experimental cycle. When it is used in the construction of immortalized cell lines, it will carry out precise and fixed-point modification of the genome in the smallest range, avoiding the greatest extent. When viruses are used as vectors for gene transfer to construct immortalized cells, they may cause cancerous changes to cells. Therefore, the construction of immortalized urine-derived cell lines using gene editing technology can retain the biological characteristics of primary urine-derived cells to the greatest extent.

Biobank is called Biobank, which mainly refers to the standardized collection, processing, storage and application of biological macromolecules, cells, tissues and organs of healthy or diseased organisms (including human organ tissues, whole blood, plasma, etc. ) and clinical, pathological, treatment, follow-up, informed consent and other data related to these biological samples and their quality control, information management and application systems. Biological sample banks include various types, in addition to common tissue and organ banks, such as blood banks, cornea banks, bone marrow banks, etc., also include cell lines of normal cells, genetic mutation cells, tumor cells and hybridoma cell lines (lines) (Department) Banks, these biological sample banks (Biobank) have played a very important role in promoting the research of major diseases such as blood diseases, immune system diseases, diabetes, and malignant tumors. Compared with European countries, my country's biological sample bank is not yet perfect and needs to be improved. At present, there is no establishment of related cell banks for immortalized urine-derived cell lines, and the acquisition of urine-derived cells is more convenient than other cells and has the characteristics of no trauma to the donor. Therefore, constructing a urine-derived cell line The cell bank of cell lines and the corresponding STR typing and HLA typing of the cells entering the bank, and the establishment of cell information files that can be tested, that is, the urine-derived cell bank, are very important for improving the construction of my country's biological sample bank. The establishment of a urine-derived cell bank can store human DNA biological data, genetic information systems, and information on the unique characteristics of human genotypes, and can provide high-quality samples for research on human diseases. It can analyze the genetic status of related diseases, analyze their causes and mechanisms, and provide scientific basis for the control, prevention and clinical treatment of diseases such as cancer, heart disease, diabetes and senile dementia. In addition, over the past 10 years, with the rapid development of frontier technologies such as genomics, functional genomics, high-throughput biochips, next-generation sequencing, bioinformatics, and high-throughput RNA, urine-derived cell banks can also be used for transformation An important tool for medical research, it can not only provide physical samples and standardized data information for basic research, but also provide reliable biological samples and data for the research and development of various drugs, diagnostics, biological products, health products, cosmetics and other products Information and analysis monitoring services, etc.

Therefore, in summary, it is necessary and significant to establish an immortalized urine-derived cell bank.

Contents of the invention

The purpose of the present invention is to provide an immortalized urine-derived cell line and its construction method and application.

The technical scheme that the present invention takes is:

An immortalized urine-derived cell line, the cell line is derived from a urine sample, and has the ability to proliferate infinitely or nearly infinitely in vitro.

Preferably, the immortalized urine-derived cell strain is constructed by introducing exogenous immortalized genes or DNA fragments into the urine-derived cells, and the immortalized genes or DNA fragments include SV40LT, Bmi1, E6, E7, At least one of p53 mutant, Myc, c-Jun and Mdm2.

Preferably, the immortalization gene is SV40LT or Myc.

Since SV40LT is a DNA sequence derived from the SV40 virus genome, it exists in the cell for a long time after being stably integrated with the host cell DNA, which may change the original characteristics of the primary cell and is potentially dangerous. The present invention has screened and studied some existing transfer methods, among which the method of gene editing is the best, which can well transfer the immortalized gene SV40LT into urine-derived cells, and pass them stably for 35 generations, while maintaining urine Normal karyotype of source cells.

A method for constructing an immortalized urine-derived cell strain comprises the following steps: transfecting a recombinant plasmid carrying an immortalized gene or a DNA fragment into a urine-derived cell to construct an immortalized urine-derived cell strain.

Preferably, the immortalization gene or DNA fragment includes at least one of SV40LT, Bmil, E6, E7, p53 mutant, Myc, c-Jun and Mdm2.

Preferably, the immortalization gene is SV40LT or Myc.

Preferably, the method of transfecting the recombinant plasmid carrying the immortalization gene or DNA fragment into the urine-derived cells includes using at least one of retroviral vectors, lentiviral vectors, and gene editing (ie, "site-directed gene knock-in") kind.

Preferably, use gene editing to construct immortalized urine-derived cells: find sgRNA, construct sgRNA into gRNA Cloning Vector (Plasmid 41824, Adegene) plasmid, then transform into DH5α competent cells, and pick monoclonal strains as colonies PCR, the strains with positive PCR results were inoculated into small extracts and then sent for sequencing, and the sequenced correct strains were then extracted into plasmids. Through recombination, the SV40LT gene was inserted into the Donor plasmid, and NruI enzyme digestion was performed to recover the SV40LTDonor fragment to obtain SV40LT recombination plasmid. The SV40LT recombinant plasmid was introduced into primary urine-derived cells by electroporation to obtain immortalized urine-derived cell lines. The applicant has verified that the immortalized urine-derived cell line obtained by recombining the immortalized gene SV40LT into urine-derived cells by gene editing (CRISPR) is the best, and can be stably passed down for 35 generations while maintaining urine Normal karyotype of fluid-derived cells.

Preferably, the specific target sgRNA in gene editing is as follows:

sgRNA-AAVS1-1: 5'TATAAGGTGGTCCCCAGCTCG 3' (SEQ ID NO: 1);

sgRNA-AAVS1-2: 5'AGGGCCGGTTAATGTGGCTC 3' (SEQ ID NO: 2).

Preferably, the sgRNA-AAVS1 guides the Cas9 nuclease to precisely cut DNA during the gene editing process. AAVS1 is a validated safety site that ensures the intended function of the transferred DNA fragment. This site is an open chromosome structure, which can ensure the normal transcription of the transgenic gene. The CRISPR-Cas9 system targeting the AAVS1 site can specifically cut the AAVS1 site on human chromosome 19, generate DNA double-strand breaks, trigger the natural repair mechanism of DNA, and induce the occurrence between the site and the AAVS1 donor DNA clone Homologous recombination, which integrates a DNA segment from a donor clone into a safe site on the genome.

Preferably, the Cas9 nuclease is expressed by the Cas9-D10A expression plasmid during gene editing. The Cas9-D10A nickase paired with a pair of sgRNAs can reduce unintended off-target genetic modifications. It creates nicks on the two opposite DNA strands, forming functional double-strand breaks. This design minimizes off-target effects while maintaining efficient and specific gene modification.

Preferably, during the gene editing process, the DNA damage is repaired at the DNA break position by the Donor recombination fragment. The Donor recombination fragment can more precisely insert the target fragment into the safe site.

Preferably, the immortalized urine-derived cell line is constructed using a lentiviral vector: the SV40LT gene fragment is obtained from the 293T/17 cell gene by PCR technology, and the SV40LT gene fragment is combined with the pSin-EF2 vector plasmid by the method of gene recombination technology, Construct pSin-EF2-SV40LT, and identify the recombinant plasmid by enzyme digestion. The recombinant plasmid is introduced into the urine-derived cell by the method of calcium phosphate transfection, so as to obtain an immortalized urine-derived cell line.

Preferably, utilize the retroviral vector to construct the immortalized urine-derived cell line: obtain the SV40LT gene fragment from the 293T/17 cell gene by PCR technology, combine the SV40LT gene fragment with the pBabe-puro vector plasmid by the method of genetic recombination technology , constitute pBabe-puro-SV40LT, and identify the recombinant plasmid by enzyme digestion. The recombinant plasmid is introduced into the urine-derived cell by the method of calcium phosphate transfection, so as to obtain an immortalized urine-derived cell strain.

The application of the immortalized urine-derived cell line described in any one of the above in the construction of a bank of the immortalized urine-derived cell line.

The beneficial effects of the present invention are:

The applicant of the present invention transfected the recombinant plasmid containing the immortalized gene or DNA fragment into the urine-derived cell, constructed the immortalized urine-derived cell line, and applied it to the construction of the immortalized urine-derived cell line.

Through comparison, the applicant chose a safer and more reliable gene editing method to transfect the immortalized gene SV40LT into urine-derived cells to obtain the immortalized urine-derived cell line GE-HUC. When the immortalized urine-derived cell line GE-HUC of the present invention is cultured to the 35th generation, the cell morphology is very young, and there is still no obvious aging phenomenon. However, when the primary urine-derived cells were cultured to the 4th passage, the cell morphology was obviously aging. The immortalized urine-derived cell line GE-HUC cultured to the 35th generation in the present invention almost shows logarithmic growth, which is three times the proliferation speed of the primary urine-derived cell cultured to the 4th generation. Through the analysis of karyotype identification and tumorigenicity detection, it is found that all indicators of the immortalized urine-derived cell line GE-HUC obtained by gene editing in the present invention are normal. After it was frozen, it was revived and cultured after 1 month. The adherence rate of the cells after revival was as high as 95%, and they could grow and pass passage stably for a long time.

Description of drawings

Figure 1 is a morphological diagram of the collection and culture process of primary urine-derived cells;

Figure 2 is a cell morphology diagram of primary urine-derived cells cultured to the fourth generation;

Figure 3 is a cell morphology diagram of the immortalized urine-derived cell line GE-HUC cultured to the 35th generation;

Figure 4 is a cell morphology diagram of the immortalized urine-derived cell line LV-HUC cultured to the 35th generation;

Figure 5 is a cell morphology diagram of the immortalized urine-derived cell line RV-HUC cultured to the 35th generation;

Figure 6 is a comparison chart of MTT results;

Fig. 7 is the normal karyotype diagram of the immortalized urine-derived cell line;

Figure 8 is a diagram of the abnormal karyotype of the immortalized urine-derived cell line LV-HUC;

Figure 9 is a map of the abnormal karyotype of the immortalized urine-derived cell line RV-HUC;

Figure 10 is the primary urine cell β-galactosidase staining diagram;

Figure 11 is a staining diagram of immortalized urine-derived cell line GE-HUCβ-galactosidase;

Figure 12 is a staining diagram of immortalized urine-derived cell line LV-HUCβ-galactosidase;

Figure 13 is the staining diagram of the immortalized urine-derived cell line RV-HUCβ--galactosidase;

Detailed ways

The present invention will be further described below in conjunction with the examples, but not limited thereto.

The specific gene accession numbers and product related information in the present invention are as follows:

Table 1 Gene accession number

Table 2 Information about the products used

name brand Item No. p53 mutant Prospec PRO-301

Example 1, Collection and Culture of Primary Urine-derived Cells

Collect 10ml-2L of urine, centrifuge (1010g, 5min) to discard the supernatant, resuspend the pellet with PBS containing double antibody and centrifuge again to discard the supernatant, then use the urine-derived cell culture medium to resuspend the pellet to the cells Culture plate (24-well plate), and add 100 μg/ml primocin, then place it in a 37°C, 5% CO ₂ cell incubator for culture, do not perform other operations on it during the period, change the medium after 5 days, and wait for the cell clone to grow to A certain size for digestion and passage. The morphology of the primary urine-derived cells collected in culture is shown in Figure 1. In Figure 1, A is the adherent state of the urine-derived cells collected and cultured on the fifth day under the microscope, and in Figure 1, B is the urine-derived cells The morphology of the cells cultured to the 12th day was collected, and it can be seen from the figure that the cells were in the shape of rice grains.

The collected primary urine cells were cryopreserved with cryopreservation solution (fetal bovine serum and dimethyl sulfoxide at a ratio of 9:1), and then placed in -196°C liquid nitrogen for cryopreservation after gradient cooling. Simultaneous identification of STR typing and HLA typing.

Embodiment 2, the construction of the immortalized urine-derived cell line

Methods for introducing foreign genes or DNA fragments into cells and integrating them into the cell genome include methods using gene editing, lentiviral vectors, retroviral vectors, and the like.

1) Gene editing method

1.1) Find out the specific target sgRNA

sgRNA-AAVS1-1: 5'TATAAGGTGGTCCCCAGCTCG 3' (SEQ ID NO: 1);

sgRNA-AAVS1-2: 5'AGGGCCGGTTAATGTGGCTC 3' (SEQ ID NO: 2).

1.2) Construct sgRNA into vector plasmid gRNA Cloning Vector

1.2.1) Vector preparation: digest gRNA Cloning Vector (Plasmid 41824, Adegene) with AflII, then use mung bean nuclease (#M0250S, NEB) to eliminate the protruding end, and finally dephosphorylate the end of the vector.

1.2.2) Design and prepare the annealed fragment of sgRNA

sgRNA-AAVS1-1: 5'TATAAGGTGGTCCCCAGCTCG 3' (SEQ ID NO: 1);

①. Clone-sgRNA-AAVS1-1-a: 5'TTGTGGAAAGGACGAAACACCGTATAAGGTG 3' (SEQ ID NO: 3);

②. Clone-sgRNA-AAVS1-1-b: 5'GTCCCAGCTCGGTTTTAGAGCTGAAATAGCAA 3' (SEQ ID NO: 4);

③. Clone-sgRNA-AAVS1-1-c: 5'CGGTGTTTCGTCCTTTCCCAA3' (SEQ ID NO: 5);

④. Clone-sgRNA-AAVS1-1-d: 5'TTGCTATTTCTAGCTCTAAAACCGAGCTGGGACCACCTTATA3' (SEQ ID NO: 6);

sgRNA-AAVS1-2: 5'AGGGCCGGTTAATGTGGCTC 3' (SEQ ID NO: 2);

⑤. Clone-sgRNA-AAVS1-2-a: 5'TTGTGGAAAGGACGAAACACCGAGGGCCGGT 3' (SEQ ID NO: 7);

⑥.Clone-sgRNA-AAVS1-2-b: 5'TAATGTGGCTCGTTTTAGAGCTAGAAATAGCAA 3'(SEQ ID NO: 8);

⑦. Clone-sgRNA-AAVS1-2-c: 5'CGGTGTTTCGTCCTTTCCCAA3' (SEQ ID NO: 9);

⑧. Clone-sgRNA-AAVS1-2-d: 5'TTGCTATTTCTAGCTCTAAAACGAGCCACATTAACCGGCCCT3' (SEQ ID NO: 10).

①②③④ and ⑤⑥⑦⑧ are annealed respectively, and then the end is treated with phosphorus.

1.2.3) Construction of SV40LT recombinant vector

Connect the vector and the annealed product and transform it into DH5a competent cells, spread evenly on the plate containing antibiotics after incubation, culture overnight in the incubator, pick the monoclonal strains for colony PCR, and the strains with positive electrophoresis results are inoculated with small extracts Sent for sequencing. After the sequencing is correct, the plasmid is extracted. After purification, the SV40LT gene fragment is obtained. Through recombination, the SV40LT gene is inserted into the Donor plasmid (with puromycin resistance gene Puro(R)), and digested with NruI SV40LT-Puro Donor fragments were recovered after treatment.

1.3) Transform SV40LT-Puro Donor into cells

1.3.1) Resuscitate the primary urine-derived cells into a 10 cm dish, and perform electroporation when the cells are cultured to a confluence of about 90%.

1.3.2) The primary urine-derived cells were digested with 0.25% trypsin, and terminated with serum-containing medium. After the termination of the cells, 3 million cells were transferred to a 15ml centrifuge tube, and the supernatant was discarded by centrifugation.

1.3.3) Add 82 μl Basic Nucleofector Solution For Mammalian Epithelial Cells and 18 μl supplement 1 and mix gently, then add plasmid Cas9D10A (5 μg), sgRNAclone AAVS1 (3.5 μg), SV40LT-puro (3.5 μg). Then use a 200 μl medium gun to mix the whole system and transfer the system to the electroporation cup, then transfer the electroporation cup to the electrotransfer instrument and use the T-020 program for electroporation.

1.3.4) Transfer the electroporated cells to a 6-well plate and culture them with 3ml of primary urine cell culture medium. After the transfer, place the cells in a 37°C, 5% CO ₂ incubator for culture.

1.4) Cell purification

Replace the urine-derived cells after electroporation with the urine cell culture medium containing 1.5 μg/ml puromycin (puromycin) for selection and culture. The urine-derived cells that have not been transferred to the SV40LT Donor fragment will gradually die. The urine-derived cells of the SV40LT Donor fragment can continue to grow in the presence of puromycin, and an immortalized urine-derived cell line was successfully established, which was named GE-HUC.

2) Lentiviral vector method

2.1) Construction of SV40LT gene eukaryotic expression vector

Using the 293T/17 cell genome as a template by PCR technology, the SV40LT gene fragment was obtained, and the SV40LT gene fragment was recombined with the pSin-EF2 vector by gene recombination technology to construct the pSin-EF2-SV40LT plasmid, and the recombinant plasmid was identified by sequencing.

2.2) Cell transfection

2.2.1) Inoculate 293T/17 cells into two wells of a six-well plate, and transfect after 24 hours when the cell attachment rate reaches about 70%.

2.2.2) Configure the following transfection system.

Add each component according to the above system, add 2xHBS at the end, quickly blow 30 times with a pipette, and add 500 μl per well of 293T/17 to be transfected after standing for 2 minutes. Before transfection, replace each well with fresh MEF medium 1.5 ml.

2.2.3) Change the medium 6 hours after transfection, add fresh medium, and place in a 37°C, 5% CO ₂ incubator to continue culturing.

2.3) Cell infection

2.3.1) Inoculate the urine-derived cells into a 6 cm dish, and infect after 24 hours when the cell adhesion rate reaches about 30%.

2.3.2) After 24 hours of transfection, collect the virus-containing medium supernatant and filter it with a 0.45 μm filter membrane, take 3.5ml of urine cells infected for the first time, and add polybrane at a ratio of 1:1000. Change 1.5ml of urine cell culture medium, add fresh MEF medium to 293T/17 to continue culturing, replace the fresh medium after 6 hours of urine cell infection, and continue culturing in a 37°C, 5% CO ₂ incubator.

2.3.3) After 48 hours of transfection, collect the virus-containing supernatant again, filter and infect the urine cells for the second time, replace with fresh medium after 6 hours, and place in a 37° C., 5% CO ₂ incubator to continue culturing.

2.4) Cell purification

The urine-derived cells infected with the virus were screened and cultured with a urine cell culture medium containing 1.5 μg/ml puromycin (puromycin), and the urine-derived cells that were not transferred to the pSin-EF2-SV40LT plasmid would gradually die, successfully The urine-derived cells transformed with the pSin-EF2-SV40LT plasmid can continue to grow in the presence of puromycin, and an immortalized urine-derived cell line was successfully established, which was named LV-HUC.

3) Retroviral vector method

3.1) Construction of SV40LT gene eukaryotic expression vector

The SV40LT gene fragment was obtained by using the 293T/17 cell genome as a template by PCR technology, and the SV40LT gene fragment was recombined with the pBabe-puro vector by gene recombination technology to construct the pBabe-puro-SV40LT plasmid, and the recombinant plasmid was identified by sequencing.

3.2) Cell transfection

3.2.1) Inoculate 293T/17 cells at 1.2×10 ⁶ into a 10 cm dish, and transfect after 24 hours.

3.2.2) Configure the following transfection system.

Add each component according to the above system, and finally add 2xHBS drop by drop, let it stand for 20 minutes, add 293T/17 to be transfected, and replace P100 with 9ml of fresh MEF medium before transfection.

3.2.3) Change the medium 6 hours after transfection, add fresh medium, and place in a 37°C, 5% CO ₂ incubator to continue culturing.

3.3) Cell infection

3.3.1) Inoculate primary urine-derived cells into a 6 cm dish, and infect after 24 hours when the cell adhesion rate reaches about 30%.

3.3.2) After 24 hours of transfection, collect the virus-containing medium supernatant and filter it with a 0.45 μm filter membrane, take 3.5ml of urine cells infected for the first time, and add polybrane at a ratio of 1:1000. Change 1.5ml of urine cell culture medium, add fresh MEF medium to 293T/17 to continue culturing, replace the fresh medium after 6 hours of urine cell infection, and continue culturing in a 37°C, 5% CO ₂ incubator.

3.3.3) After 48 hours of transfection, collect the virus-containing supernatant again, filter and infect the urine cells for the second time, replace with fresh medium after 6 hours, and place in a 37° C., 5% CO ₂ incubator to continue culturing.

3.4) Cell purification

Replace the virus-infected urine-derived cells with the primary urine cell culture medium containing 1.5 μg/ml puromycin for screening and culture, the urine-derived cells that have not been transferred to the pBabe-puro-SV40LT plasmid will gradually die, and successfully transferred to The urine-derived cells carrying the pBabe-puro-SV40LT plasmid can continue to grow in the presence of puromycin, and an immortalized urine-derived cell line was successfully established, which was named RV-HUC.

Immortalized urine-derived cell lines can also be obtained by replacing the SV40LT gene with Myc.

Embodiment 3, monoclonal culture of immortalized urine-derived cell lines

The immortalized urine-derived cell lines GE-HUC, LV-HUC and RV-HUC constructed in the above-mentioned Example 2 were respectively subjected to single-cell clone culture.

1) Digest the immortalized urine-derived cell line that has been cultured to the fourth generation to a single cell line with trypsin, centrifuge the supernatant after stopping with the medium, resuspend with the medium for counting, and dilute the cells to 500/ ml, take 0.2ml and suspend in 15ml medium, seed the plate with a row gun, and add 150μl cell suspension to each well of a 96-well plate.

2) After 2 hours, observe under a microscope after the cells adhere to the wall, and record the position of the well with only one cell.

3) After 24 hours, observe the marked wells under a microscope, and confirm that the wells with only one clone are single-cell clones.

4) Observe single-cell clones every day, change the medium every other day, and transfer to a larger culture dish to continue culturing after the number of cells increases, so as to obtain single-cell clones of immortalized urine-derived cell lines.

Culture results: Under the culture of urine cell culture medium containing puromycin, GE-HUC obtained 25 monoclones of immortalized urine cell lines, LV-HUC obtained 32 monoclones of immortalized urine cell lines, RV- HUC obtained 30 monoclonal immortalized urine cell lines.

Example 4. Morphological observation and verification of immortalized urine-derived cell lines

1) Morphological observation of the immortalized urine-derived cell line cultured to passage 35

The immortalized urine-derived cell lines were cultured in culture dishes with the urine cell culture medium containing puromycin, and the culture medium was replaced every two days. After the cells grew to about 80%, they were passaged, and the comparison culture was observed under a microscope. Morphology of primary urine-derived cells up to passage 4 and immortalized urine-derived cells cultured up to passage 35.

Figure 2 shows the cell morphology of primary urine-derived cells cultured to the 4th passage. It can be clearly seen that the cells are in poor adherence state and have aged. Figure 3, Figure 4, and Figure 5 are the immortalized urine-derived cell lines GE -HUC, LV-HUC, and RV-HUC were cultured to the 35th generation of cell morphology, and the immortalized urine-derived cell lines were all smaller in shape, adhered to the wall in a good state, and still had no obvious aging phenomenon.

2) MTT method to detect cell proliferation rate

The principle of MTT detection is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit in the cells, while dead cells have no such function. DMSO can dissolve formazan in cells, and its light absorption value is measured with a microplate reader. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of living cells. The number of living cells was judged according to the measured absorbance value (OD value).

The primary urine-derived cells cultured to the 4th passage and the immortalized urine-derived cells GE-HUC, LV-HUC and RV-HUC cultured to the 35th passage were seeded in ⁹⁶ -well plates at 2x103 cells per well , 150 μl medium per well, 5 duplicate wells in each group, after culturing 24h, 48h, 72h, 96h and 120h respectively, add 15μl tetramethylazozolium salt (MTT, 5mg/ml) to each well, and continue to cultivate for 4 Discard all the medium after 1 hour, add 150 μl dimethyl sulfoxide (DMSO), shake on a shaker for 10 minutes, measure the OD value of each well with a microplate reader, and calculate the average value of each group for comparison.

The results of MTT (Figure 6) show that: Group A is the 4th generation of primary urine-derived cells, and groups B, C, and D are the 35th generation of immortalized urine-derived cells GE-HUC, LV-HUC, and RV-HUC, respectively , the results showed that the growth of cells in group A slowed down significantly in the later period, and the cells in groups B, C, and D almost showed logarithmic growth, and the OD values of groups B, C, and D were almost three times that of group A by the fifth day.

3) Karyotype identification

Single-cell clones of the immortalized urine-derived cell lines cultured in Example 3 were randomly selected, seven GE-HUC, LV-HUC and RV-HUC each, and cultured to the 35th generation for karyotype identification.

3.1) Cell preparation

The growth state is good, and the growth density is between 80% and 90%.

3.2) Colchicine treatment

Before the termination of the culture, add colchicine with a final concentration of 0.2 μg/mL to the amount of the culture medium, and treat in a 37° C. incubator for 100 to 130 minutes.

3.3) Hypotonic treatment

After the colchicine treatment, first discard the culture medium, wash twice with PBS, add 0.25% trypsin to digest, gently tap the culture dish to make the unshedding cells fall off, add MEF to stop the digestion, and transfer to 15mL with a pipette Centrifuge the tube, centrifuge (1200rpm, 5min) to collect the cells, then add 7mL of 0.075mol/L KCL solution preheated at 37°C, pipette into a cell suspension, and place in a 37°C water bath for 18-28min.

3.4) Pre-fixation and fixation

Freshly prepare Carnot's fixative solution (methanol and glacial acetic acid at a ratio of 3:1), and add about 1 mL of fixative solution with a rubber dropper for pre-fixation for 10 min. After pre-fixation, centrifuge (1200rpm, 5min) to discard the supernatant, add about 7mL of fresh fixative, mix gently with a rubber dropper, and fix in a 37°C water bath for 40min.

3.5) Drop tablets

After the fixation is completed, centrifuge (1200rpm, 5min) and use a plastic dropper to discard most of the fixation solution, leave a part of the fixation solution (determine the amount of solution according to the amount of cells) to resuspend the cells, and put it 30cm above the frozen clean glass slide. The left and right distances are used to drop the tablets.

3.6) Baked slices

Immediately after dropping the slides, move the slides into an oven at 75°C for 3 hours.

3.7) Staining (G banding)

Add 0.03g of trypsin powder to 55mL of normal saline, shake gently, and adjust its pH to about 7.2 with 3% Tris. Put the preparation in trypsin digestion solution for 8 seconds, then quickly put in normal saline to stop its digestion, then put in Giemsa staining solution for staining for 5-10 minutes, then take out the slide with tweezers, rinse both sides gently with tap water, and leave at room temperature. Dry or blow dry.

3.8) Microscopic analysis

After the slides are dry, check under a microscope, first use a low-power lens to find a good division phase, and then use a high-power oil lens to observe, analyze the number of chromosomes and band patterns, and analyze a total of 20 division fields for each cell.

Analysis results: the karyotype of the immortalized urine-derived cell line GE-HUC was normal, and there were 2 abnormalities in the karyotype results of the immortalized urine-derived cell line LV-HUC (the abnormal rate was 28.6%). There was one abnormality in the karyotype results of the urine-derived cell line RV-HUC (the abnormality rate was 14.3%). Figure 7 is the result of normal karyotype of the immortalized urine-derived cell line, Figure 8 is the result of abnormal karyotype of the immortalized urine-derived cell line LV-HUC, and Figure 9 is the result of the immortalized urine-derived cell line RV - Abnormal karyotype result map of HUC.

4) Tumorigenicity detection

The primary urine-derived cells and A-498 cells cultured to the fourth generation in the logarithmic growth phase were taken, and then the immortalized urine-derived cells GE-HUC, LV- Seven strains of HUC and RV-HUC were digested with trypsin and prepared into single cell suspension with serum-free DMEM. 10 ⁷ /ml of single cell suspension was injected into the muscle or subcutaneous of NOD-SCID mouse limbs respectively, and then observed and cultured. One month later, the tumor formation or death of mice can be observed as follows:

Table 3 is the tumorigenicity detection of mice

The tumorigenicity test results in Table 3 show that the immortalized urine-derived cells (RV-HUC) obtained by the retroviral vector method have tumorigenic risks, and the immortalized urine-derived cells obtained by the lentiviral vector method The cells (LV-HUC) did not show tumorigenic risk in this tumorigenicity detection test, but would cause death to the mice, while the immortalized urine-derived cells (GE-HUC) obtained by the gene editing method did not Tumorigenicity and will not cause death in mice, indicating that the immortalized urine-derived cells obtained by gene editing methods can better maintain the original urine-derived cells than cells obtained by lentiviral vector method or retroviral vector method characteristic.

5) β-galactosidase activity

The 4th passage primary urine-derived cells and the 35th passage immortalized urine-derived cells GE-HUC, LV-HUC and RV-HUC were respectively digested and counted, and ^2x103 cells were plated in each well (24-well plate). Urine cell culture medium was used for culture, and Day7 was used for senescence detection. When performing aging detection, first add PBS for washing, and then wash with PBS again after fixation, then add freshly prepared β-galactosidase staining solution, incubate overnight at 37°C for staining, and observe the staining under a microscope the next day. Figure 10 is a staining diagram of primary urine cells, and Figures 11, 12, and 13 are staining diagrams of immortalized urine-derived cells GE-HUC, LV-HUC and RV-HUC, respectively. It can be observed from the figure that the primary urine-derived cells are already aging at an earlier generation, while the immortalized urine-derived cells are still relatively young after more generations of culture.

6) Detection of resuscitation efficiency

The immortalized urine-derived cells GE-HUC, LV-HUC and RV-HUC were routinely cultured in a urine cell culture medium containing puromycin in a 37°C, 5% CO ₂ incubator, and the cells were digested with 0.25% trypsin Passaging, cryopreservation of cells with cryopreservation solution (fetal bovine serum and dimethyl sulfoxide at a ratio of 9:1), and place the cells in liquid nitrogen at -196°C after gradient cooling. After 1 month, it was revived and cultured, and the adherence rate of the cells after revival was about 95%, and they could grow and pass passage stably for a long time.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Haoshenglai Biopharmaceutical Co., Ltd., Wang Linli

<120> An immortalized urine-derived cell line and its construction method

<130>

<160> 10

<170> PatentIn version 3.5

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Claims (7)

1. a kind of urine derived cell strain of immortalization, it is characterised in that：The cell line derives from urine specimen, possesses external Infinite multiplication or the ability close to infinite multiplication.

2. the urine derived cell strain of immortalization according to claim 1, it is characterised in that：The urine of the immortalization is come Source cell strain is that importing external source immutalizing gene or DNA segment are built-up in urine derived cell, the immutalizing gene Or DNA segment includes at least one of SV40LT, Bmi1, E6, E7, p53 mutant, Myc, c-Jun and Mdm2.

3. the construction method of the urine derived cell strain of a kind of immortalization, which is characterized in that comprise the following steps：It will carry forever The Transfected Recombinant Plasmid of biochemical gene or DNA fragmentation is into the urine derived cell strain that immortalization is built into urine derived cell.

4. construction method according to claim 3, it is characterised in that：The immutalizing gene or DNA fragmentation include At least one of SV40LT, Bmi1, E6, E7, p53 mutant, Myc, c-Jun and Mdm2.

5. according to claim 3-4 any one of them construction methods, it is characterised in that：Immutalizing gene or DNA will be carried The method of the Transfected Recombinant Plasmid of segment into urine derived cell includes the use of retroviral vector, slow virus carrier, gene At least one of editor.

6. construction method according to claim 5, it is characterised in that：The weight of immutalizing gene or DNA fragmentation will be carried Group plasmid transfection is into urine derived cell and to be integrated into the method for cellular genome be preferably gene editing.

7. the urine derived cell strains of any one of the claim 1-2 immortalizations is built in the urine derived cell strain of immortalization Application in storehouse.