CN106497882A - The cell strain of overexpression R spondin1 and Noggin and its construction method and application simultaneously - Google Patents

Abstract

The invention belongs to the field of molecular biology technology, and in particular relates to co-overexpressing R-spondin1 and Noggin cell line (CHO-Rspon-Noggin) and its construction method and application in intestinal stem cell culture. The cell line of the present invention overexpresses two genes of human R-spondin1 and human Noggin at the same time, and can stably secrete two cytokines R-spondin1 and Noggin in its cell supernatant, and the medium prepared with the supernatant can be cultivated Mouse intestinal stem cells and human intestinal stem cells and intestinal tumors, and grow into organoid "mini intestines" consistent with the source. "Mini intestines" have unique advantages in the study of intestinal stem cells, the development and metastasis of intestinal tumors, and the treatment of intestinal tumors including radiotherapy, chemotherapy, targeted therapy, etc. "Cultivation and follow-up research have broad application prospects.

Description

A cell line overexpressing R-spondin1 and Noggin and its construction method and application

technical field

The invention belongs to the technical field of molecular biology, and specifically relates to a co-overexpressing R-spondin1 and Noggin cell line (CHO-Rspon-Noggin) and its construction method and application in intestinal stem cell culture.

Background technique

As a member of the R-spondin family, R-spondin1 is a secreted protein that can promote the division of intestinal gland epithelial cells by regulating the Wnt/β-catenin signaling pathway, accelerate the regeneration of intestinal glands and small intestinal villi, and maintain the small and large intestine. The integrity of the intestinal mucosa can also regulate the in vitro 3D growth of intestinal crypts. Noggin is an inhibitor of BMP signaling pathway, and the expressions of Smad1, Smad5 and Smad8 in the BMP signaling pathway can be detected in the differentiated mature columnar epithelial cells on the surface of the intestinal cavity. As an inhibitor of BMP signaling pathway, Noggin can maintain the intestinal stem cell Dryness.

Intestinal organoids, or "mini intestines", are obtained from extracted intestinal crypts or tumor tissues in vitro using Matrigel to grow organoids similar to the source. Organoids can help us conduct research on intestinal stem cells, intestinal tumor development, and intestinal tumor treatment methods, including traditional radiotherapy, chemotherapy, and biological targeted therapy, and even emerging gene therapy research. However, the current culture of organoids mainly relies on expensive commercial cytokines (R-sponding1, Noggin, etc.), resulting in too high overall culture costs, which seriously hinders the research and promotion of organoids. The present invention involves a co-overexpression of R-spondin1 and Noggin cell line (namely CHO-Rspon-Noggin), which can simultaneously secrete these two cytokines into the culture supernatant and keep them active for a long time. The conditioned medium can replace commercial factors for the culture of intestinal organoids, and has broad application prospects in organoid culture and subsequent research.

Contents of the invention

The object of the present invention is to provide a cell line overexpressing R-spondin1 and Noggin at the same time and its construction method and application, including carrying out genetic engineering transformation on R-spondin1 and Noggin and performing lentiviral packaging and infecting CHO cells, and Method for collecting culture supernatant containing R-spondin1 and Noggin cytokines and application of the supernatant in intestinal stem cell culture.

The cell line provided by the present invention that can simultaneously overexpress R-spondin1 and Noggin, denoted as CHO-Rspon-Nog, can simultaneously overexpress two genes of human R-spondin1 and human Noggin, and can simultaneously and stably secrete the R-spondin1 and human Noggin Noggin two genes into its cell culture supernatant, and the medium prepared from the supernatant can stably culture mouse crypts and human intestinal normal crypts and tumor tissues. As shown in Figure 1 and Figure 2.

The method for constructing a cell line capable of simultaneously overexpressing R-spondin1 and Noggin provided by the invention, the specific steps comprising:

Two lentiviruses, pCDH-GFP-R-Spondin and pCDH-puromycin-Noggin, were packaged. 293T cells were used as tool cells for lentivirus packaging, and psPAX and pMD2.G were selected as auxiliary plasmids. The cells were collected 48 hours after transfection of the plasmids into 293T cells Supernatant, that is, lentivirus RsponV-GFP and NogginV-puro. CHO cells were infected with RsponV-GFP, and CHO-Rspon cells with high expression of GFP-Rspondin were screened by flow cytometry, and then CHO-Rspon cells were infected with NogginV-puro, and the screening pressure of 200ug/ml puromycin was used to select CHO cells with high expression of Noggin -Rspon-Noggin cells, and overexpression verification at mRNA and protein levels. As shown in Figure 3 and Figure 4.

The present invention also provides a construction and purification method of two lentiviral plasmids pCDH-GFP-R-Spondin and pCDH-puromycin-Noggin, said method comprising: construction of lentiviral vectors of two genes of human R-spondin1 and human Noggin: selection Two restriction sites, EcoRI and BamHI, amplify the CDS regions of R-spondin1 and Noggin genes, in which:

The upstream primer for PCR amplification of R-spondin1 gene is R-spondin F (5'-CCGGAATTCATGCGGCTTGGGCTGTGTGT-3'(SEQ.ID.NO.1), EcoRI)

The downstream primer for PCR amplification of R-spondin1 gene is R-spondin R (5'-GCGGGATCCCTAGGCAGGCCCTGCAGATG-3' (SEQ.ID.NO.2), BamHI);

The upstream primer for PCR amplification of Noggin gene is Noggin F (5'-CCGGAATTCATGGAGCGCTGCCCCAGCCT-3'(SEQ.ID.NO.3), EcoRI);

The downstream primer for PCR amplification of Noggin gene is Noggin R (5'- GCGGGATCCCTAGCACGAGCACTTGCACT-3' (SEQ.ID.NO.4), BamHI);

The full lengths of these two genes were connected to the expression plasmids pCDH-CMV-MCS-EF1-GFP and pCDH-CMV-MCS-EF1-puromycin respectively, and Escherichia coli DH5α was used to transform and amplify the plasmids; and they were named pCDH-GFP- Rspondin and pCDH-puromycin-Noggin.

The present invention also provides the application of the above cell line (CHO-Rspon-Noggin). include:

The method for collecting R-spondin1 and Noggin two cytokines, the method includes normal culture of CHO-Rspon-Noggin cells, after the cells grow to 100%, the ratio of 1:10 is passed, after the cells grow to 100%, the culture medium Change to a serum-free medium, continue culturing for one week, collect the medium, filter the culture solution with a 0.22um filter, and store it at -80°C.

The cell supernatant collected from the CHO-Rspon-Nog cell line was used to prepare the culture medium, and the method of culturing mouse and human intestinal crypts and tumor tissues was as follows: Prepare Advanced DMED/F12 medium containing 50% of the collected supernatant, And add other cytokines for 3D culture of mouse intestinal crypts; or prepare medium containing 20% collected supernatant for 3D culture of human intestinal normal crypts and tumor tissues. By culturing the two to grow into organoid tissues similar to the source - "mini intestines", and then carrying out gene interference on the "mini intestines", such as overexpression or knockout, etc., to carry out research on intestinal stem cells and intestinal tumors Research on the occurrence, development, metastasis and prognosis of intestinal tumors; or use drugs or radiation to treat the "mini intestines", and conduct research on the treatment of intestinal tumors, including chemotherapy, radiotherapy, targeted therapy, etc. Therefore, the CHO-Rspon-Noggin cell line has broad application prospects in the cultivation of "mini intestines" and subsequent research.

Description of drawings

Figure 1 is a schematic diagram of mouse crypts cultured with supernatants collected from CHO-Rspon-Nog cells and commercial cytokines.

Figure 2 is a schematic diagram of human intestinal normal crypts and tumor tissues cultured with the supernatant collected from CHO-Rspon-Nog cells.

Figure 3 is a schematic diagram of the expression of mRNA levels in CHO-Rspon-Nog cells.

Figure 4 is a schematic diagram of protein expression in CHO-Rspon-Nog cells.

detailed description

Below adopt specific embodiment to further illustrate content of the present invention.

The following content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Example 1: Construction method of lentiviral vectors pCDH-GFP-R-Spondin and pCDH-puromycin-Noggin

Through the method of high-fidelity PCR, two restriction sites of EcoRI and BamHI were selected, and the CDS regions of R-spondin1 and Noggin genes were amplified:

The upstream amplification primer of R-spondin is R-spondin F: (5'-CCGGAATTCATGCGGCTTGGGCTGTGTGT-3', EcoRI),

The downstream amplification primer of R-spondin is R-spondin R: (5'-GCGGGATCCCTAGGCAGGCCCTGCAGATG-3', BamHI),

Noggin’s upstream amplification primer is Noggin F (5’-CCGGAATTCATGGAGCGCTGCCCCAGCCT-3’, EcoRI),

The downstream amplification primer of Noggind is Noggin R (5'- GCGGGATCCCTAGCACGAGCACTTGCACT-3', BamHI

Then use the molecular cloning method to clone the full length of these two genes into two vectors, pCDH-CMV-MCS-EF1-GFP and pCDH-CMV-MCS-EF1-puromycin, respectively, and transform the recombinant vector into DH5α. For the selection pressure, transfer the recombinant strain to the LB solid medium containing ampicillin selection pressure, culture at 37°C for 12-16 hours, and select a single clone of the recombinant transfer strain to the LB liquid medium containing ampicillin selection pressure, Shake culture at 37°C for 12-16 hours, extract the plasmid and send it for sequencing. After confirmation, the full-length R-spondin1 and Noggin genes were cloned into pCDH-CMV-MCS-EF1-GFP and pCDH-CMV-MCS-EF1- puromycin two vectors, and named pCDH-GFP-Rspondin and pCDH-puromycin-Noggin.

Example 2. Construction method of CHO-Rspon-Noggin cell line overexpressing R-spondin1 and Noggin simultaneously

CHO-Rspon-Noggin cells were constructed by lentivirus infection, and 293T cells were used as lentivirus packaging cells. When the 293T cells grow to 50-60%, add the target gene plasmids (pCDH-GFP-Rspondin and pCDH-CMV-Noggin respectively), packaging plasmids psPAX and pMD2.G in the ratio of 4:3:1 to 293T In the cells, the virus supernatants of 293T cells were collected after 48 hours, named RsponV and NogginV respectively, and the virus supernatants were filtered with a 0.45um filter for subsequent experiments.

Taking CHO cells as the target cells, first infect the CHO cells with the virus supernatant containing RsponV, and select the cells with high expression of GFP by flow sorting. This is the CHO-Rspon cells with high expression of R-spondin1, and then the The virus supernatant containing NogginV was used to infect CHO-Rspon cells, and the CHO-Rspon-Noggin cells with high expression of Noggin were selected by the screening pressure of 200ug/ml puromycin, and Real-time PCR and Western Blotting were used to detect the mRNA and protein levels Verify the overexpression of R-spondin1 and Noggin.

Example 3. Collection of medium containing two cytokines R-spondin1 and Noggin

CHO-Rspon-Noggin cells were cultured in DMEM medium containing 10% FBS and 10ug/ml puromycin. After the cells grew to 100%, they were subcultured at a ratio of 1:10. Cultured in DMEM medium containing 10% FBS until the cells grew to 100% Afterwards, the medium was replaced with Advanced DMED/F12 serum-free medium containing 1% Glutamax and 1% Hepes, and the cell supernatant was collected after continuing to culture for one week, filtered with a 0.22um filter, and stored at -80°C.

Example 4. Application of two cytokine medium containing R-spondin1 and Noggin

Mouse crypts were collected, and mouse crypts were cultured in Matrigel with a medium containing 100% collected R-spondin1 and Noggin supernatant, and mouse crypts could grow into organoid tissues----"Mini intestines".

The normal crypts and tumor tissues of the large intestine of patients with colon cancer were collected and cultured in Matrigel with a medium containing 20% of the collected R-spondin1 and Noggin supernatant, which can also grow adult normal intestinal organoids and human Adenocarcinoma tissue.

SEQUENCE LISTING

<110> Fudan University Cancer Hospital Affiliated to Fudan University

<120> Cell line overexpressing R-spondin1 and Noggin and its construction method and application

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gcgggatccc taggcaggcc ctgcagatg 29

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ccggaattca tggagcgctg ccccagcct 29

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Claims (5)

1. a kind of cell strain of energy simultaneously overexpression R-spondin1 and Noggin, is designated as CHO-Rspon-Nog, and its feature exists In, while two genes of overexpression people R-spondin1 and people Noggin, can Simultaneous Stabilization secrete this R-spondin1 and Into its cell culture supernatant, the culture medium that is prepared by the supernatant can stably cultivate mice crypts to two genes of Noggin With the normal crypts of people's intestinal and tumor tissues.

2. a kind of energy as claimed in claim 1 is while the CHO-Rspon-Nog cells of overexpression R-spondin1 and Noggin The construction method of strain, it is characterised in that concrete steps include：

Packaging two slow viruss of pCDH-GFP-R-Spondin and pCDH-puromycin-Noggin, and successively by the two diseases Poison infection Chinese hamster ovary celI, to enable Chinese hamster ovary celI simultaneously high to express two genes of people R-spondin1 and people Noggin, and Verify on mRNA and protein level.

3. construction method according to claim 1, it is characterised in that include：People R-spondin1 and people Noggin two The slow virus carrier of gene builds：Two restriction enzyme sites of EcoRI and BamHI are selected, by two bases of R-spondin1 and Noggin Because CDS areas expand out, wherein：

PCR expands the forward primer of R-spondin1 genes：SEQ.ID.NO.1；

PCR expands the downstream primer of R-spondin1 genes：SEQ.ID.NO.2；

PCR expands the forward primer of Noggin genes：SEQ.ID.NO.3；

PCR expands the downstream primer of Noggin genes：SEQ.ID.NO.4；

The two full length genes are connected respectively to expression plasmid pCDH-CMV-MCS-EF1-GFP and pCDH-CMV-MCS-EF1- Puromycin, carries out conversion amplification plasmid using bacillus coli DH 5 alpha；And be respectively designated as pCDH-GFP-Rspondin and pCDH-puromycin-Noggin.

4. the application of CHO-Rspon-Noggin cell strains as claimed in claim 1, it is characterised in that collect R-spondin1 With two cytokines of Noggin, including normal culture CHO-Rspon-Noggin cells, 1 after cell growth to 100%, is pressed:10 Ratio is passed on, and culture medium is changed into after cell growth to 100% culture medium of serum-free, collects culture after continuing culture one week Base, with the filter broth filtrate of 0.22um, preserves in -80 DEG C.

5. the application of CHO-Rspon-Noggin cell strains according to claim 1, it is characterised in that adopt CHO- The cell supernatant that Rspon-Nog cell strains are collected prepares culture medium, culture mice and people's intestinal crypts and tumor tissues, including The 3D that crypts is carried out according to different proportion preparation crypts culture medium is cultivated and is grown into the organoid tissue similar to source.