Immortalized human umbilical cord mesenchymal stem cell line, and construction method and application thereof

A technology of mesenchymal stem cells and stem cell lines, applied in the field of immortalized human umbilical cord mesenchymal stem cells, can solve the problems of limited cell division and difficulty in obtaining stem cells, and achieve the effect of high efficiency of lentivirus

Inactive Publication Date: 2013-03-13
中国疾病预防控制中心病毒病预防控制所 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of this invention is to screen out immortalized human umbilical cord mesenchymal stem cells to solve the problems of dif

Method used

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  • Immortalized human umbilical cord mesenchymal stem cell line, and construction method and application thereof
  • Immortalized human umbilical cord mesenchymal stem cell line, and construction method and application thereof
  • Immortalized human umbilical cord mesenchymal stem cell line, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Establishment and Identification of Human Umbilical Cord Mesenchymal Stem Cell Line

[0046] 1. Cell isolation and culture

[0047] Human umbilical cord mesenchymal stem cells were isolated from the umbilical cord by Percoll density gradient centrifugation; AlphaMEM+10%FBS was routinely cultured, the cells grew to 80%-90% and passaged at 1:3.

[0048] 2. Construction and identification of recombinant lentiviral vector plvx-htert

[0049]High-purity pEGFP-htert and plvx were extracted, respectively, and identified by restriction endonucleases EcoRI and XbaI. Purified htert and linearized plvx fragments were recovered from pEGFP-htert plasmid and plvx plasmid digestion products with a gel extraction kit. T4 ligase ligation at 16°C, transformed DH5α Escherichia coli was inoculated in ampicillin-resistant LB medium, and cultured overnight at 37°C. Bacteria were picked and cultured overnight, plasmids were extracted, identified by double enzyme digestion with Ec...

Embodiment 2E

[0074] Example 2 Expression application of EBV adenovirus vaccine Adv-lmp2 in hMSC-hTERT

[0075] LMP2 is a latent protein in EBV virus. It has been confirmed that LMP2 can induce a strong specific CTL response and can be used for the prevention and treatment of nasopharyngeal carcinoma. Adv-lmp2 is used to infect hMSC-hTERT cells, so that they can continuously express LMP2, and can continuously induce specific CTL (cytotoxic T lymphocyte) responses.

[0076] The hMSC-hTERT cell line was cultured in α-MEM medium containing 10% FBS. One day before the experiment, the cells were inoculated into a six-well plate. The next day, the cells reached 70-90%, and the recombinant adenovirus Vector Adv-lmp2 infected cells according to MOI 10:1. Change the medium after 4h. On the third day after infection, the cells were collected by trypsinization and made into cell drops. The expression of LMP2 protein in hMSC-hTERT cell line was detected by immunoenzyme method, and the results showed...

Embodiment 3

[0078] Example 3 Study on the expression of 2G12 broad-spectrum anti-HIV neutralizing antibody in hMSC-hTERT

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Abstract

The invention acquires an immortalized human umbilical cord mesenchymal stem cell line. Through the construction of a recombinant lentiviral plvx-hteret vector, a catalytic subunit hTERT of human telomerase is introduced into umbilical cord mesenchymal stem cells, so as to screen out a stem cell line capable of stably expressing hTERT. According to cell culture and identifications at various levels, it is proved that an immortalized umbilical cord mesenchymal stem cell line is obtained, and the cell line maintains the capacity of differentiation. The invention also confirms biological activity and function of the cell line through a variety of methods, and it is demonstrated that the cell line can be used as an effective vector for gene therapy. Through a mouse tumorigenicity experiment, the invention also indicates that the cell line is safe and has no tumorigenic tendencies.

Description

Technical field: [0001] The invention relates to an immortalized human umbilical cord mesenchymal stem cell, in particular to a stem cell line capable of stably expressing human telomerase catalytic subunit hTERT. The invention also relates to the establishment method and application of the stem cell line. [0002] The invention also relates to the construction of a recombinant lentiviral vector containing human telomerase catalytic subunit gene htert. Background technique: [0003] Human mesenchymal stem cells (Human Mesenchymal Stem Cells, hereinafter referred to as hMSCs) are targeted cells that can be used in regenerative medicine and gene therapy, and have broad clinical application prospects in cell replacement and gene therapy. [0004] However, there are many problems to be solved in hMSCs, especially the insufficient quantity of autologous hMSCs in transplantation application, which limits the clinical application. The main way to solve this problem should include...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/66A61K48/00A61P31/22C12Q1/02
Inventor 曾毅郝彦哲滕智平马晶张晓梅李东升
Owner 中国疾病预防控制中心病毒病预防控制所
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