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Controllable immortalized retrovirus vector and human umbilical cord mesenchymal stem cell and construction method thereof

A retrovirus and a construction method technology, applied in the field of retroviral vectors and human umbilical cord mesenchymal stem cells and their construction, can solve problems such as insecurity, and achieve the effects of high immortalization efficiency, high expression, and high infection efficiency

Pending Publication Date: 2019-08-09
广州辉园苑医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of this, it is necessary to provide a controllable immortalized retroviral vector and human umbilical cord mesenchymal stem cells and its construction method to overcome the defects of the existing immortalized umbilical cord mesenchymal stem cells. Solving the unsafety caused by the continuous and uncontrollable expression of the immortalization factor TERT

Method used

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  • Controllable immortalized retrovirus vector and human umbilical cord mesenchymal stem cell and construction method thereof
  • Controllable immortalized retrovirus vector and human umbilical cord mesenchymal stem cell and construction method thereof
  • Controllable immortalized retrovirus vector and human umbilical cord mesenchymal stem cell and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of controllable immortalized umbilical cord mesenchymal stem cells

[0045] 1. Primary isolation and culture of umbilical cord mesenchymal stem cell lines

[0046] Take a 2-3cm umbilical cord, place it in a 100mm petri dish, wash the surface of the umbilical cord with PBS containing double antibodies (double antibody, TBD, PS2004HY; dPBS powder, Boster, PYG14190), and then peel off the umbilical vein and umbilical cord with sterile forceps For arteries, remove blood clots, remove the epidermis, transfer the remaining tissue to a sterile plate, cut it into 1-2mm tissue pieces with scissors, and culture them with UltraCULTURE serum-free medium, when the cell density reaches 80-90% , according to 1:2-1:3 passage, the cell density is not too thin, otherwise the cells are easy to age.

[0047] The calculation formula of cell population doubling (PD) is as follows:

[0048] PD=△t×Lg2 / (LgNt-LgN0)

[0049] △t: the counting interval days;

[0050] LgNt...

Embodiment 3

[0103] Embodiment 3 PCR and RT-PCR detection

[0104] In order to detect whether the TERT gene can be expressed in hUMSCs cells, hUMSCs were collected after adding or not adding doxycycline induction culture for 7 days, ihUMSCs-TERT(+) in doxycycline-induced group, ihUMSCs-TERT(+) in non-doxycycline-induced group TERT(-) cells were precipitated, and total RNA was extracted from three groups of cells. TERT was amplified using primers P11 and P12, and GAPDH was used as an internal reference to detect the mRNA expression level of TERT. The primer sequences are as follows:

[0105] TERT-F(P11):5'-ATGCCGCGCGCTCCCCGCTG-3'

[0106] TERT-R(P12):5'-TCAGTCCAGGATGGTCTTGA-3'

[0107] GAPDH-F(P11):5'-GAAGGTGAAGGTCGGAGTC-3'

[0108] GAPDH-R(P11):5'-GAAGATGGTGATGGGATTTC-3'

[0109] The results of fluorescent quantitative PCR at the mRNA level showed that the expression of TERT gene in the cells of the ihUMSCs-TERT(+) group was higher at the mRNA level, and no expression of the TERT gene w...

Embodiment 4

[0110] Example 4 Detection of Telomerase Activity

[0111] According to the telomerase activity detection kit ( RT Telomerase Detection Kit, Millipore, catalog number: S7710) Instructions for detecting telomerase activity in hUMSCs, ihUMSCs-TERT(-) and ihUMSCs-TERT(+) cells.

[0112] ihUMSCs-TERT(+) cells not only detected the expression of TERT, but also had telomerase activity, while hUMSCs and ihUMSCs-TERT(-) cells did not detect telomerase activity, the results were as follows Figure 7 shown.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a controllable immortalized retrovirus vector and human umbilical cord mesenchymal stem cell and a construction method thereof. The construction method comprises the steps that a Tet-on 3G system is used for constructing a controllable immortalized retrovirus vector containing immortalized factors, the retrovirusis packaged for infecting the umbilical cord mesenchymal stem cell, expression of the immortalized factor gene is induced by adding an inductor, and the gene is converted into the immortalized cell with the high multiplication capacity; when the cell needs to be used for normal physiologic or disease treatment, the inductor is removed, the immortalized factor gene is in the transcription closed state, the cell recovers the characteristics of a primary umbilical cord mesenchymal stem cell, and the cell has the great important significance in gene function research or disease treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a controllable immortalized retrovirus vector, human umbilical cord mesenchymal stem cells and a construction method thereof. Background technique [0002] Human umbilical cord mesenchymal stem cells (hUMSCs) are a kind of stromal cells derived from mesoderm with multi-lineage differentiation potential. They have low immunogenicity and immune regulation function. They are used in gene therapy and cell replacement therapy, It has broad clinical application prospects in immune diseases. However, hUMSCs, like other normal cells in the body, after a limited number of cell passages, cells stop proliferating, senescence and death occur, that is, cell senescence. Studies have shown that hUMSCs undergo cell aging after about 10-30 passages in vitro, which hinders the clinical application and mechanism research of hUMSCs. If hUMSCs can be immortalized so that it can avoid aging ...

Claims

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Application Information

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IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/10043
Inventor 张娇邹翠云张蓓江福能钟惟德朱建国
Owner 广州辉园苑医药科技有限公司
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