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Bone marrow derived mesenchymal stem cell line for stable expression of exogenous EX-4 gene

A technology for stable expression and cell line, which is applied in the field of bone marrow-derived mesenchymal stem cell lines and their preparation, and can solve problems such as poor therapeutic effect

Inactive Publication Date: 2017-12-22
CHINA PHARM UNIV
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Problems solved by technology

The GLP-1 analog exendin EX-4 is a hormone substance found in the saliva of the Gila monster growing in the southwestern United States and the deserts of Mexico. It is also a GLP-1R agonist and interacts with GLP-1R. The affinity is stronger than GLP-1, but its half-life in vivo is only 3-4h, and the therapeutic effect is not good

Method used

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  • Bone marrow derived mesenchymal stem cell line for stable expression of exogenous EX-4 gene
  • Bone marrow derived mesenchymal stem cell line for stable expression of exogenous EX-4 gene
  • Bone marrow derived mesenchymal stem cell line for stable expression of exogenous EX-4 gene

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specific Embodiment approach

[0026] 2. The overexpression of EX-4 can enhance the ability of MSCs to resist apoptosis, proliferation, and migration; the specific implementation is as follows:

[0027] 1) MTT detects MSCs cell viability

[0028] Spread MSCs, MSCs-HA, and MSCs-EX-4 on a 96-well plate, 5×10 per well 3 Cells, 6 replicate wells in each group, with a final concentration of 0.3mM H 2 O 2 Process for 12h. After removing the supernatant, add 10μl of 5mg / ml MTT solution to each well, incubate for 4h, then remove the supernatant, add 150μl of DMSO to each well, shake at low speed for 10min on a shaker to fully dissolve the crystals, and determine OD490.

[0029] 2) RNA level detection of Bax expression

[0030] The expression level of Bax mRNA in different groups was detected by RT-PCR, showing that the overexpression of EX-4 group was significantly lower than that of the model group.

[0031] 3) Western Blot detects the expression of Beclin-1, Cleaved-caspase-9 and p-ULK1

[0032] Western Blot detected the e...

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Abstract

The invention provides an MSCs (mesenchymal stem cells) line for stable expression of EX-4. The MSCs line for stable expression of EX-4 is obtained by lentivirus infection. The lentivirus used by the invention is obtained by transfection of 293T cells with a PLVTH-pol-IRES-puro lentivirus overexpression system, and the lentivirus overexpression system comprises PLVTH-EX-4 expression plasmid, coating plasmid pMD2.G, and packaging plasmid p8.91. The PLVTH-EX-4 is obtained by double enzyme digestion of the lentivirus overexpression vector plasmid PLVTH-pol-IRES-puro and EX-4 target gene and then connection to T4 ligase. The cell line obtained by the invention on the one hand can enhance the anti anti-apoptosis ability, multiplication capacity and migration ability of MSCs itself, and on the other hand the EX-4 overexpressed MSCs group has better effects of inhibiting apoptosis and promoting proliferation on pancreatic beta cells than the MSCs group. Through the two aspects of effects, the cell line provided by the invention can recover the functions of pancreatic beta cells to a normal level, and improve the disadvantages of MSCs in treatment of diabetes.

Description

Technical field [0001] The present invention relates to a bone marrow-derived mesenchymal stem cell line stably expressing exogenous Exendin-4 (EX-4) gene and a preparation method thereof, and this cell line can effectively inhibit pancreatic β-cell apoptosis and promote proliferation. effect. Background technique [0002] Mesenchymal stem cells (MSCs) are stem cells with multi-differentiation potential, which can play a role in treating diabetes in a variety of ways, directly or indirectly. Recent studies have found that MSCs have reduced survival, proliferation, and differentiation capabilities under severe conditions such as high glucose, high fat, and hypoxia. After MSCs are cultured in vitro, the expression of inflammation-related molecules such as CCR2 on their surface decreases, which makes MSCs move to the inflammation site. The reduced migration capacity greatly limits the therapeutic effect of MSCs. [0003] Recently, it has been found that MSCs express GLP-1R on the su...

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N5/071
CPCC07K14/605C12N5/0663C12N5/0676C12N15/86C12N2500/84C12N2740/15043
Inventor 黄凤杰陈秋华刘俊俊
Owner CHINA PHARM UNIV
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