Gene engineering stem cell for treating Type 2 diabetes

A type 2 diabetes and genetic engineering technology, applied in the field of genetically engineered stem cells for the treatment of type 2 diabetes and its preparation, can solve the problems of type 2 diabetes and improve the prognosis

Inactive Publication Date: 2014-11-26
高连如
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the analysis, it is the para-autocrine effect of Huatong glue-derived mesenchymal stem cells, which regulates the immune function of the body, partially improves insu

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of mesenchymal stem cells derived from umbilical cord wharton jelly

[0024]Umbilical cord Huatong jelly-derived mesenchymal stem cells were prepared according to the method recorded in Chinese Patent No. ZL200910157731.6. The specific method is as follows: take a fresh umbilical cord, wash off the residual blood of the umbilical cord with isotonic balanced salt solution or serum-free medium, remove the umbilical artery, umbilical vein and umbilical cord adventitia, peel off Huatong glue and cut it into small pieces, and then After washing with isotonic balanced salt solution or serum-free culture solution, soak in 0.05-0.3% collagenase solution, place in 35-37.5°C incubator for 10-30 hours, the tissue block is digested, and the liquid becomes viscous. Then add the medium containing fetal bovine serum equivalent to 1-10 times of the original solution to stop the digestion, divide it into culture bottles or petri dishes, and place in CO 2 Incubato...

Embodiment 2

[0026] Example 2 AAV6-betatrophin cDNA was transfected into WJMSCs to prepare AAV6-betatrophin-WJMSCs

[0027] The second or third generation of umbilical cord WJMSCs (WJMSCs) was collected and diluted with complete cell culture medium after counting. The concentration of WJMSCs was 2×10 5 cells / ml at 75cm 2 Add 10 ml of WJMSCs suspension to the culture flask, mix well, and place it at 37°C, saturated humidity, containing 5% CO 2 cultured in a cell culture incubator. When the adherent cells reach 60% to 80% confluence, discard the medium in the culture flask, add 10 ml of AAV6-betatrophin cDNA transfection medium, and after culturing for 48 hours, discard the AAV6-betatrophin cDNA transfection medium, add phosphoric acid After lightly washing WJMSCs twice with salt buffer solution, add 4 ml of 0.125% trypsin to digest for 1 to 2 minutes, observe under the microscope that the transfected umbilical cord Huatong jelly-derived mesenchymal stem cells completely detach from the ...

Embodiment 3

[0030] Example 3 Experimental Observation on the Effectiveness and Safety of Betatrophin Genetically Engineered Stem Cells in the Treatment of Type 2 Diabetes Mellitus

[0031] Rats were used as experimental animals, and the experimental observation was carried out according to the following steps.

[0032] ① Synthesize betatrophin cDNA plasmid;

[0033] ② Build AAV6-betatrophin;

[0034] ③ Three generations of WJMSCs were prepared according to the method of Example 1;

[0035] ④ Prepare AAV6-betatrophin-WJMSCs according to the method of Example 2, and set aside;

[0036] ⑤ Establishment of type 2 diabetes model in rats: Rats were fed with high-fat and high-sugar diet plus a small dose of levostreptocin (STZ) intraperitoneally injected to establish a type 2 diabetes model in rats. The high-fat and high-sugar feed ingredients are: 18% lard, 20% sucrose, 3% egg yolk, and 59% basic feed. Insulin resistance was induced in rats fed with high-sugar and high-fat diet for 2 mont...

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PUM

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Abstract

The invention relates to a gene engineering stem cell which is prepared by the following steps: transforming betatrophin gene cDNA (complementary deoxyribonucleic acid) plasmid into mesenchymal stem cell by using gland related virus AAV6 as a vector for carrying, and overexpressing the betatrophin molecule to obtain the betatrophin gene engineering stem cell. The gene engineering stem cell can be input into the human body via the veins to treat Type 2 diabetes; and the gene engineering stem cell can control to stimulate the proliferation of the endogenous pancreatic island beta cell to restore the failed beta cell function, and can also display the immunoregulation and insulin resistance improvement functions of the paracrine/autocrine of the stem cell, thereby basically improving the prognoses of patients with Type 2 diabetes.

Description

technical field [0001] The invention relates to medical biomaterials and a preparation method thereof, in particular to genetically engineered stem cells for treating type 2 diabetes and a preparation method thereof. Background technique [0002] Diabetes is a disease that seriously endangers human health, and 90-95% of diabetic patients are type 2 diabetes. The pathogenesis of type 2 diabetes is due to the long-term compensation of insulin resistance by pancreatic islet? cells, resulting in? cell function damage,? cell dedifferentiation, apoptosis and loss, and eventually irreversible islet? cell failure and cell number sharply reduced. Reconstructing endogenous functional islet? cells is the only way to cure diabetes. [0003] An important discovery in cell biology research in recent years is that the generation of new?cells comes from the self-replication of endogenous?cells, not from the remaining precursor cells of the islets, indicating that the islet?cells still reta...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N5/10C12N5/0775A61K48/00A61P3/10
Inventor 高连如张宁坤杨晔朱智明
Owner 高连如
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