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Method for improving insulin secretion of islet cells through gene modification

A technology of islet cells and gene modification, applied in the field of biomedicine, can solve the problem of low insulin secretion by islet cells

Active Publication Date: 2021-04-16
山东省成体细胞产业技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the deficiencies in the prior art, the present invention provides a method for improving the secretion of insulin by islet cells through genetic modification, so as to solve the problem that the amount of insulin secreted by induced islet cells is too low

Method used

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  • Method for improving insulin secretion of islet cells through gene modification
  • Method for improving insulin secretion of islet cells through gene modification
  • Method for improving insulin secretion of islet cells through gene modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Optimizing the codons of the ERRγ gene

[0031] Based on the ERRγ gene sequence of No. XM_021197757.2 in the gene bank, codon optimization was performed to increase gene expression and generate more proteins. The optimized nucleotide sequence of ERRγ is represented as SEQ ID NO.1. At the same time, the comparison experiment was carried out with the ERRγ gene sequence of XM021197757.2.

Embodiment 2

[0032] Example 2 Construction of recombinant adeno-associated virus vector of ERRγ gene

[0033] Entrust Beijing Biomed Gene Technology Co., Ltd. to synthesize the full-length sequence of the ERRγ gene after codon optimization, and connect it to the adeno-associated virus (pAAV-IRES-hrGFP) vector, and perform sequencing after PCR verification (such as figure 1 ). After the sequence was correct, the strain bank was established, and the plasmid was extracted and purified using the plasmid purification kit of OMEGA Company to obtain the recombinant adeno-associated virus expression vector pAAV-ERRγ, and the plasmid concentration was 356ng / μl. At the same time, the recombinant adeno-associated virus expression vector pAAV-ERRγ-XM was constructed with the ERRγ gene sequence of XM021197757.2, and the plasmid concentration was 312ng / ul.

Embodiment 3

[0034] Example 3 Packaging and titer determination of adeno-associated virus

[0035] ① Recovery and passage of 293T cells

[0036] Take out the frozen 293T cells from the liquid nitrogen tank, quickly throw them into a 37°C water bath and shake them quickly, try to completely dissolve the cell solution within 1-2 minutes. Transfer the cell solution to a 50ml centrifuge tube, add physiological saline to wash DMSO, mix well and centrifuge at 1500 rpm for 5 min. Remove the supernatant, add 5 ml of fresh complete medium to resuspend the cells, transfer to T75 flasks, make up to 10ml of complete medium in each bottle. Place the culture flask steadily at 37°C, 5% CO 2 cultured in an incubator. The cell viability was observed the next day, and the medium was replaced. Afterwards, the growth of the cells was observed every day, and the cells were passaged when the cells covered 80%-90% of the bottom of the bottle. After passage, the cells were used for transfection.

[0037] ② ...

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Abstract

The invention provides a method for improving insulin secretion of pancreatic islet cells through gene modification. The pancreatic related cells are modified by adopting an ERRgamma gene. According to the invention, specific ERRgamma protein is brought into pancreatic islet cells to activate glycometabolism-related pathways, so that the content of ERRgamma protein secreted by pancreatic islet cells and the amount of insulin are increased. The pancreatic islet cells are obtained by culturing 7-9 pancreatic related cells infected by the virus containing the ERR gamma gene, and the content of secreted ERR gamma protein is 3.2-3.5 ng / ml; the content of insulin secreted under the stimulation of 22mmol of glucose is 71.2-75 ng / ml, and the content of insulin secreted under the stimulation of 2mmol of glucose is 11.6-12 ng / ml.

Description

technical field [0001] The invention relates to a method for improving insulin secretion of pancreatic islet cells through gene modification, which belongs to the technical field of biomedicine. Background technique [0002] The pancreas is an exocrine gland that secretes digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase, and an endocrine gland that secretes pancreatic hormones such as glucagon, insulin, somatostatin, and pancreatic polypeptide. Pancreatic hormones are secreted by a group of cells in the pancreas called "islets", which include four types of cells, namely alpha cells, beta cells, delta cells, and pp cells. [0003] Diabetes is a disease caused by insulin deficiency or insufficient work. Once this disease occurs in a patient, it is difficult to completely cure it. There are two main types of diabetes, insulin-dependent type 1 diabetes and insulin-independent type 2 diabetes. Type 2 diabetes is a chronic disease that occu...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/12C12N5/10C12P21/02
Inventor 刘明录韩庆梅强邦明金海锋王立新张传鹏冯建海王亮李希鹏
Owner 山东省成体细胞产业技术研究院有限公司
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