Establishment of congenital heart disease ventricular septal defect cell model by virtue of recombinant hTERT and cell bank of recombinant congenital heart disease ventricular septal defect cell model

A technology for congenital heart disease and ventricular septal defect, applied to recombinant DNA technology, cells modified by introducing foreign genetic material, libraries, etc., can solve problems such as inability to carry out research, and achieve the effect of maintaining chromosomes

Inactive Publication Date: 2015-03-18
翁炳焕
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Problems solved by technology

[0010] However, there have been no literature reports on the construction of immortal cell models and cell banks using hTERT to study the

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  • Establishment of congenital heart disease ventricular septal defect cell model by virtue of recombinant hTERT and cell bank of recombinant congenital heart disease ventricular septal defect cell model

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Embodiment Construction

[0016] 1. Extraction of hTERT: ① Digestion of pClneo-hTERT: hTERT is located between the EcoRI and SalI sites of the plasmid pClneo-hTERT, and the multiple cloning site (MCS) of the pLXSNneo vector contains EcoRI and XhoI restriction sites. Commercially purchased pCIneo-hTERT plasmid, dissolved in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2O, add restriction endonuclease EcoR I and Xho I 0.5ul each, incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also by adding 0.5mol / L EDTA) The reaction was terminated, and hTERT was amplified according to the conventional PCR method, and the amplified product was collected for electrophoresis. ②hTERT electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it into the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel makin...

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Abstract

The invention relates to a method for establishing a congenital heart disease ventricular septal defect cell model by virtue of a recombinant hTERT and a cell bank of the congenital heart disease ventricular septal defect cell model, belonging to the field of medicines. The method is characterized by comprising the following steps: carrying out double-digestion on plasmid pCIneo-hTERT and a vector pLXSNneo through endonucleases EcoR I and Xho I, connecting Ligation Mix with a digestion product of hTERT and pLXSNneo, which is subjected to PCR amplification and gel electrophoresis separation, thus establishing a pLXSNneo-hTERT recombinant, transforming DH5a competent cells so as to purify, amplify and extract the plasmid, carrying out lipofection transfection on congenital heart disease ventricular septal defect cells of in vitro passage and in logarithmic growth, integrating the recombinant with cell DNA, carrying out amplifying culture, screening cells with positive recombinants by using G418, cloning, screening cells of which the cellular morphology, the growth curve, the karyotype, the inoculated nude mice experiment result, the transfection cell telomerase activity, the hTERT mRNA expression, the immunohistochemistry, the cell generation cycle and the cell apoptosis rate meet the characteristics of immortalized cells and are identical or similar to those of primary cells as an hTERT transfected congenital heart disease ventricular septal defect cell model, and freezing and storing the cell model in liquid nitrogen. A foundation is laid for studying the pathogenesis in vitro for a long time at the level of cells is made.

Description

technical field [0001] The invention relates to recombinant hTERT (catalytic subunit of telomerase reverse transcriptase) or hTERT-mediated construction of a congenital heart disease ventricular septal defect cell model and its cell bank, which is mainly used in the field of neonatal birth defect intervention research, and is a congenital heart disease room In vitro studies of septal defects provide cell models and preserve scientific research resources. Background technique [0002] Congenital heart disease, ventricular septal defect, is one of the birth defects with the highest incidence rate and has become a serious social problem. There are 112,000-160,000 new cases of congenital heart disease each year in my country, which will cause economic losses of 10.9 billion to 15.6 billion; the treatment cost of congenital heart disease is as high as 12 billion yuan per year, causing serious psychological burden and spiritual burden to the society and families. Suffering has ser...

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕舒静李晓张燕沈国松黄荷凤
Owner 翁炳焕
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