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Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse

A technology for gene transcription and inhibition of liver cancer, applied in the field of GPC-3 related gene technology, can solve the problems of no simple and efficient method for liver cancer nude mice transplantation, and the verification of liver cancer nude mice is not easy enough.

Inactive Publication Date: 2014-04-23
AFFILIATED HOSPITAL OF NANTONG UNIV
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Problems solved by technology

In the prior art, there is no simple and efficient method based on silencing GPC-3 gene transcription to inhibit liver cancer xenografts in nude mice, and the verification of the ability of silencing GPC-3 gene transcription to liver cancer xenografts in nude mice is not simple enough

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  • Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse
  • Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse
  • Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse

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Embodiment

[0052] 1.1 Preparation of materials:

[0053]Human HepG2 cell line was purchased from Shanghai Institute of Cells, Chinese Academy of Sciences; fetal bovine serum, trypsin solution and phosphate buffer saline (PBS) were purchased from Invitrogen Company of the United States; dimethyl sulfoxide and blasticidin were purchased from Sigma Company of the United States; GenJetTM DNA In Vitro Transfection Reagent was purchased from SignaGen Company of the United States; mouse anti-human GPC-3 antibody was purchased from Abcam Company of the United States; β-catenin, p-GSK3β, cyclinD1 and β-actin rabbit anti-human antibodies were purchased from CST Company of the United States; Oxidase-labeled goat anti-rabbit and goat anti-mouse IgG antibodies were purchased from Everest Biotech, UK.

[0054] 1.2 Preparation of experimental animals:

[0055] Eighteen BALB / c nude mice (provided by the Experimental Animal Center of Nantong University), aged 4-6 weeks, weighing 18-20g, male and female,...

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Abstract

The invention discloses a method for detecting the inhibiting ability of silent GPC-3 (glypican-3) gene transcription on a hepatocellular carcinoma transplanted tumor in a nude mouse. The method includes: 1) designing and synthesizing 4 pairs of miRNA oligomeric single-strand DNA specific to a target gene consensus sequence, then synthesizing the corresponding dsDNA, inserting the dsDNA into a vector to construct 4 recombinant plasmids, and picking out the recombinant plasmid with the highest interference efficiency through fluorescence quantitative PCR; 2) transfecting the recombinant plasmid with the highest interference efficiency into HepG2 cells to construct transfected HepG2 cells, and performing screening, thus obtaining a stably transfected HepG2 cell; 3) inoculating the stably transfected HepG2 cell to the nude mouse subcutaneously to construct a human hepatocellular carcinoma tumor-bearing nude mouse model; and 4) then determining the growth state of the stably transfected HepG2 cell in the human hepatocellular carcinoma tumor-bearing nude mouse model. The method provided by the invention can be employed to detect the inhibiting ability of silent GPC-3 gene transcription on a hepatocellular carcinoma transplanted tumor in the nude mouse, and can be used for research of GPC-3 on transplanted tumors.

Description

technical field [0001] The invention relates to GPC-3 related gene technology, in particular to a method for detecting the ability of silencing GPC-3 gene transcription to inhibit liver cancer transplanted tumors in nude mice. Background technique [0002] As a cell surface protein, phosphatidylinositol proteoglycan-3 (glypican-3, GPC-3) is highly expressed in hepatocellular carcinoma (hepatocellular carcinoma, HCC), and is considered to be an early diagnosis and specific treatment for liver cancer. potential targets. Glypican-3 (GPC-3) belongs to the family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. The GPC-3 gene is located on human chromosome X26.10, because the mutation or abnormal expression of this molecule is often associated with diseases, it has received extensive attention. In the prior art, there is no simple and efficient method based on silencing GPC-3 gene transcription to inhibit liver cancer xenografts in nude mice, and the veri...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/535C12N15/85
CPCC12N15/85G01N33/5011
Inventor 姚登福陈洁姚敏王理
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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