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In Vitro Study of Treating Human Primary Hepatocellular Carcinoma by AFP/tk/angio Fusion Gene

Author: ZouHao
Tutor: ZhangXiaoWen
School: Kunming Medical College
Course: Surgery
Keywords: Human hepatocellular carcinoma Human alpha-fetoprotein promoter Human herpes simplex virus thymidine kinase gene type Ⅱ Angiostatin gene Gene therapy
CLC: R735.7
Type: Master's thesis
Year: 2005
Downloads: 67
Quote: 0
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Abstract


Objective To observe the human alpha-fetoprotein (AFP) promoter regulated herpes simplex virus thymidine kinase gene type Ⅱ (HSV-Ⅱ-tk) gene with angiostatin (Angio) fusion gene on human hepatocellular carcinoma in vitro therapeutic effect. Methods PCR technique was cloned from a clinical cancer specimens AFP promoter ; by gene recombination technology , eukaryotic expression vector pcDNA3/AFP/tk/Angio; Liofectamine by cationic liposome -mediated transfection in vitro human liver cells hepatoma cell line SMMC-7721 and Bel7402. Join GCV, cell culture . By RT-PCR, light microscopy , transmission electron territory , MTT, flow cytometry was used to detect HSV-Ⅱ-tk gene and Angiostatin SMMC-7721 and Bel 7402 cells and the cell-killing effect. Results 1. Obtained by alpha-fetoprotein (AFP) promoter 300bp; 2. Were confirmed by sequencing AFP promoter inserted correctly pcDNA3/tk/Angio; 3. Expression of AFP Bel 7402 cells obtained by RT-PCR gene (HSV- Ⅱ-tk) the same size (1100bp) of the specific bands , SMMC-7721 cells, no any bands appear ; MTT assay showed that , Bel 7402 experimental group OD 570 values ??significantly lower than controls group, and there was a significant (P lt; 0.05), Bel 7402 experimental group OD 570 values ??of the experimental group was significantly lower than SMMC 7721 , and there were significant (P lt; 0.05); SMMC-7721 each groups OD 570 values ??no significant difference (P gt; 0.05); Flow cytometry showed , Bel 7402 experimental group showed significant apoptosis peak , while the control group and each group SMMC-7721 did not apoptotic peak appeared ; transmission electron habitat experimental observation Bel 7402 cells apoptotic bodies . Conclusion cloned AFP promoter ; successfully constructed the eukaryotic expression vector pcDNA3/AFP/tk/Angio; Liofectamine through cationic liposome -mediated , HSV-Ⅱ-tk expression of AFP in Bel 7402 cells can be obtained , and its expression product combined with GCV can induce human hepatocellular carcinoma cell apoptosis.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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