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目的 构建携带反义二型基质金属蛋白酶 (MMP2 )基因的重组腺病毒。方法 从新鲜肝癌组织中提取总RNA ,用RT PCR法合成MMP2cDNA序列中 5′端转录起始位点附近长约 5 0 0bp的基因片段 ,将此片段反义克隆到腺病毒载体 (AdEasy)系统的多克隆位点 ,经转染 2 93细胞生成携带反义MMP2基因的重组腺病毒Ad MMP2 AS。结果 成功构建并包装携带反义MMP2基因片段的重组腺病毒Ad MMP2 AS,病毒滴度达 1× 10 8/ml。结论 构建的重组腺病毒Ad MMP2 AS可望能有效地将反义MMP2基因片段导入人肝癌细胞株 ,为进一步研究肝癌浸润和转移机理以及探讨抑制肝癌浸润和转移的方法提供实验基础。
Objective To construct a recombinant adenovirus carrying the antisense MMP2 gene. Methods Total RNA was extracted from fresh HCC tissues. A gene fragment of approximately 500 bp near the 5’ transcription initiation site in the MMP2 cDNA sequence was synthesized by RT PCR. The antisense cDNA was cloned into the AdEasy system. The multi-cloning site was transfected with 293 cells to generate the recombinant adenovirus Ad MMP2 AS carrying the antisense MMP2 gene. Results The recombinant adenovirus Ad MMP2 AS carrying the antisense MMP2 gene fragment was successfully constructed and packaged with a titer of 1×10 8 /ml. Conclusion The constructed recombinant adenovirus Ad MMP2 AS is expected to effectively introduce the antisense MMP2 gene fragment into human hepatoma cell lines, providing experimental basis for further research on the mechanism of invasion and metastasis of hepatocellular carcinoma and to explore methods for inhibiting invasion and metastasis of hepatic carcinoma.