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目的构建HPV16 E7(E7)真核表达载体,观察E7过表达对A431细胞增殖和侵袭的影响。方法从Caski细胞中抽提总RNA,针对E7序列设计特异引物,进行逆转录和聚合酶链式反应扩增得到E7cDNA。应用基因工程方法将得到的PCR片段克隆至真核表达载体pEGFP-N1的多克隆位点。重组质粒经双酶切鉴定和测序后,使用LipofectamineTM 2000转染A431细胞。Western blot检测E7在细胞中的表达。WST-1和Transwell系统分别检测细胞增殖和侵袭能力。流式细胞术检测转染前后细胞周期的变化。ELISA检测TGF-β分泌情况。结果经酶切、测序鉴定,E7基因正确插入到pEGFP-N1多克隆位点,获得pEGFP-E7;Western blot检测结果显示:与未转染组相比,转染组E7蛋白高表达。过表达E7的A431细胞增殖和侵袭能力均显著增强,细胞周期中S期细胞数量增多,G1期细胞数量相对减少。TGF-β分泌增多。结论E7过表达可改变细胞周期,提高皮肤鳞癌细胞的增殖和侵袭能力,而分泌增多的TGF-β并不能抑制肿瘤的增殖和侵袭。
Objective To construct eukaryotic expression vector of HPV16 E7 (E7) and observe the effect of E7 overexpression on the proliferation and invasion of A431 cells. Methods Total RNA was extracted from Caski cells, specific primers were designed according to E7 sequence, and E7 cDNA was amplified by reverse transcription and polymerase chain reaction. The obtained PCR fragment was cloned into the multi-cloning site of the eukaryotic expression vector pEGFP-N1 using genetic engineering method. Recombinant plasmids were identified by double restriction enzyme digestion and sequenced. A431 cells were transfected with LipofectamineTM 2000. Western blot was used to detect the expression of E7 in cells. WST-1 and Transwell systems were used to detect cell proliferation and invasion respectively. The changes of cell cycle before and after transfection were detected by flow cytometry. ELISA detection of TGF-β secretion. Results E7 gene was inserted into pEGFP-N1 multi-cloning site correctly after digestion and sequencing. The result of Western blot showed that E7 protein was highly expressed in transfected cells compared with untransfected cells. The proliferation and invasion of A431 cells over-expressing E7 were significantly increased, the number of S phase cells in the cell cycle increased, and the number of G1 phase cells decreased. TGF-β secretion increased. Conclusion E7 overexpression can change the cell cycle and enhance the proliferation and invasion of skin squamous cell carcinoma cells. However, the increased secretion of TGF-β does not inhibit the proliferation and invasion of tumor cells.