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目的 人乳头瘤病毒 16型 E6 E7区基因编码的蛋白具有转导人上皮细胞 ,使之获得永生化特性的作用。为建立卵巢癌永生细胞系 ,我们对 HPV16 E6 E7转导卵巢癌细胞的方法及转导细胞对顺铂的耐药性进行了研究。 方法 采用脂质体方法 ,将含有 HPV16 E6 E7的重组逆转录病毒载体 p L XSN16 E6 E7,转染逆转录病毒包装细胞 PT6 7,经 G418筛选 ,病毒滴度测定 ,筛选出 1株高滴度 (1.9× 10 7/m l)的产病毒克隆 ,将之转导卵巢癌细胞系SKOV3、3AO,各筛选出 1株稳定表达 HPV16 E6 E7的克隆 ,SKOV3/E6 E7、3AO/E6 E7。经 RT- PCR、免疫细胞化学检测证实 HPV16 E6 E7的表达 ,之后采用 MTT法检测了 HPV16 E6 E7转导的卵巢癌细胞系对顺铂耐药性的变化。结果 HPV16 E6 E7转导的卵巢癌细胞系 SKOV3/E6 E7、3AO/E6 E7对顺铂的耐药性无改变。 结论 HPV16 E6 E7转导的卵巢癌细胞没有发生顺铂耐药性的改变。本研究为采用此法建立卵巢癌永生细胞系 ,特别是建立耐药卵巢癌细胞系奠定了基础。
The protein encoded by the human papillomavirus type 16 E6 E7 region gene has the effect of transducing human epithelial cells to obtain immortalized properties. To establish an immortal ovarian cancer cell line, we studied the method of transfection of ovarian cancer cells by HPV16 E6 E7 and the resistance of transduced cells to cisplatin. Methods The recombinant retroviral vector pL XSN16 E6 E7 containing HPV16 E6 E7 was transfected into retroviral packaging cell PT6 7 by liposome method. After screening by G418 and virus titer, a high titer (1.9 × 10 7 / ml) were transfected into ovarian cancer cell lines SKOV3 and 3AAO, and one clone, SKOV3 / E6 E7,3AO / E6 E7, stably expressing HPV16 E6 E7 was screened out. The expression of HPV16 E6 E7 was confirmed by RT-PCR and immunocytochemistry. The changes of resistance to cisplatin in HPV16 E6 E7 transduced ovarian cancer cell line were detected by MTT assay. Results The resistance of cisplatin to SKOV3 / E6 E7,3AO / E6 E7 transduced by HPV16 E6 E7 did not change. Conclusion There is no change of cisplatin resistance in HPV16 E6 E7 transduced ovarian cancer cells. This study laid the foundation for the establishment of immortalized ovarian cancer cell lines by this method, especially the establishment of drug-resistant ovarian cancer cell lines.