角质形成细胞转染人乳头瘤病毒后c-jun、c-fos及MDM2基因的表达
Expression of c-jun, c-fos and MDM2 mRNA in cultured kerotinocytes through transfecting HPV16
目的 研究正常角质形成细胞转染人乳头瘤病毒(HPV) 16后,c-fos、c-jun、MDM2 mRNA表达情况。方法 在无血清的M154培养基上培养角质形成细胞,适当时进行传代培养。采用转染试剂FuGENETM6将质粒pSV2-neo/HPV16转入正常角质形成细胞,应用逆转录聚合酶链反应(RT-PCR)的方法检测转染后角质形成细胞中c-jun、c-fos、MDM2 mRNA表达。结果 转染质粒pSV2-neo/HPV16后角质形成细胞形态有所改变,细胞变大,胞核结构疏松,胞浆出现颗粒。转染细胞株中,检测到HPV16 mRNA的表达,扩增产物为110 bp;同时检测到HPV16 DNA片断(7.9 kb)。转染后角质形成细胞中有c-jun、c-fos、MDM2 mRNA的表达,其产物大小分别为196 bp,332 bp,548 bp;而正常角质形成细胞中未检测到c-jun、c-fos、MDM2 mRNA的表达。结论 体外试验提示HPV感染可能通过c-jun、c-fos、MDM2的表达而促进细胞的增生。
更多Objectives To study the expression of c-jun, c-fos and MDM2 mRNA in cultured kerotinocytes through transfecting HPV16. Methods Keratinocytes were cultured in serum-free medium, and secondary cultures continued in a suitable time. Plasmid pSV2-neo/HPV16 was transfected into cultured kerotinocytes using FuGENETM6. Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to detect c-jun, c-fos and MDM2 mRNA in keratinocytes of norml and transfected keratinocytes. Results After transfection, the morphology and structure of the keratinocytes were changed. The cells became larger and their nuclei became looser. There were particles in the cytoplasm. The expression of HPV16 mRNA was detected in the keratinocytes after transfecting pSV2-neo/HPV16 and the product was 110 bp fragment. The DNA fragment (7.9 kb) of HPV16 was also detected. In the transfected keratinocytes, the exprssions of c-jun, c-fos and MDM2 mRNA were detected and the products were 196 bp, 332 bp and 548 bp fragments, respectively. However, the above-mentioned expressions those were not detected in normal keratinocytes. Conclusion HPV leads to cell proliferation by expressing c-jun, c-fos and MDM2.
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