三种环境雌激素对卵巢癌PEO4细胞增殖及凋亡相关基因表达的影响

Effects of three environmental estrogens on expression of proliferation and apoptosis-associated genes in PEO4 cells

目的 探讨几种常见的环境类雌激素壬基酚 (NP)、双酚A(BisA)和邻苯二甲酸二丁酯 (DBP)对人雌激素受体阳性卵巢癌细胞PEO4增殖和凋亡相关基因PCNA ,bcl 2和baxmRNA及相应蛋白质表达的影响。方法 PEO4细胞在DMEM培养液 (含 1 0 %小牛血清 )中进行常规传代培养 ,实验前将细胞转移至无酚红DMEM(含 5 %活性碳葡聚糖苷处理过的FBS)中继续培养 5d ,收集细胞 ,PBS洗涤后接种于内置血盖片的六孔板或 75ml培养瓶 ,32× 1 0 - 7mol LBisA和NP及 32× 1 0 - 6 mol LDBP对PEO4细胞分别处理 72h ,用半定量RT PCR技术观察其对核增殖抗原PCNA、bcl- 2及baxmRNA表达的影响 ,并用免疫组化方法对结果进行验证。结果 与溶剂对照组相比 ,32× 1 0 - 7mol LNP和BisA对PEO4处理 72h可促进核增殖抗原PCNA和抗凋亡基因bcl 2mRNA表达 ,但对baxmRNA的表达没有影响 ;DBP可促进PCNA的表达 ,但对bcl 2和bax的表达均没有明显的影响。随后进行的免疫组化实验结果显示 ,NP和BisA可促进Bcl 2蛋白的表达 ,并抑制Bax蛋白的表达。结论 对PCNA和bcl 2表达的影响可能是环境类雌激素调节卵巢癌细胞PEO4增殖和凋亡的重要机制之一。

Objective This study was designed to investigate the molecular mechanisms of proliferation and apoptosis by environmental estrogens (n 4 noniphenol, NP; Bisphenol A, BisA; Dibutylphthalate, DBP) in ovarian cancer PEO4 cells.Methods PEO4 cells were maintained in DMEM medium with 10% neonatal bovine serum. Five days before the beginning of experiments, the cells were seeded in phenol red free DMEM medium containing 5% charcoal dextran treated FBS. The cells were harvested and seeded in 6 well culture plates or in 75ml flacks. After various concentration of NP, BisA and DBP treatment for 72h, the cells were harvested and detected mRNA and protein expression of PCNA, bcl 2 and bax by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Results 32×10 -7 mol/L NP and 32×10 -7 mol/L BisA could significantly up regulate PCNA and bcl-2 mRNA expression and down regulate the bax mRNA expression, and 32×10 -6 mol/L DBP could up regulate PCNA mRNA expression, but had no effect on bax and bcl 2 mRNA expression. These results were further confirmed by following immunohistochemistry. Conclusion PCNA, bcl 2 and bax pathway might involve in cell proliferation and apoptosis events by environmental estrogens in ovarian cancer PEO4 cells.