5-氮-2＇-脱氧胞苷对人卵巢癌细胞株OVCAR-3凋亡及DAPK表达影响的研究

Study on Effects of 5 - aza - 2＇ - Deoxycytine on Apoptosis of Human Ovarian Carcinoma Cell Line OVCAR - 3 and Expression of Death Associated Protein Kinase Gene

目的研究去甲基化药物5-氮-2’-脱氧胞苷（5-aza—CdR）对人卵巢癌细胞株OVCAR-3的凋亡以及对死亡相关蛋白激酶（DAPK）基因的表达水平的影响，探讨卵巢癌可能的发病机理，并为临床探索卵巢肿瘤治疗的新途径提供实验和理论基础。方法（1）培养人卵巢癌细胞株OVCAR-3至对数生长期，建立用药组及对照组，用药组用不同浓度的5-aza—CdR进行干预。（2）采用逆转录PCR（RT—PCR）技术检测用药前后DAPKmRNA的表达水平。以各泳道中DAPK与α-actin条带的光密度比值表示DAPKmRNA的相对表达量。（3）利用流式细胞仪检测细胞凋亡情况。结果（1）未经5-aza—CdR处理的OVCAR-3细胞中DAPKmRNA不表达，经5×10-8mol／L5-aza—CdR作用后DAPKmRNA表达量为0．098±0．017，并随浓度增加逐渐增强（P〈0．01），浓度为10—5mol／L时DAPKrnRNA表达量为0．471±0．023。（2）流式分析结果为5-aza—CdR处理后AnnexinV＋／PI-细胞比例升高，凋亡率明显增加（P〈0．01）。结论5-aza—CdR通过不同程度恢复DAPK表达与基因转录。5-aza—CdR有效促进细胞凋亡，抑制卵巢癌OVCAR-3细胞的生长和增殖。

Objoctive To investigate the effects of 5 - aza - 2＇ - deoxyeytine （5 - aza - CAR）on apoptosis of human ovarian carcinoma cell line OVCAR - 3 and the transcription level of death associated protein kinase gene（DAPK）, then discover the conceivable mechanism in ovarian cancer initiation process, and provide the experimental and academic groundwork for the therapy of ovarian cancer. Methods （1） Ovarian cancer cell line originated from human epithelial ovarian tumor was cultured in vitro to the period of logarithmic growth.Study groups were treated with 5 - aza- CdR in different concentrations,and control group was not treated with 5 - aza- CdR. （2）OVCAR- 3 cells were treated by methylation inhibitor,5 - aza- CdR.The transcriptions of DAPK gene in different study groups were analyzed by reversed transcription PCR（ RT - PCR）. The relative expression level of DAPK mR- NA was indieated by the ratio of the optical density value of DAPK to that of β- actin. （3）Flow cytometry was used to study OV- CAR- 3 ceils stained by Annexin V - FITC. Results （1）The expression of DAPK mRNA was absent in OVCAR - 3, it was reversed in different degree after treated with 5 - aza- CdR.From 5 ×10^-8moL/L group to 10-5mol/L group, it increased from 0.098 ±0.017 to 0.471 ±0.023（ P 〈 0.01 ）. （2）With the flow eytometry detecting the transloeation of phosphatidyl serine（PS）, the result showed that the proportion of AnnexinV＋/PI- cell increases and 5 - aza- CdR（5×10^-8 mol/L - 10-Smol/L）ean induce apoptosis of OVCAR - 3 cells. Conclusion 5 - aza - CdR can renew the expression of DAPK mRNA. 5 - aza - CdR can inhibit the growth of OVCAR - 3 cells.