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目的寻找一种能够全面、客观地反映卵巢癌细胞耐药程度变化的指标。方法采用人类上皮性卵巢癌细胞株A2780和由它诱导的耐顺铂细胞株AD4,在四甲基偶氮唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide,MTT)法测定耐药倍数的基础上,以逆转录聚合酶链反应(ReverseTranscriptionPolyrmeraseChainReaction,RT-PCR)检测耐药基因mdrl、TopoⅡα和GSTπ的表达情况,并以G3PDH作内参,进行半定量分析;同时进行DNA梯度电泳及流式细胞仪检测细胞凋亡情况以反映耐药程度的变化,并比较几种方法用于检测耐药的优劣。结果经MTT测定卵巢癌敏感细胞株A2780的IC50为19.2,耐药细胞AD4的IC50为66μg/ml,耐药倍数为3.4。RT-PCR检测A2780和AD4中mdrl的表达都非常低,与G3PDH的比值分别为0.09±0.03及O.10±O.02,且在敏感及耐药细胞株中的表达差异无显著性;TopoⅡα在敏感细胞中的表达为2.60±0.12,耐药细胞为O.11±O.03,两组比较差异有显著性;而GSTπ则相反,在敏感细胞中的表达低于耐药细胞,比值分别为0.11±0.03和3.13±0.14,差异也有显著性,证实了卵巢癌对顺铂耐药主要是通过TopoⅡα和GSTπ等途径,而非mdrl途径。但在所取时间点(6h、12h、24h)内各基因表达未发现明显差异。可见RT-PCR虽可特异性检测几种耐药基因的表达,但由于耐药机制的多样性,检测往往不够全面,同时受细胞增殖周期的限制,此方法不能在短时间内反映耐药程度的变化;用DNA梯度电泳方法检测凋亡,在顺铂作用A2780及AD424h、48h后均有180-200bp整数倍的均匀梯度产生,此方法虽对凋亡的检测较具特异性,但只能检测晚期凋亡且只能做定性分析;流式细胞仪检测凋亡在用药后几小时即可发现各实验组耐药性的差异,实现了对耐药进行早期、定量检测的目标;并且通过检测凋亡反映耐药程度的变化不受耐药机制的限制,使结果较为全面、客观。结论以流式细胞仪检测凋亡程度而反映耐药,避免了只检测某种耐药基因时可能产生的漏诊,是一个能够较全面、客观地反映卵巢癌细胞耐药程度变化的指标,同时它还具有检测早期耐药性变化及能够定量分析等优点,虽仍有一些问题存在,但通过我们进一步深入研究,并改善标本取材及实验操作方法后将对临床上诊断耐药提供一个可信度较高的客观指标,有望结束目前对耐药的诊断仅评主观判断的历史。
Objective To find a kind of index that can comprehensively and objectively reflect the change of drug resistance in ovarian cancer cells. Methods The human epithelial ovarian cancer cell line A2780 and the cisplatin-resistant cell line AD4 induced by it were cultured in the medium of 4- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl -2H-tetrazoliumbromide (MTT) assay, the expressions of mdrl, TopoⅡα and GSTπ were detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR), and G3PDH was used as internal reference Semi-quantitative analysis; DNA gradient electrophoresis and flow cytometry were used to detect the apoptosis of cells to reflect the change of drug resistance, and compared several methods to detect the advantages and disadvantages of drug resistance. Results The IC50 of A2780, a sensitive ovarian cancer cell line assayed by MTT assay, was 19.2. The IC50 of drug-resistant cells, AD4, was 66 μg / ml, with a resistance multiple of 3.4. The expression of mdrl in A2780 and AD4 was very low by RT-PCR, the ratio of GDNF to G3PDH was 0.09 ± 0.03 and O.10 ± 0.02, respectively. There was no significant difference in the expression of mdrl between the sensitive and resistant cell lines. In sensitive cells was 2.60 ± 0.12, drug-resistant cells were O.11 ± O.03, the difference was significant between the two groups; while GSTπ in contrast, the expression in sensitive cells was lower than the resistant cells, the ratio of respectively 0.11 ± 0.03 and 3.13 ± 0.14, the difference was also significant, confirmed that ovarian cancer resistance to cisplatin is mainly through TopoⅡα and GSTπ other pathways, rather than the mdrl pathway. However, no significant difference was found in the expression of all the genes at the time points (6h, 12h, 24h). Although RT-PCR can specifically detect the expression of several drug resistance genes, due to the diversity of drug resistance mechanisms, the detection is often not comprehensive enough and is limited by the cycle of cell proliferation. This method can not reflect the degree of drug resistance in a short time . Apoptosis was detected by DNA gradient electrophoresis. A2780 and AD424h after cisplatin and 48h after cisplatin all produced a uniform gradient of 180-200bp. This method is more specific for the detection of apoptosis, Detection of late apoptosis and can only do qualitative analysis; apoptosis detected by flow cytometry in a few hours after treatment can be found in the experimental group differences in drug resistance, to achieve early detection of drug resistance, quantitative detection of the target; and by Detection of apoptosis reflects changes in drug resistance is not limited by the mechanism of resistance, the result is more comprehensive and objective. Conclusion Flow cytometry can detect the degree of apoptosis and reflect the drug resistance, which can avoid the misdiagnosis which may occur only when detecting a drug resistance gene. It is an index that can reflect the change of drug resistance in ovarian cancer cells more completely and objectively It also has the advantages of detecting changes in early drug resistance and being capable of quantitative analysis. Although there are still some problems, it will provide a credible evidence for clinical diagnosis of drug resistance through further study and improvement of sample preparation and experimental methods Objective indicators of higher degrees, is expected to end the current diagnosis of resistance only to judge the history of subjective judgments.