[bcl-2反义寡脱氧核苷酸对卵巢癌细胞系A2780/DDP顺铂耐药逆转的研究]

[Study of bcl-2 antisense oligodeoxynucleotides on reversal of cisplatin resistance in ovarian cancer cell line A2780/DDP].

作者信息

Li Min, Yu Li-li, Cai Li-qiong, Wang Hong-bo, Wang Zhi-hua, Wang Ze-hua

机构信息

Department of Obstetrics and Gynecology, Union Hospital of Tongji Medical College, Huazhong Science and Technology University, Wuhan 430022, China.

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2006 Feb;41(2):121-5.

PMID:16640863

Abstract

OBJECTIVE

To evaluate the influence of bcl-2 antisense oligodeoxynucleotides (AODN) on reversing cisplatin (DDP) resistance in ovarian carcinoma cell lines.

METHODS

In the experiment groups, cisplatin resistant ovarian carcinoma cell line A2780/DDP cells were treated with cisplatin after oligodeoxynucleotides were transfected by lipofectamine. Cell proliferation was determined by counting the number of the cells continuously for six days. The expression level of bcl-2 mRNA was detected with reverse transcription polymerase chain reaction (RT-PCR) and protein was tested by immunofluorescence technique. The apoptosis of ovarian cancer cell lines was measured by DNA ladder assay. The inhibitory rate of cells was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The cells treated with culture media were used as the negative control, and the untreated cells were used as the blank.

RESULTS

The proliferation of AODN transfected cells was inhibited significantly compared with that in the control groups, and the logarithmic growth delayed for 24h. The expression of bcl-2 mRNA and bcl-2 protein in AODN transfected cells was 0.76 +/- 0.01 and 171.3 +/- 3.1 respectively. Compared with the expression of bcl-2 mRNA 1.56 +/- 0.03 and bcl-2 protein 105.3 +/- 1.7 in the blank, the difference was significant (P < 0.01), but compared with the same concentration of sense oligodeoxynucleotides (SODN) or random-oligodeoxynucleotides (RODN) transfected cells, the difference was not significant (P > 0.05). The AODN transfected cells were sensitive to DDP again, and the inhibitory rate was (97.49 +/- 1.00)%, which was higher than that of the negative control (P < 0.01). In the cells treated with 16 microg/ml AODN for 72 h followed by DDP, DNA ladder was obviously observed with agarose gel electrophoresis, but in the RODN and SODN transfected cells, DNA ladder could not be seen.

CONCLUSION

Through inhibiting the expression of bcl-2 mRNA and protein and increasing the apoptosis, AODN can reverse the resistance to cisplatin in ovarian cancer cell line A2780/DDP.

摘要

目的

评估bcl - 2反义寡脱氧核苷酸（AODN）对逆转卵巢癌细胞系顺铂（DDP）耐药性的影响。

方法

在实验组中，用脂质体转染寡脱氧核苷酸后，顺铂耐药的卵巢癌细胞系A2780/DDP细胞再用顺铂处理。通过连续六天计数细胞数量来测定细胞增殖。用逆转录聚合酶链反应（RT-PCR）检测bcl - 2 mRNA的表达水平，并用免疫荧光技术检测蛋白质。通过DNA梯状条带分析检测卵巢癌细胞系的凋亡。用3-(4,5 - 二甲基噻唑 - 2 - 基)-2,5 - 二苯基四氮唑溴盐（MTT）检测细胞的抑制率。用培养基处理的细胞作为阴性对照，未处理的细胞作为空白对照。

结果

与对照组相比，AODN转染细胞的增殖明显受到抑制，对数生长期延迟24小时。AODN转染细胞中bcl - 2 mRNA和bcl - 2蛋白的表达分别为0.76±0.01和171.3±3.1。与空白组中bcl - 2 mRNA 1.56±0.03和bcl - 2蛋白105.3±1.7的表达相比，差异有统计学意义（P < 0.01），但与相同浓度的正义寡脱氧核苷酸（SODN）或随机寡脱氧核苷酸（RODN）转染细胞相比，差异无统计学意义（P > 0.05）。AODN转染细胞对DDP再次敏感，抑制率为（97.49±1.00）%，高于阴性对照（P < 0.01）。在16μg/ml AODN处理72小时后再用DDP处理的细胞中，琼脂糖凝胶电泳明显观察到DNA梯状条带，但在RODN和SODN转染细胞中未见DNA梯状条带。