bcl-2反义寡核苷酸对肾癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用

Inhibition of Bcl-2 Protein Expression and Induction of Apoptosis in Renal Cell Carcinoma Cells by Antisense Oligodeoxynucleotide Targeting the bcl-2 Gene

目的:评价bcl-2反义寡核苷酸对肾细胞癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用。方法:合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1与翻译起始端互补,AS2与编码区互补),以阳离子脂质体Lipofectin为转染载体,MTS比色实验测定细胞活率,反转录-聚合酶链反应(RT-PCR)测定bcl-2 mRNA的表达,Western杂交法测定Bcl-2蛋白表达,碘化丙啶(PI)染色流式细胞术测定凋亡细胞。结果:所测定的5个肾癌细胞株均有稳定的。bcl-2 mRNA表达;转染的反义寡核苷酸对ACHN细胞Bcl-2蛋白的表达有明显抑制作用,而正义寡核苷酸无明显影响,AS2的抑制作用大于AS1;反义寡核苷酸可诱导ACHN细胞的凋亡,AS1和AS2的诱导率分别为32.1％和43.2％。结论:bcl-2反义寡核苷酸可抑制肾癌细胞Bcl-2蛋白的表达,并进而诱导细胞的调亡。

Objective: To evaluate the effects of antisense oligodeoxynucleotides (ODNs) (AS1 complementary to the translation initiation region and AS2 complementary to the coding region) targeted to bcl-2 oncogene on Bcl-2 protein expression and apoptosis of human renal cell carcinoma (RCC) cells. Methods; Expression of bcl-2 mRNA in RCC cell lines was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The ODNs were transfected with Lipo-fectin into RCC cell lines. The expression of Bcl-2 protein in ACHN tumor cells was examined by Western blot analysis, and the apoptosis of those cells was determined by flow cytometric analysis. Results: Expression of bcl-2 mRNA was detected in all five RCC lines. Transfected bcl-2 antisense ODNs, but not sense ODNs, inhibited Bcl-2 protein expression in ACHN cells. The AS2 antisense ODN showed a superior effect compared with AS1 ODN. The apoptosis of ACHN cell could been induced by bcl-2 antisese ODNs , and percentage of apoptotic cells was noted 32. 1% and 43. 2% treated with AS1 and AS2, respectively. Conclusions: Treatment of human RCC cells with antisense ODNs targeting bcl-2 gene inhibits expression of Bcl-2 protein and induce apoptosis.