Bcl-2硫代反义寡核苷酸对人恶性黑色素瘤A375细胞增殖和凋亡的影响

Effects of bcl-2 antisense oligodeoxynucleotide on proliferation and apoptosis of human melanoma A375 cell line

目的:研究bcl-2硫代反义寡核苷酸(bcl-2ASODN)对人恶性黑色素瘤细胞A375增殖和凋亡的影响及其作用机制。方法:采用MTT法、激光共聚焦显微镜、TUNEL法和AnnevinV/PI双染法检测bcl-2ASODN对A375细胞增殖和凋亡的影响。应用RT-PCR方法,观察bcl-2ASODN处理前后bcl-2mRNA在A375细胞表达水平的变化。结果:MTT法检测显示bcl-2ASODN抑制A375细胞增殖,并呈时间和剂量依赖性;经30μmol/LASODN作用48h,激光共聚焦显微镜观察细胞呈现典型凋亡特征;TUNEL染色可见ASODN组多数A375细胞核被标记呈棕褐色,而SODN组和对照组细胞核未被明显标记;AnnexinV/PI检测ASODN组凋亡细胞比例升高,与SODN组和对照组均有显著性差异(P<0.001);RT-PCR结果显示bcl-2mRNA表达水平下降。结论:bcl-2ASODN可抑制A375细胞增殖和诱导A375细胞凋亡,其诱导凋亡的机制是下调bcl-2mRNA的表达。

AIM： To investigate effects of bcl-2 fully phosporothioated antisense oligodeoxynucleotide （ bcl-2 ASODN） on proliferation and apoptosis of human melanoma A375 cell, and its possible mechanism. METHODS： Proliferation and apoptosis of A375 cell with bcl-2 ASODN treatment were evaluated by MIF colorimetric assay, laser scanning confocal microscop （ LSCM）, TUNEL and Annexin V/ propidium iodide（PI）, and the level of bcl-2 mRNA expression in A375 cell was detected by RT-PCR before and after being treated by bcl-2 ASODN. RESULTS： MTT assay demonstrated that bcl-2 ASODN could inhibit the proliferation of the cells in both time and concentration dependent manner. Characteristic morphologic apoptosis changes were observed by LSCM after incubated with bcl-2 ASODN for 48 h. Most nuclears were labeled in brown by TUNEL in ASODN group, but not markedly labeled both in SODN and control group. The apoptosis rate of A375 cells in 30 μmol/L bcl-2 ASODN group was significantly higher than that in bcl- 2 SODN and in control group. The bcl-2 ASODN-induced apoptosis of A375 cells, which was accompanied by declined expression of bcl-2 mRNA was distinctly lower than that in SODN and control groups. CONCLUSION： These results suggest that bcl-1 ASODN can not only inhibit proliferation but also induce apoptosis of human melanoma A375 cells in vitro, and the apoptosis-induced mechanism is down-regulating expression of bcl-2 mRNA.