生存素反义寡核苷酸促进顺铂诱导骨肉瘤细胞凋亡

Promotion of antisurvivin oligonucleotides to cisplatin inducing apoptosis in osteosarcoma cells in vitro

目的:探讨生存素(survivin)反义寡核苷酸(antisenseoligonucleotide,ASODN)对顺铂(diamminedichloroplatinum,DDP)诱导骨肉瘤细胞凋亡的促进作用。方法:通过合成靶向survivin的ASODN转染骨肉瘤OS732细胞并与DDP联合作用,同时设空白对照组、正义(SODN)组及DDP组进行比较。采用RTPCR、免疫细胞化学法检测各组细胞survivinmRNA和蛋白表达状态,流式细胞仪(flowcytometry,FCM)、吖啶橙/溴化乙锭(acridineorange/ethidiumbromide,AO/EB)染色法检测各组细胞凋亡水平和形态,MTT法检测细胞生长抑制情况。结果:与空白对照组、SODN组及DDP组相比,ASODN转染组细胞survivinmRNA及蛋白表达明显减弱,细胞凋亡水平较空白对照组、SODN组相应提高,细胞萎缩、染色质浓缩呈典型凋亡改变,细胞生长相对受抑;上述指标在ASODN+DDP组变化更为明显,其细胞凋亡指数(apoptoticindex,AI)和细胞生长抑制率(inhibitionratio,IR,61.36%)明显高于各单“药”组(SODN组6.81%、ASODN组31.82%和DDP组41.35%)及SODN+DDP组(45.45%),P<0.05。结论:ASODN能够特异性封闭骨肉瘤OS732细胞中survivin基因表达,使其功能相应受抑,增加细胞对DDP敏感性,进而增进DDP诱导骨肉瘤细胞凋亡效应。

OBJECTIVE：To study the effect of antisense oligonucleotides （ASODN） of survivin, a member of a gene family of inhibitors of apoptosis, on the ability of diamminedichloroplatinum （DDP） to induce apoptosis in osteosarcoma cells. METHODS： ASODNs of survivin were synthesized and transfected into osteosarcoma OS-732 cells. The effects of ASODN alone and with DDP as well as the effect of sense oligonucleotides （SODN） with and without DDP were examined. The reverse tanscriptionase-polymerase chain reaction （RT-PCR） was utilized to detect the expression of survivin mRNA in each group of OS-732 cells. The cells were examined by flow cytometry （FCM） and stained with acridine orange/ ethidium bromide （AO/EB） to determine the morphology of the cells and the level of apoptosis in each group. The MTT assay was used to estimate the condition of cell growth. RESULTS： In comparing the cells transfected with ASODN and the control, SODN and DDP groups, the expression of survivin mRNA was significantly decreased （P 〈 0.01 ）, and the level of apoptosis enhanced. ASODN-treated cells appeared atrophic with condensed chromatin, characteristics of typical apoptotic changes. Cell growth was relatively inhibited in the ASODN groups. Furthermore, the apoptotic index （AI） and cell growth inhibition ratio （IR, 61.36 % ） were significantly higher in ASODN＋ DDP group compared with each group treated with a single agent （SODN group 6.81%, ASODN group 31.82%, DDP group 41.35%） and SODN＋DDP group （45.45%, P〈0.05）. CONCLUSIONS.. ASODN can specifically inhibit the expression of the survivin gene in OS-732 cells, and correspondingly suppress survivin function, resulting in an increase in sensitivity to DDP and thus the ability of DDP to induce apoptosis in osteosarcoma cells.