摘要
目的探讨存活素反义寡核苷酸(ASODN)体外转染对耐顺铂人肺腺癌细胞A549/ CDDP凋亡及其对顺铂(CDDP)敏感性的影响。方法常规体外培养A549/CDDP细胞,以脂质体包裹的survivin ASODN转染细胞,采用RT-PCR法和免疫细胞化学法检测survivin mRNA及蛋白表达。通过形态学观察、DNA琼脂糖凝胶电泳、caspase-3酶活性及细胞凋亡率(AI)的测定,评价细胞凋亡程度。采用MTT法测定细胞存活率和生长抑制率,计算半效抑制浓度(IC_(50))及耐药倍数(RI)。结果转染组survivin mRNA及蛋白表达下调明显,分别达41．56%和0．864±0．045,与其他各组比较,差异均有统计学意义(P＜0．05)。细胞形态学显示有细胞凋亡改变,表现为细胞核固缩、核边集和核碎裂等；DNA凝胶电泳可见DNA梯形条带；ASODN转染组细胞凋亡率和caspase-3相对活性分别增高至34．03%和1．1298±0．2502,而ASODN+CDDP组增高更为明显,分别达65．85%和1．6805±0．2758,与其他各组比较,差异均有统计学意义(P＜0．05)。ASODN转染组和ASODN+CDDP组生长抑制率分别增高至59．3%和83．7%(P＜0．05)；而ASODN转染组细胞对CDDP的IC_(50)由对照组的(225．03±10．59)μmol/L减低至(158．84±4．26)μmol/L,RI由11．9减至8．4。结论survivin ASODN通过下调survivin的表达,自身诱导了细胞凋亡,并逆转细胞对CDDP诱导的细胞凋亡的耐受,降低凋亡阈值,从而增强了人肺腺癌细胞对CDDP的敏感性。
Objective To investigate the effects of survivin antisense oligudeoxynucleoties （ASODN） transfection mediated by cytofectin on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells in vitro. Methods A549/CDDP cells were cultured routinely in RPMI-1640 medium. Survivin ASODN mediated by eytofectin was transfected into the A549/ CDDP cells. Reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry SABC assays were performed to determine the regulation of survivin expression by ASODN. The influence of ASODN transfection on apoptosis was determined by fluoroscence microscopy and Hoechst staining, agarose gel electrophoresis, flow cytometry and caspase-3 colorimetric assay. MTT assay was performed to detect the cell viability, half-maximum inhibitory concentration （IC50） and cisplatin resistance index （RI） were thereby calculated. Results Transfected by survivin ASODN for 48 h, down-regulation of survivin expression was measured, of which mRNA and protein expression was significantly down-regulated to 41.56% and 0. 864 ±0. 045, respectively （ P 〈 0.05 ）. Transfection with survivin ASODN caused typical apoptotic changes, including characteristic chromatin condensation, nuclear shrinkage, nuclear cleavage and the cells grew more regularly, and some cells were floating. Typical DNA ladder pattern was observed by agarose gel electrophoresis. Furthermore, apoptotic index and caspase-3 activity was enhanced to 34.03% and 1. 1298 ± 0.2502, respectively （P 〈 0.05 ）. It was significantly different as compared with the control group. While combination with ASODN and 10 μmol/L cisplatin caused far more distinctive apoptotic alterations, of which AI and caspase-3 activity reached to 65.85% and 1. 6805 ± 0.2758, respectively （ P 〈 0.05 ）, and even compared with the single ASODN group, the difference was still significant （ P 〈 0. 05 ）. Transfected with survivin ASODN only or with combination of cisplatin for 48 h, the inhibitory rate of cell growth was enhanced to 59.3% and 83.7% （P 〈0.05）, respectively, while inversely, the cell viability reduced to a lowest value. The half-maximum inhibitory concentration of cisplatin was reduced from 225. 03 ± 10. 59 μmol/L to 158. 84 ± 4. 26 μmol/L, and the resistant index was conversely reduced from 11.9 to 8.39. Non-sense oligodeoxynucleotides （NSODN） and liposome had no effect on the cells growth （P 〉 0. 05）. Conclusion Transfection with survivin ASODN can to a great extent reverse the cisplatin resistance in human cisplatin resistant lung adenocarcinoma cells A549/CDDP in vitro, and can thereby significantly inhibit the growth of the cells. The mechanism of reversal of resistance to cisplatin by this transfection can be associated with specific downregulation of survivin expression, which decreases the threshold of apeptosis, induces more pronounced apeptosis, and reverses the resistance to apeptosis induced by cisplatin in A549/CDDP cells in vitro.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2006年第6期408-412,共5页
Chinese Journal of Oncology
