survivin 反义寡核苷酸逆转顺铂诱导的人肺腺癌细胞凋亡耐受作用

 目的 探讨survivin反义寡核苷酸逆转人肺腺癌细胞对顺铂诱导的细胞凋亡耐受的效应。方法 1.常规体外培养A549/CDDP细胞,以脂质体包裹的survivin反义寡核苷酸（ASODN）体外转染细胞。2.采用二苯胺反应法（DPA）测定DNA片段化、激酶法检测Caspase-3酶活性及流式细胞术检测细胞凋亡率（AI）,评价细胞凋亡程度。3.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（IC50）及耐药倍数（RI）。结果 1.单用ASODN转染组DNA片段化比率、细胞凋亡率和Caspase-3相对活性分别增高至（43.55±6.07）%、（34.03±2.25）%和1.1298±0.2502,和各对照组相比较,均有显著性变化（P〈0.01）;而ASODN和CDDP联用组上述凋亡指标增高更为明显,分别达（76.73±2.04）%、（65.85±5.47）%和1.6805±0.2758（P〈0.05）。2.转染组A549/CDDP细胞生长抑制显著,单用转染组和联合CDDP组生长抑制率分别增高达59.3%和83.7%（P〈0.05）。3.ASODN转染后CDDP对细胞IC50由（225.03±10.59）μmol/L减低至（158.84±4.256）μmol/L,其RI由11.9减为8.39。结论 survivin反义寡核苷酸体外转染可降低耐顺铂人肺腺癌细胞的凋亡阈值,从而较大程度上逆转其对顺铂诱导的细胞凋亡耐受。

Antisense Oligodeoxynucleotides Targeting survivin Gene Reverse Apoptotic Resistance Induced by Cisplatin in Human Lung Adenocarcinoma Cells

Objective 　The acquisition of resistance to apoptosis is a major reason behind t reatment failures.Ectopic expression of some apoptosis related factors , such as bcl-2 family and inhibitor apoptosis family proteins ( IAPs) ,can result in inhibition of apoptosis induced by chemotherapy agent s and thereby confer to drug resistance in malignancies. Regulation of those gene expressions may reverse the apoptosis resistance to chemical agents. In the present study , we t ransfect cisplatin-resistant human lung adeno-carcinoma A549/ CDDP cell lines to explore whether antisense oligodeoxynucleotides (ASODN) targeting survivin gene can reverse apoptotic resistance induced by cisplatin in human lung adenocarcinoma cells.Methods 　A549/ CDDP cell lines were cultured routinely with RPMI-1640 medium. survivin ASODN mediated by cytofectin was t ransfected into the A549/ CDDP cells . The influence of ASODN t ransfection on apoptosis was determined by Diphenylamine assay(DPA) ,caspase-3 colorimet ric assay and flow cytometry. 3-(4 ,5-dimethylthiazol-2-yl)-2 ,5-diphenyl tetrazolium bromide (MTT) assay was performed to detect the cell viability , half-maximum inhibitory concent ration ( IC₅₀ ) and cisplatin resistant index ( R I) was thereby calculated , respectively. Results 　In the A549/ CDDP cells t ransfected by survivin ASODNfor 48hr , the percentage of DNA f ragmentation , apoptotic index and Caspase23 activity was enhanced to (43. 55 ±6. 07) % , (34. 03 ±2. 25) % and 1. 1298 ±0. 2502 , respectively ( P < 0. 05) , it was obviously significant ,compared to the controls. While combination with ASODN and 10μmol/ L cisplatin caused far more obvious apoptotic alterations in the cells , of which the percentage of DNA fragmentation , AI and Caspase-3 activity was reached to (76. 73 ±2. 04) % , (65. 85 ±5. 47) % and 1. 6805 ±0. 2758 ( P < 0. 05) , respectively ,and even compared to single ASODN group , it was still significant ( P < 0. 05) . Transfected with survivin ASODN single and with combination of cisplatin for 48hrs ,the rate of cell growth inhibition in the resistant cells was enhanced to 59. 3 % and 83. 7 % ,respectively( P < 0. 05) , while cell viability inversely had the lowest reduction. And the half-maximum inhibitory concent ration of cisplatin was reduced from 225. 03 ±10. 59μmol/ L to 158. 84 ±4. 256μmol/ L ,while the resistant index was conversely reduced from 11. 9 to 8. 39. Non-sense oligodeoxynucleotides (NSODN) and liposome had no effect on the cells ( P> 0. 05) . Conclusion 　Transfection of antisense oligodeoxynucleotides targeting survivin gene can reduce the apoptosis threshold , thereaf ter to great extent reverse apoptotic resistance induced by cisplatin in cisplatin-resistant human lung adenocarcinoma in vitro.