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 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.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.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 单用ASODN转梁组DNA片段化比率、细胞凋亡率和Caspase3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＜0.05);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).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] 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 transfected into the A549/CDDP cells .The influence of ASODN transfection on apoptosis was determined by Diphenylamine assay (DPA) ,caspase-3 colorimetric 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 concentration (IC50) and cisplatin resistant index(RI) were thereby calculated, respectively. Results] In the A549/CDDP cells transfected by survivin ASODN for 48 hrs, the percentage of DNA fragmentation, apoptotic index and caspase-3 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 10 μmol/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 48 hrs, 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 concentration of cisplatin was reduced from (225.03±10.59)μmol/L to(158.84±4.256) μmol/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, thereafter to great extent reverse apoptotic resistance induced by cisplatin in cisplatin-resistant human lung adenocarcinoma in vitro.