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(英文)

Antisense oligodeoxynucleotides targeting survivin gene reverse apoptotic resistance induced by cisplatin in human lung adenocarcinoma cells

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转染组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。

[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 viabihty, 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.