摘要
目的探讨bc l-2表达上调对人小细胞肺癌(SCLC)多药耐药的影响及相关机制。方法通过定向克隆方法,构建bc l-2正义RNA真核表达载体pLXSN-bc l-2;同时,体外合成bc l-2反义硫代磷酸寡核苷酸(AS-PS-ODNs),然后采用脂质体分别将其转染至人SCLC H446细胞及H446/DDP细胞,用逆转录-聚合酶链反应(RT-PCR)和W estern b lot方法检测转染细胞中bc l-2 mRNA和蛋白的表达水平;流式细胞仪DNA倍性分析观察顺铂(DDP)诱导转染细胞凋亡情况。转染时细胞分3组,pLXSN-bc l-2转染组[或bc l-2 AS-PS-ODNs转染组(AS-PS-ODNs转染组)]、pLXSN转染组[或bc l-2无义硫代磷酸寡核苷酸转染组(NS-PS-ODNs转染组)]和对照组(H446细胞组、H446/DDP细胞组)。结果(1)W estern b lot检测结果表明,对照H446细胞组、pLXSN转染组、pLXSN-bc l-2转染组间Bc l-2蛋白表达水平差异有统计学意义(F=11 221,P<0.05)。转染pLXSN-bc l-2的H446细胞强表达Bc l-2蛋白(灰度值0.854±0.016),与对照H446细胞组(灰度值为0.103±0.005)相比差异有统计学意义(t=2.45,P<0.01);NS-PS-ODNs转染组的H446/DDP细胞,AS-PS-ODNs转染组的H446/DDP细胞,对照H446/DDP细胞组间Bc l-2蛋白表达水平差异有统计学意义(F=11 028,P<0.01)。转染bc l-2AS-PS-ODNs的H446/DDP细胞表达Bc l-2蛋白水平(灰度值为0.695±0.014),与对照H446/DDP细胞组(灰度值为0.942±0.018)相比,显著下降(t=2.26,P<0.01),但仍高于亲代H446细胞Bc l-2表达水平(t=2.31,P<0.01)。(2)流式细胞仪DNA倍性分析结果显示,转染pLXSN-bc l-2的H446细胞,经5μg/m l DDP诱导后凋亡率为(20.9±0.2)%,明显低于对照H446细胞组的(31.1±0.21)%,差异有统计学意义(t=2.45,P<0.01);5μg/m l DDP诱导转染AS-PS-ODNs的H446/DDP细胞的凋亡率为(31.5±0.4)%,对照H446/DDP细胞组的凋亡率为(9.0±0.1)%,转染细胞的凋亡率显著增加(P<0.05)。结论靶向性抑制抗凋亡相关基因bc l-2的表达可能是克服SCLC化疗耐药的重要的基因治疗方法之一。
Objective To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer （SCLC） cell subline H446/DDP. Methods After the construction of pLXSN-bcl-2,in which the full length of bcl-2 cDNA amplified from total RNA of H446/DDP cells was integrated into the mammalian expression vector pLXSN, allowing transcription of a bicistronic mRNA, and the synthesis in vitro of 20-mer ODNs targeting the coding region of bcl-2 mRNA and the scrambled ODNs used as the control, the cationic lipid DOTAP was used to transfect them into H446 and H446/DDP cells, respectively. When H446 cells were transfected with mammalian expression vector pLXSN, the cells were divided into 3 groups, including cells transfected with the recombinant pLXSN-bcl-2, cells transfected with the vector pLXSN and cells untransfected （control） ; when H446 cells and H446/DDP cells were transfected with PS-ODNs, the cells were divided into 3 groups, including cells transfected with AS-PS-ODNs, cells transfected with NS-PS-ODNs and cells untransfected （control）, repectively. After transfection, Western blot were carried out to detect the expression level of bcl-2 in the control cells and the transfected cells, respectively. Meanwhile flow cytometry （FCM） was used to detect cell apoptosis in the control cells and the transfected cells. Results （ 1 ） The data from Western blot showed that compared with the control H446 cells （ gray-scase value 0. 103 ± 0.005 ）, the expression level of bcl-2 in the pLXSN-bcl-2 transfected H446 cells （ gray-scare value 0. 854 ± 0. 016） was increased significantly （ P 〈 0. 01 ） ; and the apoptosis from DNA content analysis decreased significantly in the pLXSN-bcl-2 transfected H446 cells [ （20.9 ±0.2 ）% ] compared with that of the control H446 cells [ （31.1± 0.2）% ] by DNA content analysis （P 〈 0. 01 ）. （2） The results from Western blot showed that bcl-2 expression in the AS-PS-ODNs transfected H446/DDP cells （gray-scare value 0. 695 ± 0. 014） was significantly reduced compared with that of the control H446/DDP cells （gray-scale value 0.942 ± 0.018）, although not totally reduced to the level of the control H446 cells （P 〈 0. 01 ） ; and the data from DNA content analysis indicated that the sensitivity of the AS-PS-ODNs transfected H446/DDP cells to 5 μg/ml DDP-induced apoptosis was strongly increased as compared with that of the control H446/DDP cells （P 〈 0. 01 ）. Conclusion Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2006年第3期156-160,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家自然科学基金资助项目(30200119)
中国博士后科学基金资助项目(200303365)
