RNA干扰沉默Livin基因对裸鼠移植瘤生长的影响  

Effects of human Livin gene down-regulation by RNA interference on xenograft of lung adenocacinoma SPC-A-1 cells in BALB/C nude mice

作　　者：陈愉生[1] 李友堂[1] 李鸿茹[1] 陈小岩[2] 林立芳[3] 

机构地区：[1]福建医科大学省立临床医学院呼吸科,福州350001 [2]福建医科大学省立临床医学院病理科,福州350001 [3]福建省心血管病重点实验室

出　　处：《中华结核和呼吸杂志》2011年第1期43-47,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：福建省自然科学基金（2008J0074）

摘　　要：目的 利用RNA干扰（RNA interference,RNAi）技术沉默凋亡抑制蛋白基因Livin基因在人肺腺癌细胞株SPC-A-1中的表达,探讨Livin基因表达沉默对SPC-A-1裸鼠移植瘤生长的影响.方法 以慢病毒为载体,将携带针对Livin基因短发夹RNA（short hairpin RNA,shRNA）及无关序列片段的shRNA转染SPC-A-1细胞,转染成功后将转染Livin shRNA的SPC-A-1细胞（实验组）、转染无关序列的SPC-A-1细胞（阴性对照组）及未转染任何序列的SPC-A-1细胞（空白对照组）分别接种于BALB/C裸鼠右腋皮下,复制SPC-A-1裸鼠皮下移植瘤模型,观察各组细胞在裸鼠体内的成瘤时间、成瘤率、瘤体体积及重量,绘制肿瘤生长曲线并计算抑瘤率;RT-PCR、免疫组织化学法分别检测Livin mRNA及蛋白的表达情况,原位末端转移酶标记法（TdT-mediated dUTP nick end labeling,TUNEL）检测移植瘤组织凋亡情况.结果 与空白对照组、阴性对照组比较,实验组裸鼠移植瘤成瘤时间晚,瘤体生长缓慢（F=70.509,P＜0.01）,体积抑瘤率达（59.5±3.4）%;瘤体重量明显减轻（F=12.821,P＜0.01）,瘤重抑瘤率达（71.1±5.6）%.实验组Livinα mRNA表达水平为（37.2±1.6）%,Livinβ mRNA表达水平为（29.4±1.1）%,明显低于空白对照组[（63.3±3.8）%,（53.2±3.4）%]及阴性对照组[（66.1±2.6）%,（52.3±3.1）%],差异有统计学意义（Fα=45.309,Fβ=30.076,均P＜0.01）.实验组Livin蛋白表达水平为（15.3±2.8）%,明显低于空白组的（51.3±2.1）%和阴性对照组的（52.5±2.5）%（F=78.92,P＜0.01）.实验组瘤组织细胞凋亡明显增多,凋亡率为（35.4±3.2）%,明显高于空白对照组的（5.4±1.3）%和阴性对照组的（8.6±1.5）%,差异有统计学意义（F=14.509,P＜0.01）.结论 沉默SPC-A-1细胞中Livin基因表达可有效抑制人肺癌裸鼠移植瘤的生长,Livin基因有望成为肺癌治疗的靶点之一.Objective To explore the in vivo inhibitory effect of Livin gene silencing by RNA interference on xenograft of lung adenocarcinoma SPC-A-1 cells in BALB/C nude mice. Methods Three different BALB/C nude mice models were established by subcutaneously inoculating differently treated SPC-A-1 cells into 3 nude mice groups： the blank control group was inoculated with blank SPC-A-1 cells,while the negative group was inoculated with cells transfected with lentivirus-delivered negative shRNA, the experimental group was inoculated with cells with lentivirus-delivered Livin shRNA. Then the growth of tumors was observed, and the volume and weight of the tumors were measured at different time points. The curve of tumor growth was then described, and the inhibition rate was calculated. Livin gene expression in the tumor tissues was determined by RT-PCR and Inmunohistochemistry. Cell apoptosis of tumor tissues was detected by TUNEL. Results Slower tumor growth, smaller tumor volume and lighter tumor weight were observed in the experimental group as compared to the blank and negative groups （ F = 70. 509, P ＜ 0. 01; F = 12. 821, P ＜ 0. 01 ）. The inhibition rate of tumor volume was （ 59. 5 ± 3.4 ） % , and the inhibition rate of tumor weight was （71. 1 ±5.6）%. Livinα mRNA and Livinβ mRNA expressions in the experimental group were significantly lower than the 2 control groups [ （ 37. 2 ± 1.6 ） % versus （ 63.3 ± 3.8 ） % , （ 66. 1 ±2.6）% ;（29.4±1.1）% versus （53.2 ±3.4）% ,（52.3 ±3. 1）% （Fα =45.309, P ＜0.01 ;Fβ =30.076,P ＜ 0. 01 ） ] . Livin protein expression level was also significantly lower than the blank and the negative groups [ （ 15.3 ± 2. 8 ） % versus（51.3 ± 2. 1 ） %, （ 52. 5 ± 2. 5 ） %, F = 78. 92, P ＜ 0. 01 ]. The apoptosis rate in the experimental group was significantly higher than that in the 2 control groups [ （ 35.4 ± 3.2 ） %versus（5.4±1.3）%,（8.6 ± 1.5）%,F= 14.509, P＜0.01]. Conclusion The lentivirus-delivered Livin shRNA was sh

关 键 词：基因 RNA干扰 移植瘤 SPC-A-1细胞株 

分 类 号：R734.2[医药卫生—肿瘤]