中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2011年
1期
43-47
,共5页
陈愉生%李友堂%李鸿茹%陈小岩%林立芳
陳愉生%李友堂%李鴻茹%陳小巖%林立芳
진유생%리우당%리홍여%진소암%림립방
基因%RNA干扰%移植瘤%SPC-A-1细胞株
基因%RNA榦擾%移植瘤%SPC-A-1細胞株
기인%RNA간우%이식류%SPC-A-1세포주
Gene%RNA interference%Transplantation tumor%SPC-A-1 cell line
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.
目的 利用RNA榦擾(RNA interference,RNAi)技術沉默凋亡抑製蛋白基因Livin基因在人肺腺癌細胞株SPC-A-1中的錶達,探討Livin基因錶達沉默對SPC-A-1裸鼠移植瘤生長的影響.方法 以慢病毒為載體,將攜帶針對Livin基因短髮夾RNA(short hairpin RNA,shRNA)及無關序列片段的shRNA轉染SPC-A-1細胞,轉染成功後將轉染Livin shRNA的SPC-A-1細胞(實驗組)、轉染無關序列的SPC-A-1細胞(陰性對照組)及未轉染任何序列的SPC-A-1細胞(空白對照組)分彆接種于BALB/C裸鼠右腋皮下,複製SPC-A-1裸鼠皮下移植瘤模型,觀察各組細胞在裸鼠體內的成瘤時間、成瘤率、瘤體體積及重量,繪製腫瘤生長麯線併計算抑瘤率;RT-PCR、免疫組織化學法分彆檢測Livin mRNA及蛋白的錶達情況,原位末耑轉移酶標記法(TdT-mediated dUTP nick end labeling,TUNEL)檢測移植瘤組織凋亡情況.結果 與空白對照組、陰性對照組比較,實驗組裸鼠移植瘤成瘤時間晚,瘤體生長緩慢(F=70.509,P＜0.01),體積抑瘤率達(59.5±3.4)%;瘤體重量明顯減輕(F=12.821,P＜0.01),瘤重抑瘤率達(71.1±5.6)%.實驗組Livinα mRNA錶達水平為(37.2±1.6)%,Livinβ mRNA錶達水平為(29.4±1.1)%,明顯低于空白對照組[(63.3±3.8)%,(53.2±3.4)%]及陰性對照組[(66.1±2.6)%,(52.3±3.1)%],差異有統計學意義(Fα=45.309,Fβ=30.076,均P＜0.01).實驗組Livin蛋白錶達水平為(15.3±2.8)%,明顯低于空白組的(51.3±2.1)%和陰性對照組的(52.5±2.5)%(F=78.92,P＜0.01).實驗組瘤組織細胞凋亡明顯增多,凋亡率為(35.4±3.2)%,明顯高于空白對照組的(5.4±1.3)%和陰性對照組的(8.6±1.5)%,差異有統計學意義(F=14.509,P＜0.01).結論 沉默SPC-A-1細胞中Livin基因錶達可有效抑製人肺癌裸鼠移植瘤的生長,Livin基因有望成為肺癌治療的靶點之一.
목적 이용RNA간우(RNA interference,RNAi)기술침묵조망억제단백기인Livin기인재인폐선암세포주SPC-A-1중적표체,탐토Livin기인표체침묵대SPC-A-1라서이식류생장적영향.방법 이만병독위재체,장휴대침대Livin기인단발협RNA(short hairpin RNA,shRNA)급무관서렬편단적shRNA전염SPC-A-1세포,전염성공후장전염Livin shRNA적SPC-A-1세포(실험조)、전염무관서렬적SPC-A-1세포(음성대조조)급미전염임하서렬적SPC-A-1세포(공백대조조)분별접충우BALB/C라서우액피하,복제SPC-A-1라서피하이식류모형,관찰각조세포재라서체내적성류시간、성류솔、류체체적급중량,회제종류생장곡선병계산억류솔;RT-PCR、면역조직화학법분별검측Livin mRNA급단백적표체정황,원위말단전이매표기법(TdT-mediated dUTP nick end labeling,TUNEL)검측이식류조직조망정황.결과 여공백대조조、음성대조조비교,실험조라서이식류성류시간만,류체생장완만(F=70.509,P＜0.01),체적억류솔체(59.5±3.4)%;류체중량명현감경(F=12.821,P＜0.01),류중억류솔체(71.1±5.6)%.실험조Livinα mRNA표체수평위(37.2±1.6)%,Livinβ mRNA표체수평위(29.4±1.1)%,명현저우공백대조조[(63.3±3.8)%,(53.2±3.4)%]급음성대조조[(66.1±2.6)%,(52.3±3.1)%],차이유통계학의의(Fα=45.309,Fβ=30.076,균P＜0.01).실험조Livin단백표체수평위(15.3±2.8)%,명현저우공백조적(51.3±2.1)%화음성대조조적(52.5±2.5)%(F=78.92,P＜0.01).실험조류조직세포조망명현증다,조망솔위(35.4±3.2)%,명현고우공백대조조적(5.4±1.3)%화음성대조조적(8.6±1.5)%,차이유통계학의의(F=14.509,P＜0.01).결론 침묵SPC-A-1세포중Livin기인표체가유효억제인폐암라서이식류적생장,Livin기인유망성위폐암치료적파점지일.
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 shown to inhibit the proliferation of transplantation tumor of lung carcinoma effectively, and Livin may be a target for gene therapy in lung cancer.
