摘要
目的观察针对hTERT基因的短发夹状RNA(shRNA)表达载体pSilencer-hTERT对人肾癌细胞及移植瘤生长的抑制作用。方法(1)pSilencer-hTERT转染人肾癌Ketr-3细胞,1、3、5、7d后逆转录-聚合酶链反应(RT-PCR)、蛋白印迹技术检测hTERT mRNA、蛋白表达,MTT法检测细胞增殖,原位末端标记法检测凋亡。(2)BALB/C-nu裸鼠接种Ketr-3细胞成瘤,瘤内分别注射pSilencer- hTERT(50μg)、空质粒pSilencer 1.0-U6(50μg)及等体积(100μl)生理盐水,隔日1次,共7次。治疗结束后第3天处死小鼠,取瘤组织检测肿瘤体积,免疫组织化学染色检测hTERT表达。结果(1) pSilencer-hTERT转染3 d后Ketr-3细胞hTERT mRNA表达(43.2±4.4)%、蛋白表达(42.6±5.6)%最低,细胞增殖抑制率(37.3±6.6)%、凋亡细胞阳性率(30.5±4.7)%最高,分别与空质粒对照组[(98.8±4.7)%、(98.0±3.7)%、(3.3±0.9)%、(10.4±2.4)%]比较,差异均有统计学意义(P<0.01)。(2)pSilencer-hTERT处理组小鼠肿瘤体积(62.4±36.5)mm^3减小,hTERT表达率(65.7±4.7)%降低,分别与空质粒对照组(83.2±38.7)、(90.7±4.2)%比较,差异均有统计学意义(P<0.05)。结论pSilencer-hTERT能有效、持续抑制人肾癌Ketr-3细胞及裸鼠肾癌移植瘤生长。
To evaluate the anti-tumor effects of vector-mediated small hairpin interfering RNA （shRNA）-targeted hTERT gene, namely pSilencer-hTERT, on human renal carcinoma in vitro and in vivo. Methods （ 1 ） The pSilencer-hTERT was transfected into human renal carcinoma Ketr-3 cells. At 1 st, 3rd, 5th, 7th day posttransfection, the effects of pSilencer-hTERT on the hTERT expression of Ketr-3 cells were detected by RT-PCR and Western blot. The proliferation of Ketr-3 cells was measured by MTr assay. The apoptosis of Kerr-3 cells was assayed by TUNEL assay. （2） The xenografts tumor models were established and animals were randomly divided into PBS group （n = 10） , pSilencerl. 0-U6 group （ n = 10）, pSilencer-hTERT group （ n = 10）. A daily dose of 100 /μl PBS, 50 μg pSilencerl. , 0-U6 in 100μl PBS and 50μg pSilencer-hTERT in 100 μl PBS was administrated intratumorally every other days for 7 times respectively. At the end of the experiment, the nude mice were killed and the tumor sizes were measured to calculate the tumor volume. The hTERT expression in tumor was detected by immunohistochemical technique. Results （ 1 ） The inhibitory effect of pSilencer-hTERT was greatest at 3rd day post-transfection, and the expression level of hTERT mRNA and protein was （43.2 ± 4.4 ） % and （42.6 ± 5.6 ）%, proliferation inhibition rate was （37.3 ± 6. 6 ）%, the average apoptosis rate was （30.5 ± 4.7）%, respectively. There were statistically significant differences when compared with the control plasmid pSilencer-U6 group （ P 〈 0.01 ）. （2） In pSilencer-hTERT group, tumor volume and hTERT-positive staining was （62.4 ± 36.5 ） mm3 and （ 65.7 ＋ 4.7） %, respectively, significantly less than in pSilencerl. 0-U6-treated group [ （83.2 238.7） mm^3, （90.7 24.2）% respectively]. There were statistically significant differences between the pSilencer-hTERT-treated group and the pSilencerl. 0-U6treated group （P 〈 0.05）. Conclusion The pSilencer-hTERT can effectively inhibit
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第10期1236-1238,共3页
Chinese Journal of Experimental Surgery
基金
卫生部科学研究基金(WKJ2005-2-026)
江苏省青年创新人才基金(BK2005429)
"青蓝工程"基金
南开大学教育部重点实验室开放课题
