端粒酶锤头状核酶抑制肺癌细胞端粒酶活性及细胞增殖的实验研究  

Attenuation of telomerase activity and cell proliferation of A549 lung cancer cells by telomerase hammerhead ribozyme

作　　者：何霞[1] 周俊宜[1] 朱振宇[1] 黄小荣[1] 谢金卫[1] 王于[1] 柏芸[1] 米洋[1] 陶莎[1] 

机构地区：[1]中山大学基础医学院生化教研室,广东广州510080

出　　处：《中华肿瘤防治杂志》2007年第18期1372-1375,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省科技计划项目(2006B35502007);广东省自然科学基金(04105351)

摘　　要：目的:探讨端粒酶特异性核酶对A549肺癌细胞端粒酶活性、增殖和凋亡的影响。方法:构建人端粒酶锤头状核酶真核表达质粒,稳定转染人A549肺癌细胞株。RT-PCR法检测核酶的表达以及hTERT mRNA、hTR的含量,TRAP法测定细胞端粒酶活性,MTT法、流式细胞仪等测定细胞增殖及凋亡。结果:稳定转染核酶重组体的细胞克隆,均可以检测到核酶的表达,端粒酶活性被抑制,clonehTERT1～6和clonehTR(1、3、5)的平均端粒酶活性分别是空白对照组的(27±18)%和(36±13)%,P值分别为0.000和0.013;clonehTERT1～6hTERT mRNA及clonehTR1～6hTR含量下降,分别是空白对照组的(30±19)%和(49±17)%,P值分别为0.000和0.001;clonehTERT1、clonehTR3生长较空白对照组及阴性对照组减缓,凋亡率增加;而clonehTERT1又较clonehTR3生长慢,凋亡率高(凋亡率分别为21.5%和16.1%)。结论:端粒酶特异性核酶能明显抑制端粒酶活性和细胞增殖,促进细胞凋亡;端粒酶hTERT mR-NA可能是比端粒酶RNA更为理想的端粒酶抑制剂的靶点。OBJECTIVE：To study the effect of telomerase hammer head ribozymes on the telomerase activity, proliferation and apoptosis of A549 lung cancer cells. METHODS： Two telomerase hammerhead ribozyme expression recombinants were constructed. The recombinants were stably transfected into A549 cells. The expression of ribozymes, hTERT mRNA and hTR were detected by RT-PCR. The telomerase activities were detected by TRAP method. The cell growth curves were measured by MTT method. The apoptosis rates were determined by the flow cytometry assay and Hoechst 33342 staining. RESULTS： The ribozyme was stably expressed in transfected clones. Telomerase activities was attenuated. The average telomerase activities of clonehTERT1-6 and clonehTR （1, 3, 5） were （27±18）% and （36±13）% of the blank control level, respectively. The difference was statistically significant, P = 0. 000, P = 0. 013. The hTERTmRNA level of clonehTERT1-6 and hTR level of clonehTR1 6 were decreased and the levels were （30±19）% and （49 ± 17）% of the blank control level, respectively, P= 0. 000, P=0. 001. The difference was statistically significant. ClonehTERT1 and clonehTR3 grew more slowly than negative control and blank control, while clonehTERT1 grew more slowly than clonehTR3. The apoptosis rates of clonehTERT1 and clonehTR3 were higher than those of the blank control and negative control, while the apoptosis rate of clonehTERT1 was higher than that of clonehTR3 （apoptosis rates were 21.5% and 16.1% respectively）. CONCLUSIONS： Telomerase activity and cell proliferation are attenuated and cell apoptosis is induced by telomerase hammerhead ribozyme, hTERTmRNA may be the more ideal target than hTR as telom erase inhibitor.

关 键 词：端粒 末端转移酶 端粒 RNA 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]