摘要
目的探讨RNA干扰人端粒酶逆转录酶(hTERT)基因对Hep-2细胞生长增殖的抑制作用及诱导凋亡作用.方法根据hTERT cDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1、pshRNA2.随机选取一段与人类基因无同源性的碱基序列构建表达shRNA的含荧光素基因的质粒pshRNA3.构建不表达shRNA的含荧光素基因的对照质粒pshRNA4.实验分为5组:A(pshRNA1)、B(pshRNA2)、C(pshRNA3)、D(pshRNA4)、E(空白培养液).酶切后电泳分析鉴定质粒pshRNA1～3.采用共聚焦显微镜检测质粒转染后细胞荧光表达情况;以蛋白印迹法研究hTERT表达变化;以TRAP-PCR ELISA 法研究端粒酶活性变化;以四甲基偶氮唑盐(MTT)法、倒置相差显微镜及原位细胞凋亡法观察各质粒抑制细胞增殖及诱导凋亡作用.结果(1)质粒pshRNA1～3 SalⅠ酶切后电泳见400 bp小带,与预期插入的目的基因大小一致.共聚焦显微镜下见大量的细胞表达绿色荧光.(2)pshRNA1、2转染细胞后hTERT表达显著降低;细胞端粒酶活性明显受到抑制,A组细胞活性为:0.159±0.039、B组细胞活性为0.163±0.028、E组细胞活性为1.512±0.076.A、B组与E组比较,差异均有统计学意义(P<0.01).(3)与E组细胞吸光度(A)值比较,A、B组细胞各时间点均减小,P值均小于0.01.同等培养条件下,A、B组脱落瓶壁的死亡细胞显著增多,凋亡率明显升高.结论 (1)靶向hTERT mRNA的shRNA真核表达质粒能有效转染 Hep-2细胞;(2)RNA干扰hTERT能显著地抑制Hep-2细胞的生长增殖并诱导细胞凋亡.
Objective To investigate the effect of RNA interference by targeting human telomerase reverse transeriptase （hTERT） mRNA in the larynx cancer cell line, Hep-2. Methods The primary structures of hTERT cDNA were found in GenBank. Then the structure analysis were done according to RNAi strategy which determined the specific base sequences to design shRNA plasmid. Two types of plasmid, pshRNA1 and pshRNA2, involved in fluoreseein gene were synthesized based on the specific base sequences. Control pshRNA3, a random sequence, and control pshRNA4, without additional specific sequence were also constructed. Cells were treated daily with pshRNA1-4 or normal culture medium respectively. The pshRNA1-3 was identified by eleetrophoresis. After administration of pshRNA1-4, fluorescence expression was detected by eonfoeal microscopy, the expression of hTERT of the transfeeted cells was determined by Western blotting, telomerase activity was measured by TRAP-PCR ELISA, cell viability was determined by MTT assay, morphological changes and apoptosis were examined by inverted microscope and TUNEL respectively. Results There was a 400 bp balteum in pshRNA1-3 after cut by Sal Ⅰ ,which was identical with the size of the objective gene. Many cells presented green fluorescence after being treated by pshRNA1-4, but there are much more dead green fluorescent cells in the pshRNA1 and pshRNA2 group, hTERT protein and telomerase activity was significantly decreased after treated by pshRNA1 or pshRNA2. It was observed that treatment with pshRNA1 or pshRNA2 in the presence of a valid transfeetion reagent could reduce cell viability of Hep-2 cells within 96 h （ P 〈 0. 01 ）. Under the same culture conditions, cells grew more sparsely and the number of apoptotic cell increased significantly. Conclusions shRNA plasmid directed against human telomerase reverse transcriptase can effectively transfect Hep-2 ceils, shRNA targeted hTERT gene can significantly inhibit the growth and proliferation of Hep-2 cells, which results in apoptotic c
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2005年第12期796-800,共5页
Chinese Journal of Pathology
基金
国家自然科学基金资助项目(30471873)
湖北省教育厅资助项目(301140180)
