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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hTERTcDNA序列构建表达hTERTmRNA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pshRNA1~3SalⅠ酶切后电泳见400bp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hTERTmRNA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 inhibitory effect of human telomerase reverse transcriptase (hTERT) gene on Hep-2 cell proliferation and apoptosis induced by RNAi. Methods According to the hTERT cDNA sequence, we constructed shRNA eukaryotic expression plasmids pshRNA1 and pshRNA2 containing hTERTmRNA specific and containing luciferase gene. Randomly select a base sequence homologous to the human gene to construct a shRNA expression plasmid containing the fluorescein gene pshRNA3. The control plasmid pshRNA4 containing the fluorescein gene was constructed. The experiment was divided into five groups: A (pshRNA1), B (pshRNA2), C (pshRNA3), D (pshRNA4), E (blank medium). After digestion electrophoresis analysis identified plasmid pshRNA1 ~ 3. The expression of hTERT was detected by Western blotting. The changes of telomerase activity were detected by TRAP-PCR ELISA. The MTT assay was used to invert the expression of hTERT. Phase-contrast microscope and in situ cell apoptosis were used to observe the effect of each plasmid on cell proliferation and apoptosis induction. Results (1) The plasmid pshRNA1 ~ 3SalI electrophoresis showed 400bp band, consistent with the expected size of the inserted gene. A large number of cells expressed green fluorescence under a confocal microscope. (2) The expression of hTERT in pshRNA1,2 transfected cells was significantly decreased; the telomerase activity was significantly inhibited in group A, the cell activity in group A was (0 · 159 ± 0 · 039), the cell activity in group B was 0 · 163 ± 0 · 028 , Group E cell activity was 1 · 512 ± 0 · 076. There was significant difference between group A, group B and group E (P <0.01). (3) Compared with the value of cell absorbance (A) in group E, the cells in group A and group B decreased at all time points, P values were less than 0.01. Under the same culture conditions, the number of dead cells in the bottle wall of A, B group increased significantly, and the apoptosis rate was significantly increased. Conclusions (1) shRNA eukaryotic expression plasmid targeting hTERT mRNA can effectively transfect Hep-2 cells; (2) RNA interference hTERT can significantly inhibit Hep-2 cell proliferation and induce apoptosis.