论文部分内容阅读
目的:观察人端粒酶逆转录酶基因(human telomerase reverse transcriptase,hTERT)启动子驱动的肿瘤抑素(tamsta-tin)基因在肝癌细胞HepG2内特异性表达及其体外抗血管形成的作用。方法:构建phTERT-tumstatin、pCMV-tumstatin(阳性对照)、phTERT-EGFP(阴性对照)质粒,脂质体介导转染HepG2肝癌细胞、L-02正常肝细胞,荧光显微镜检测EGFP的表达。Western blotting检测tumstatin在HepG2细胞内的表达,MTS法检测稳定转染后HepG2细胞的增殖以及含或不含tumstatin蛋白的条件培养基对人脐静脉内皮细胞(human umbilical vascular endothelial cell,HUVEC)增殖的影响。通过计数管样分支数检测tumstatin蛋白对HUVEC管道结构形成的影响。结果:成功构建phTERT-tumstatin、pCMV-tumstatin和phTERT-EGFP质粒,质粒转染后tumstatin基因在肝癌细胞HepG2中特异地表达,在正常肝细胞L-02中无表达。phTERT-tumstatin和phTERT-EG-FP转染均不影响HepG2细胞的增殖。含tumstatin蛋白的条件培养基CM-T抑制HUVEC的增殖[抑制率为(56.49±0.33)%];CM-T与不含tumstatin蛋白的条件培养基CM-N、CM-NT相比显著抑制了HUVEC血管结构的形成[(3.33±1.53)%vs(24.44±3.11)%、(23.94±2.92)%,P<0.01)]。结论:phTERT基因启动子可驱动tumstatin靶向表达于肝癌细胞,并抑制HUVEC血管管道结构形成。
OBJECTIVE: To observe the specific expression of tamsta-tin gene driven by the human telomerase reverse transcriptase (hTERT) promoter in HepG2 hepatoma cells and its antiangiogenic effects in vitro. Methods: The plasmids of phTERT-tumstatin, pCMV-tumstatin (positive control) and phTERT-EGFP (negative control) were constructed and transfected into HepG2 hepatocarcinoma cells and L-02 normal hepatocytes by lipofectamine. The expression of EGFP was detected by fluorescence microscopy. The expression of tumstatin in HepG2 cells was detected by Western blotting. The proliferation of HepG2 cells and the proliferation of human umbilical vascular endothelial cells (HUVECs) were detected by MTS assay. influences. The effects of tumstatin on the formation of HUVECs were investigated by counting the number of tube-like branches. Results: The plasmids phTERT-tumstatin, pCMV-tumstatin and phTERT-EGFP were successfully constructed. The tumstatin gene was specifically expressed in HepG2 hepatoma cells after transfection, and was not expressed in normal hepatocytes L-02. Transfection of both phTERT-tumstatin and phTERT-EG-FP did not affect the proliferation of HepG2 cells. The CM-T containing tumstatin inhibited the proliferation of HUVEC [(56.49 ± 0.33)%]; CM-T significantly inhibited the proliferation of CM-N and CM-NT compared with the control medium without tumstatin The formation of vascular structure of HUVEC [(3.33 ± 1.53)% vs (24.44 ± 3.11)%, (23.94 ± 2.92)%, P <0.01)]. Conclusion: The phTERT gene promoter can drive the expression of tumstatin in hepatocellular carcinoma cells and inhibit the formation of vascular ducts in HUVECs.