You Yong-ping, Fu Zhen, Zhao Peng, Wang Cun-zu, Liu Ning, Lu Ai-lin
Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, P.R. China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2006 Dec;23(6):605-9.
To study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo.
To construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods.
After transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited.
Growth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.
研究反义人端粒酶逆转录酶（hTERT）/PTEN联合基因治疗对恶性胶质瘤的体内外抑制效果。
利用细菌中的高效同源重组系统构建分别含有反义hTERT和野生型PTEN的两种腺病毒重组体。将这两种腺病毒重组体分别或联合转染至体外培养的U251人恶性胶质瘤细胞及体内的肿瘤组织。采用MTT法和流式细胞术检测体外U251细胞增殖情况，通过测量裸鼠胶质瘤体积检测体内肿瘤生长情况。采用端粒重复序列扩增法（TRAP）检测端粒酶活性。用蛋白质印迹法检测hTERT和PTEN蛋白表达。
体外转染后，U251细胞生长明显受到抑制。抑制作用呈时间依赖性。联合转染组抑制作用最强，第6天时，存活率为37.6%，端粒酶活性（仅28.8 TPG）明显受到抑制，hTERT蛋白表达也明显受到抑制，为0.2106，但PTEN蛋白表达明显增加，为0.9630。在体内，肿瘤生长也受到有效抑制。
反义hTERT和PTEN联合转染后，恶性胶质瘤细胞的生长在体内外均受到有效抑制。
