目的： 构建稳定表达人 CD133 基因的脑胶质瘤U251细胞株，并探讨 CD133 对U251细胞生物学行为的影响。 方法： 将人 CD133 全长cDNA构建入逆转录病毒表达载体pEGZ-Term ，包装成逆转录病毒pEGZ-Term-CD133，进而感染脑胶质瘤U251细胞株。流式细胞术及Real-time PCR检测感染后U251细胞CD133分子的表达。细胞计数法、神经球形成实验观察CD133过表达对U251细胞体外的增殖和神经球形成的影响。裸鼠皮下成瘤法检测感染后U251细胞的体内致瘤性。 结果： 成功构建pEGZ-Term-CD133逆转录病毒表达载体，并获得稳定表达 CD133 的U251细胞。相比U251-mock、U251细胞，U251-CD133细胞高表达 CD133 mRNA \[（7 400.2±5 003.4) vs (2.0±1.1)、(1.0±2.2)，均P=0.0007）和蛋白。感染pEGZ-Term-CD133对U251细胞的体外增殖并无影响（P＞0.05）；但在无血清神经干细胞培养条件下，U251-CD133细胞所形成的神经球数量显著高于U251-mock和U251细胞\[（34.0±7.5） vs（14.6±2.3）、（11.5±1.3）个，均P<0.01\]。接种量为1×105 个细胞时，U251-CD133细胞在裸鼠体内的成瘤时间（32 d）少于U251-mock细胞（38 d）、成瘤率更高（100% vs 30%），在第41天时，肿瘤体积显著增大\[（180.3±146.8） vs （4.0±0.0）mm3，P=0.003\]。 结论： CD133分子不影响脑胶质瘤U251细胞的体外增殖，但可促进U251细胞神经球的形成和致瘤性。

Objective: To study the impact of CD133 on biological characteristics of human glioma U251 cells by construction a U251 cell line stably overexpression human CD133 gene. Methods: The full length human CD133 cDNA was sub-cloned into retroviral expressing vector pEGZ-Term to obtain recombinant pEGZ-Term-CD133 retrovirus. Then, U251 cells were infected by pEGZ-Term-CD133 retrovirus. The expression of CD133 on infected U251 cells was detected by flow cytometry and real-time PCR. By cell counting and neurosphere formation analysis, the impact of CD133 overexpression on the proliferation and neurosphere formation of U251 cells in vitro was determined. Tumorigenicity was evaluated by subcutaneous injection of CD133 infected U251 cells into the nude mice. Results: The pEGZ-Term-CD133 retrovirus expression vector was constructed successfully and a CD133 stably infected U251 cell line was obtained. Compared with U251-mock and U251 cells, the expressions of CD133 mRNA (\[7 400.2±5 003.4\] vs \[2.0±1.1, 1.0±2.2\]; all P=0.0007) and CD133 protein were significantly increased in U251-CD133 cells. pEGZ-Term-CD133 retrovirus infection showed no effect on the proliferation of U251 cells (P>0.05). However, the neurosphere formation of U251-CD133 cells was obviously higher than U251-mock and U251 cells in the presence of serum free neurosphere medium (\[34.0±7.5\] vs \[146±23\], \[11.5±13\]; all P<0.01). Compared with the U251-mock cells, the tumor formation time of U251-CD133 cells was shorter (32 d vs 38 d), the tumorigenesis ratio was higher (100% vs 30%) and the tumor volume was significantly larger (\[180.3±146.8\] vs \[4.0±0.0\] mm3, P=0.003) at 41 d when subcutaneous inoculated 1×105 cells. Conclusion: CD133 has no influence on proliferation of glioma U251 cells, but could enhance neurosphere formation and tumorigenicity of U251 cells. 

国家自然科学基金资助项目（No. 31200661）；苏州市自然科学基金资助项目（No. SYS201106）