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目的探讨脂质体介导 PTEN 基因转染人胆管癌细胞(QBC939),联合化疗药物奥沙利铂(L-OHP),分别进行人胆管癌细胞体外培养和体内接种生长,观察分析该基因对胆管癌细胞生长的抑制情况,探索人类胆管癌的生物治疗的可行性和方法。方法将携带 PTEN 基因的真核表达载体 pBP-PTEN 和不含该基因的空载体。转胆管癌 QBC939细胞,嘌呤霉素抗性筛选克隆、扩增培养。绘制细胞生长曲线及 MTT 细胞活性观察;利用免疫组化检测转染前后 PTEN 阳性表达率;透射电镜扫描观察转染前后及联合奥沙利铂后细胞的超微结构变化;流式细胞分析细胞的周期变化和细胞凋亡情况;体外细胞侵袭力抑制试验;裸鼠体内肿瘤生长,病理学及电镜扫描的观测。结果PTEN 基因转染后 QBC939细胞稳定表达,阳性表达率升高(P<0.05);肿瘤细胞活性下降(P<0.05),细胞周期 G_1～S期抑制,细胞凋亡率增加(P<0.01);透射电镜细胞较成熟、分化好;细胞侵袭力明显抑制(P<0.05);裸鼠体内肿瘤未转染组生长早,加入奥沙利铂后生长受抑制(P<0.05),病理证实为腺癌。结论 1.脂质体成功将 PTEN 基因转染人胆管癌 QBC939细胞,并稳定表达。2.PTEN 基因转染后细胞生长速度无明显变化;MTT 实验活性细胞数有所下降。3.转 PTEN 基因后的胆管癌细胞超微结构变化显示线粒体增多,细胞较成熟、分化好;流式细胞议分析,细胞周期 G_1期被抑制,细胞凋亡多,侵袭力受到抑制;4.接种转 PTEN 基因裸鼠的体内成瘤显著抑制;6.转基因的生物治疗联合奥沙利铂(L-OHP)对人胆管癌细胞生长具有显著的抑制作用。
OBJECTIVE: To investigate the effect of liposome-mediated PTEN gene transfection on human cholangiocarcinoma cells (QBC939) in combination with chemotherapeutic drug oxaliplatin (L-OHP), and to study the in vitro culture and in vivo growth of human cholangiocarcinoma cells. Cholangiocarcinoma cell growth inhibition, to explore the feasibility of human biopsy and cholangiocarcinoma methods. Methods The eukaryotic expression vector pBP-PTEN carrying PTEN gene and the empty vector without the gene were obtained. To cholangiocarcinoma QBC939 cells, puromycin resistance screening clone, expansion culture. The cell growth curve and the activity of MTT were observed. The expression of PTEN was detected by immunohistochemistry before and after transfection. The ultrastructural changes of cells before and after transfection combined with oxaliplatin were observed by transmission electron microscope. Flow cytometry Cycle changes and apoptosis; in vitro cell invasion inhibition test; tumor growth in nude mice, pathology and electron microscopy observations. Results After transfection of PTEN gene, QBC939 cells were stably expressed and the positive rate was increased (P <0.05). The activity of tumor cells was decreased (P <0.05), the cell cycle was inhibited in G_1 ~ S phase and the apoptosis rate was increased (P <0.01) (P <0.05). The growth of untransfected tumor in nude mice was earlier than that in oxaliplatin (P <0.05), and the pathology was confirmed as Adenocarcinoma Liposomes successfully transfected PTEN gene into human cholangiocarcinoma QBC939 cells and stably expressed. There was no significant change in the cell growth rate after transfection of PTEN gene; the number of MTT experimental cells decreased. The ultrastructural changes of cholangiocarcinoma cells after transfection of PTEN gene showed that the number of mitochondria increased, and the cells were more mature and differentiated. Flow cytometry analysis showed that cell cycle G1 phase was inhibited, apoptosis was more, and invasiveness was inhibited.4. The in vivo tumorigenicity of PTEN gene inoculated nude mice was significantly inhibited.6. Transgenic biological therapy combined with oxaliplatin (L-OHP) significantly inhibited the growth of human cholangiocarcinoma cells.