PTEN基因联合奥沙利铂对胆管癌细胞生长抑制的研究

Inhibitory effects of mediated PTEN gene in combination with L-OHP on human cholangiocarcinoma cell line

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 inhibitory effects of mediated PTEN gene in combination with L-OHP on human cholangiocarcinoma cell line. Methods A eukaryotic expression vector containing PTEN gene was transfected into human cholangiocarcinoma cell line QBC-939 under mediation of lipofectamine, and the positive cell clones were selected and amplified. The expression of PTEN gene was determined with immunohistochemistry. Cell growth curve was illustrated and MTT performed to test cell invasion. Electron microscopy was conducted to determine the effects of PTEN on cell cycle, ultrastructure and cell growth of the transfected QBC-939 cells and on tumorgenicity in nude mice. Results PTEN was significantly expressed in the transfected QEC-939 cells （P〈0. 05）. The cell growth was slower than in other groups （P〉0. 05）. The size of transfected cells changed. The growth of the transfected cells was inhibited at phase G1 to S and its apoptotic rate markedly increased （P〈0. 05）. The cell growth in nude mice was inhibited after addition of L-OHP. Conclusions The inhibitory effects of PTEN on cholangiocarcinoma cell line can be strengthened by L-OHP.