【摘要】 目的　将人血纤溶酶原Kringle 5基因导入人胰腺癌PC3细胞 ,观察其对人胰腺癌PC3细胞系的基因治疗作用。方法　将外源性Kringle 5通过脂质体转染法导入人胰腺癌PC3细胞 ,筛选阳性克隆 ,同时设空质粒组及PC3细胞为对照。采用RT PCR、免疫组织化学、MTT、电镜、流式细胞术等方法 ,鉴定及检测转染前后细胞超微结构、细胞周期及Kringle 5蛋白表达情况 ,并用血管内皮细胞增殖实验及裸鼠接种检测目的胰腺癌细胞株表达的Kringle 5蛋白抑制血管内皮细胞生长的活性及裸鼠移植瘤生长情况。结果　人血纤溶酶原Kringle 5基因及蛋白水平在胰腺癌PC3细胞系均可以稳定表达 ,能抑制ECV30 4血管内皮细胞生长及具有抑制裸鼠移植瘤生长的作用。结论　成功转染人Kringle 5基因的人胰腺癌PC3细胞可稳定表达Kringle 5蛋白 ,且具有抗血管内皮细胞增殖活性的良好作用 ,为进一步进行体内抗血管生成基因治疗奠定了基础。更多还原

【Abstract】 Objective To investigate the effects of transfection of human plasminogen Kringle 5 gene on pancreatic cancer. Methods Human pancreatic cancer cells of the line PC 3 were cultured. Recombinant plasmid RINGLE 5 containing human plasminogen Kringle 5 cDNA was transfected into the PC 3 cells mediated by lipodectAMINE. RT PCR was used to detect the expression of Kringle 5. Another 2 groups of PC3 cells: PC3 cells cultured in pure culture medium and those transfected with blank plasmids were used as controls. MTT method was used to draw the growth curves of cells. Immunohistochemistry was used to detect the expression of Kringle 5. Flow cytometry was used to observe the cell cycle and apoptosis. The morphology of cells was observed by transmission electron microscopy. Human umbilical vein endothelial cells ECV304 at logarithmic growth phase were cultured and the supernatants of the 3 groups of PC3 cells were added into the culture fluid respectively. MTT method was used to detect the absorbance and draw the growth curves. Twenty one BALB/c nude mice were randomly divided into 3 groups of 7 mice to be inoculated with these 3 different PC3 cells. The appearance and size of tumor were observed continuously. Results Kringle 5 was expressed in the PC3 cells transfected with Kringle 5. The proliferation in the PC3 cells transfected with Kringle 5 became significantly lower compared with the other groups of PC3 cells 3 or 7 days respectively after transfection ( P =0.045 or P =0.038). Four days after the addition of the different kinds of supernatant the ECV304 cells grew significantly slower in the Kringle 5 group than in the other 2 groups ( P =0.041). Immunohistochemistry showed high expression of Kringle 5 protein in the PC3 cells transfected with Kringle 5 and very weak expression in other 2 groups. The apoptotic indices of the PC3 cells, PC3/ PUCKRINGLE 5 cells, and PC3/ Kringle 5 cells were 6 6%±1 3%, 7 3%±0 9%, and 12 1%±2 3% respectively ( P =0 045) The S stage change rates of the PC3 cells, PC3/ PUCKRINGLE 5cells, and PC3/Kringle 5 cells were 33 3%±3 7%, 19 4%±2 4%, and 7 4%±3 3% respectively Transmission electron microscopy showed significant apoptosis in the PC3 cells transfected with Kringle 5 gene Since the 5th week after inoculation the tumor grew significantly slower in the Kringle 5 group than in the other 2 groups ( P =0 032) Since the 7th week the tumor growth remarkably slowed down ( P =0 05). Conclusion PC3 cell successfully transfected with K5 gene can potently inhibits endothelial cell proliferation and the growth of pancreatic cancer in nude mouse The study provides a promise for the anti angiogenesis gene therapy in vivo更多还原