【摘要】 目的　构建含转移抑制基因nm2 3H1全长的重组腺相关病毒 (rAAV nm2 3H1) ,探讨rAAV nm2 3H1对以人卵巢癌细胞株SW6 2 6所建立的转移性原位移植模型转移性逆转的可行性。方法　用基因重组方法构建含nm2 3H1全长的重组腺相关病毒骨架质粒pUF1 nm2 3H1,磷酸钙沉淀法将腺相关病毒载体系统共转染入病毒包装细胞 2 93细胞中 ,分别合成rAAV nm2 3H1和rAAV Laz ,并测定其转染效率 ,斑点杂交法测定重组病毒的滴度。建立转移性卵巢癌原位移植模型 ,观察其生物学特性 ,并用免疫蛋白印迹法检测瘤体nm2 3H1表达。肿瘤原位移植术后第 10天对空白对照组 (12只小鼠 )、rAAV Laz组 (13只小鼠 )和rAAV nm2 3H1组 (13只小鼠 )分别行腹腔注射 ,观察rAAV nm2 3H1对转移性卵巢癌模型的肝转移率和生存时间的影响。结果　成功构建并鉴定了rAAV nm2 3H1和rAAV Laz,滴度均为 1× 10 10 个病毒分子 /ml,转染效率为 70 %。生物学特性证明构建的卵巢癌模型属转移性 ;腹腔注射后 ,rAAV nm2 3H1组瘤体中nm2 3H1的表达明显高于空白对照组 (P <0 0 5 )。空白对照组、rAAV Laz组、rAAV nm2 3H1组平均生存时间分别为 :10 6 6 7d、10 7 0 6d、136 6 7d ;空白对照组与rAAV Laz组间的生存率比较P =0 0 5 9,P >0 0 5 ;rAAV nm2 3H1组和rAAV更多还原

【Abstract】 Objective To explore the feasibility of reverse effect of recombinant nm23H 1 adeno-associated virus (rAAV-nm23H 1) in ovarian carcinoma metastastic orthotopic implantation nude model. Methods Using DNA recombination technique to construct AAV main plasmid pUF 1-nm23H 1. rAAV-nm23H 1 and rAAV-Laz were produced by co-transfection of rAAV system in package cell 293 by phosphate-calcium deposit method, and then the transfection efficiency was measured. The titer was measured by dot hybridization. Ovarian carcinoma cell line SW626 was used in the establishment of the ovarian carcinoma orthotopic implantation nude model. The biologic feature of the model was observed and the expression of nm23H 1 in the tumor was measured by Western blot. Three groups of ovarian carcinoma orthotopic implantation nude model were applied by PBS (as control) (12 mice), rAAV-Laz (13 mice), rAAV-nm23H 1 (13 mice) intraperitoneally on the day 10 after transplantation. Then the effect of rAAV-nm23H 1 on survival and liver metastastic incident rates in models were observed. Results rAAV-nm23H 1 and rAAV-Laz were constructed and identified successfully. The titers were both 1×10 10 virus particles/ml. The transfection efficiency was 70%. The observation of biological feature showed the orthotopic implantation model was metastatic. The expression of nm23H 1 in AAV-nm23H 1 group was higher significantly than control andrAAV-Laz group ( P <0.05) .Survival time of rAAV-nm23H 1 group was 136.67 days, compared with blank control group 106.67 days and rAAV-Laz group 107.06 days. Kaplan-Meier analysis showed rAAV-nm23H 1 group survived longer than control and rAAV-Laz group ( P =0.0051, P =0.018 P <0.05). The control and rAAV-Laz groups showed no statistically difference in survival time ( P =0.059 P >0.05). The metastatic incident rate of control,rAAV-Laz and rAAV-nm23H 1 group was 75%,61.5%,and 30.8% respectively, the rate of rAAV-nm23H 1 group was lower significantly than that of control and rAAV-Laz group ( P =0.03, P =0.01). There was no significant difference between control and rAAV-Laz group in liver metastatic rate ( P >0.05). Conclusion SW626 line which expressed lower level of nm23H 1 showed high-frequency metastasis properties, rAAV-nm23H 1 could reverse its metastasis in ovarian carcinoma orthotopic transplantation model.更多还原