脂质体转染法与电转染法在丙型肝炎病毒RNA转染人肝癌细胞中的应用效果比较  

Application effects of electroporation and lipofection in HCV RNA transfecting human hepatoma cells

作　　者：卢莎[1,2] 谭文杰[2] 张玲[2] 邓瑶[2] 陶格斯[1,2] 蔡敏[1] 李恋[1] 沈晓玲[1] 

机构地区：[1]内蒙古医科大学,呼和浩特010110 [2]中国疾病预防控制中心病毒病预防控制所

出　　处：《山东医药》2016年第19期15-18,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81373229);传染病科技重大专项(2013ZX10004601;2013ZX10004609);内蒙古医科大学科技百万项目(NY2011BW003)

摘　　要：目的：比较脂质体转染法与电转染法在丙型肝炎病毒（ HCV ） RNA转染人肝癌细胞中的应用效果。方法培养人肝癌细胞Huh7．5-CD81并分为A、B组及对照组。 A组采用电转染质粒pHebei（E1E2）／JFH1来源的HCV RNA；B组采用脂质体转染质粒pHebei（E1E2）／JFH1来源的HCV RNA；对照组采用脂质体转染复制缺陷质粒pJFH1／GND转录获得的HCV RNA。光学显微镜下观察各组转染后细胞形态，采用免疫荧光法（ IFA）检测三组转染效率，real-time PCR法检测三组细胞培养液上清中的HCV RNA，采用TCID50法检测A、B组细胞培养液上清中的丙型肝炎病毒（HCVcc）感染滴度。结果 A组转染使用细胞数为1×106，转染后第2天细胞损伤过半，边缘毛糙；B组转染使用细胞数为2×105，转染后第2天细胞基本无损伤。 A组转染效率为9．98％±0．83％，B组转染效率为2．58％±0．39％，两组转染效率相比，P＜0．01；对照组未检出荧光阳性细胞。 A、B组转染后细胞培养液上清中HCV RNA拷贝数均呈现先下降后升高的趋势，最终稳定于106 copies／mL。 A组转染后21 d、B组转染后31 d方能检测到HCV感染滴度，A、B组收获HCV最高感染滴度均为104 ffu／mL。结论脂质体转染和电转染两种方法均可建立嵌合HCV的人肝癌细胞培养体系，脂质体转染法对细胞的损伤较小，电转染法转染效率较高、收获HCVcc的时间较短，但两种方法收获的HCVcc感染滴度无明显差异。Objective To observe the application effects of electroporation and lipofection in hepatitis C virus （ HCV） RNA transfecting human hepatoma cells .Methods Human hepatoma cells （ Huh7.5-CD81） were cultured and divided in-to groups A, B and control group.Cells in the group A were transfected with HCV RNA of pHebei （E1E2）/JFH1 by elec-troporation, cells in the group B were transfected with HCV RNA of pHebei （E1E2）/JFH1 by lipofection, and cells in the control group were transfected with HCV RNA of replicating defective pJFH 1/GND.Cell morphology was observed using an optical microscope in each group after transfection .Immunofluorescence was employed to detect the transfectional efficien-cy.RNA copies in the supernatant were tested by real-time PCR.TCID50 was applied to detect HCV infectious titer .Re-sults The number of cells used in the group A were 1 ×106 , and more than half were damaged with rough edges one day after transfection;the number of cells in the group B were 2 ×105 and almost no damage was found one day after transfec-tion.Transfectional efficiency of group A was 9.98%±0.83%, and that was 2.58%±0.39%in the group B, P＆lt;0.01. Fluorescence positive cells in the control group were not detected .Meantime, HCV RNA copy number in the supernatant of groups A and B first decreased , then increased and eventually reached at 106 copies/mL.HCV infection titer was detected successfully at 21days after transfection in the group A and at 31days in the group B.The highest infectious titer of groups A and B was both 104 ffu/mL.Conclusions Electroporation and lipofection both can be used to establish HCV cell culture system.Lipofection displays less damage to cells .Although electroporation demonstrates higher transfectional efficiency and short time of obtaining HCVcc , but there is no difference in infectious titer of obtained HCVcc .

关 键 词：丙型肝炎病毒 电穿孔 脂质体 RNA转染 

分 类 号：R373.9[医药卫生—病原生物学]