人轮状病毒G1P[8]型感染树鼩原代小肠上皮细胞模型的建立  

Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8]

作　　者：李道群[1] 彭杰[1] 甸子芩[1,2] 王文广[3] 张阿梅[1] 冯悦[1] 牛华[2] 代解杰[3] 夏雪山[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]昆明理工大学附属医院(云南省第一人民医院),昆明650032 [3]中国医学科学院/北京协和医学院医学生物学研究所树鼩标准化研究与疾病动物模型创建省创新团队,昆明650118

出　　处：《中国实验动物学报》2017年第2期111-116,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家科技支撑计划项目(编号:2014BAI01B01)

摘　　要：目的探究树鼩原代小肠上皮细胞的增殖特性,建立人轮状G1P[8]型病毒体外感染树鼩原代小肠上皮的细胞模型。方法采用胶原酶XI和中性蛋白酶I联合消化法获取树鼩原代小肠上皮细胞,经纯化和鉴定,用人轮状G1P[8]型病毒感染细胞,测定培养上清的病毒滴度和载量,并用Western blot和间接免疫荧光检测法检测人轮状病毒G1P[8]型VP6蛋白的表达情况,评价人轮状G1P[8]型病毒体外对树鼩原代小肠上皮细胞的感染性。结果分离的树鼩原代小肠上皮细胞经传代培养纯化,获得纯度达90%树鼩原代小肠上皮细胞。对树鼩原代小肠上皮细胞、原代肾细胞、HCT116细胞和MA104细胞进行轮状病毒易感性比较,确定树鼩原代小肠上皮细胞可以被人轮状病毒G1P[8]感染,培养72 h时病毒滴度可达到2.0×105TCID_(50)/m L。经Western blot和间接免疫荧光发现在人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞1~5 d均能检测到人轮状病毒VP6蛋白的表达和分布。结论确立了树鼩原代小肠上皮细胞的分离、纯化与培养方法,并建立了人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞的体外模型。Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus （RV） G1P [ 8 ] infection to these cells, and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8]. Methods The primary small intes- tinal epithelial cells were obtained by collagenase XI and dispase I digestion from tree shrew. After purification and identifi- cation, the obtained primary small intestinal epithelial cells were infected with RV. Then, culture supernatants of infected cells were collected every 12 hours after infection. Viral titer and viral load were subsequently determined. Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells. The infectivity of RV to the tree shrew primary cells was finally evaluated. Results After purification and identification of pri- mary epithelial ceils from the tree shrew, high purity above 90% primary tree shrew small intestinal epithelial cells was ob- tained. These primary small intestinal epithelial ceils could be infected with RV virus by comparing the virus infectivity to primary renal cells, HCTll6 cells and MA104 cells. The virus titer reached to 2.0 × 105TCID50/mL at 72 h after infec- tion. Using Western blot and indirect immunofluorescence observation, the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells, and were located in the cytoplasm from days 1 to 5. Conclusions The separation, purification and cultivation methods of tree shrew primary small intestinal epithelial ceils are successful, and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

关 键 词：树鼩原代小肠上皮细胞 轮状病毒 病毒增殖特性 VP6蛋白 

分 类 号：Q95-33[生物学—动物学]