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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 investigate the proliferation characteristics of primary intestinal epithelial cells of tree shrews and to establish a cell model of human primary shunt epithelial cells infected by human rotifer G1P [8] virus in vitro. Methods Primary intestinal epithelial cells of tree shrews were obtained by digestion with collagenase XI and neutral protease I. After purification and identification, the human rotaric G1P [8] virus was used to infect the cells and the virus titer and load in the culture supernatant were determined . The expression of human rotavirus G1P [8] VP6 protein was detected by Western blot and indirect immunofluorescence assay to evaluate the infectivity of human rotifer G1P [8] virus to the primary intestinal epithelial cells of tree shrew in vitro. Results The isolated tree 鼩 primary intestinal epithelial cells were subcultured and purified to obtain 90% pure epithelial cells of tree shrews. The primary intestinal epithelial cells, primary kidney cells, HCT116 cells and MA104 cells of tree shrews were compared for susceptibility to rotavirus to determine that the primary epithelial cells of tree shrews could be infected with human rotavirus G1P [8] and cultured for 72 h When the virus titer can reach 2.0 × 105TCID_ (50) / m L. Western blot and indirect immunofluorescence showed that the expression and distribution of human rotavirus VP6 protein was detected in human primary circular epithelial cells of tree shrews 1 ~ 5 days after infection with human rotarod G1P [8]. Conclusion The isolation, purification and culture methods of primary intestinal epithelial cells of tree shrews were established. In vitro model of primary intestinal epithelial cells of tree shrews infected with human rotifer G1P [8] virus was established.