HCMV感染人海马NSCs分化Astrocytes并诱导细胞凋亡的研究
发布时间：2019-06-04 23:11
【摘要】： 目的研究人类巨细胞病毒(HCMV)能否感染体外培养的由人海马神经干细胞(NSCs)分化成的星形胶质细胞(Astrocytes),并观察病毒感染对细胞凋亡的影响,为探讨先天性巨细胞病毒感染致脑发育异常的机制提供理论依据。 方法体外分离、培养人海马NSCs,添加血清诱导其定向分化为星形胶质细胞,倒置显微镜下观察细胞的形态学特点并利用间接免疫荧光染色方法通过检测其各自的标志蛋白进行鉴定。然后用HCMV AD169株感染人星形胶质细胞(MOI=1),镜下观察感染后细胞的形态学变化,利用间接免疫荧光技术检测细胞内病毒基因产物-IE蛋白的表达,采用流式细胞术PI染色和细胞涂片Annexin V-FITC/PI染色法检测感染细胞的凋亡情况。同时收集感染细胞的培养液,接种到正常人胚肺二倍体细胞(MRC-5)中,观察有无典型的HCMV致细胞病变出现。 结果体外原代培养的人海马NSCs以神经球的形式悬浮生长,绝大多数细胞都表达NSCs的标记物-巢蛋白(nestin),诱导其分化后,细胞高表达星形胶质细胞的标志物—神经胶质纤维酸性蛋白(GFAP)。HCMV AD169感染星形胶质细胞7天后,细胞出现肿胀、变大、变圆等典型病变,胞核内检测到HCMV即刻早期基因的表达产物-IE蛋白。流式细胞术PI单染结果显示:MOI=1时,感染组细胞凋亡比例为14.72%±5.78%,明显高于正常对照组7.59%±2.33%,两组比较有统计学意义(t=3.18,P0.05)。细胞涂片Annexin V-FITC/PI染色结果也显示,感染组细胞凋亡比例高于对照组。收集感染细胞的培养液并接种到HCMV的完全容许性细胞—MRC-5中,一周后细胞也出现上述典型病变,证明HCMV可在星形胶质细胞内增殖、复制并释放到胞外。 结论(1)在体外培养的由人NSCs分化成的星形胶质细胞中,HCMV AD169能够建立增殖性感染,该细胞是HCMV的容许性细胞。 (2)HCMV感染诱导体外培养的由人NSCs分化成的星形胶质细胞凋亡,这可能与先天性巨细胞病毒感染导致脑发育异常有关。
[Abstract]:Objective to study whether human cytomegalovirus (HCMV) can infect (Astrocytes), which is differentiated from human hippocampal neural stem cell (NSCs) in vitro, and to observe the effect of viral infection on apoptosis. It provides a theoretical basis for exploring the mechanism of abnormal brain development caused by congenital cytomegalovirus infection. Methods Human hippocampal NSCs, was isolated in vitro and cultured with serum to induce its directional differentiation into astrocytes. The morphological characteristics of the cells were observed under inverted microscope and identified by indirect immunofluorescence staining. Then the human astrocytes (MOI=1) were infected with HCMV AD169 strain. The morphological changes of the infected cells were observed under microscope, and the expression of IE protein, the intracellular viral gene product, was detected by indirect immunofluorescence technique. Flow cytometry PI staining and cell smear Annexin V-FITC/PI staining were used to detect the apoptosis of infected cells. At the same time, the culture medium of infected cells was collected and inoculated into normal embryonic lung diploid cells (MRC-5) to observe whether there were typical HCMV-induced cytopathic effects. Results Human hippocampal NSCs cultured in vitro was suspended in the form of neurosphere. Most of the cells expressed nestin (nestin), a marker of NSCs, to induce the differentiation of Hippocampal DNA. Seven days after the astrocytes were infected with glial fibrillar acidic protein (GFAP). HCMV AD169), a marker of high expression of astrocytes, the cells showed typical lesions such as swelling, enlargement and roundness. IE protein, an immediate early gene expression product of HCMV, was detected in the nucleus. The results of flow cytometry PI single staining showed that the proportion of apoptosis in the infected group was 14.72% 卤5.78%, which was significantly higher than that in the normal control group (7.59% 卤2.33%). There was significant difference between the two groups (t 鈮，
本文编号：2493069
[Abstract]:Objective to study whether human cytomegalovirus (HCMV) can infect (Astrocytes), which is differentiated from human hippocampal neural stem cell (NSCs) in vitro, and to observe the effect of viral infection on apoptosis. It provides a theoretical basis for exploring the mechanism of abnormal brain development caused by congenital cytomegalovirus infection. Methods Human hippocampal NSCs, was isolated in vitro and cultured with serum to induce its directional differentiation into astrocytes. The morphological characteristics of the cells were observed under inverted microscope and identified by indirect immunofluorescence staining. Then the human astrocytes (MOI=1) were infected with HCMV AD169 strain. The morphological changes of the infected cells were observed under microscope, and the expression of IE protein, the intracellular viral gene product, was detected by indirect immunofluorescence technique. Flow cytometry PI staining and cell smear Annexin V-FITC/PI staining were used to detect the apoptosis of infected cells. At the same time, the culture medium of infected cells was collected and inoculated into normal embryonic lung diploid cells (MRC-5) to observe whether there were typical HCMV-induced cytopathic effects. Results Human hippocampal NSCs cultured in vitro was suspended in the form of neurosphere. Most of the cells expressed nestin (nestin), a marker of NSCs, to induce the differentiation of Hippocampal DNA. Seven days after the astrocytes were infected with glial fibrillar acidic protein (GFAP). HCMV AD169), a marker of high expression of astrocytes, the cells showed typical lesions such as swelling, enlargement and roundness. IE protein, an immediate early gene expression product of HCMV, was detected in the nucleus. The results of flow cytometry PI single staining showed that the proportion of apoptosis in the infected group was 14.72% 卤5.78%, which was significantly higher than that in the normal control group (7.59% 卤2.33%). There was significant difference between the two groups (t 鈮，
本文编号：2493069
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