【作者】 张慧娟；

【导师】 方峰；

【作者基本信息】 华中科技大学 ， 儿科学， 2010， 硕士

【摘要】 【目的】研究鼠巨细胞病毒(MCMV)对神经干细胞Wnt信号通路上游分化相关靶基因Wnt-3和Wnt-7a蛋白表达的影响,以进一步探讨CMV致胎脑发育异常的分子机制。【方法】⑴分离培养小鼠神经干细胞(NSCs):剖宫取出孕13.5d的BALB/c胎鼠,去除脑膜,取脑组织,用PBS冲洗,过滤制备单细胞悬液,用含EGF (20ng/mL)、bFGF(20ng/mL)和B27(2%)的DMEM/F12培养基培养。第3代小鼠神经干细胞用于后续实验;⑵建立MCMV感染NSCs分化培养细胞模型:用高、中、低三种感染复数(MOI分别为5、1和0.1)的MCMV毒株感染NSCs,用含2%胎牛血清(无EGF、bFGF和B27)的DMEM/F12培养基培养诱导其分化。同期设正常NSCs分化培养作为对照;⑶MCMV感染对神经干细胞Wnt信号通路上游分化相关靶基因蛋白Wnt-3和Wnt-7a表达的影响:用Western blot法检测分化培养后0.5d、1d、2d、3d、4d和5d NSCs Wnt信号通路上游蛋白Wnt-3、Wnt-7a表达水平的动态变化。实验重复三次。【结果】⑴小鼠神经干细胞体外分离和培养成功,可连续传代,保持球形生长的能力;采用含2%胎牛血清(无EGF、bFGF和B27)的DMEM/F12培养基培养可进一步诱导其分化;⑵当MCMV感染NSCs后,镜下可见细胞贴壁受影响,已贴壁的神经球出现肿胀飘起,神经球边缘的细胞发生脱落,部分细胞崩解坏死。细胞形态改变随病毒感染复数(MOI)的增加而更加明显;⑶Western blot法检测结果: a)正常对照组Wnt-3蛋白表达呈双峰型,在分化2d和5d时达峰值,第二峰值高于第一峰值。各感染亚组Wnt-3蛋白表达亦于分化2d达峰值;MOI=0.1亚组在分化5d出现第二峰,但峰值显著低于第一峰,而MOI=1和5亚组在分化2d后表达水平逐渐下降,第二峰缺失;除MOI=0.1亚组分化2d以外,其余各感染亚组与各时间点Wnt-3蛋白表达量均显著低于正常对照组(P<0.05);MOI=1亚组分化培养0.5d和2d与MOI=5亚组分化培养1d、2d和5d表达量均明显低于MOI=0.1亚组(P<0.05); b)正常NSCs分化培养后Wnt-7a蛋白表达先增后降,在分化1d达峰值。MOI=0.1亚组Wnt-7a蛋白表达的时序性变化与正常对照组相似。而MOI=1和5亚组Wnt-7a蛋白表达量在分化0.5d和1d明显低于正常对照组和MOI=0.1亚组(P<0.05),峰值后移至分化培养3d,MOI=1亚组峰值显著低于正常对照组(P<0.05)。【结论】⑴MCMV抑制分化培养NSCs细胞的Wnt-3蛋白表达,其抑制程度随感染滴度增加和分化进程而趋于明显;MCMV在分化早期(0.5d～1d)抑制Wnt-7a蛋白表达,使其峰值后移,其抑制效应随感染滴度增加更趋明显;⑵MCMV抑制NSCs Wnt信号通路上游分化相关靶基因Wnt-3和Wnt-7a蛋白表达可能是其干扰NSCs增殖分化导致胎脑损伤的重要机制之一。更多还原

【Abstract】 Objectives To investigate the influence of MCMV (murine cytomegalovirus) infection on the protein expression of upstream-differentiation-related target genes (Wnt-3, Wnt-7a) of Wnt signaling pathway in neural stem cells (NSCs) and explore the molecular mechanisms of fetal encephalodysplasia caused by congenital CMV infection.Methods⑴The isolation and culture of murine NSCs: NSCs were isolated from fetal brain of BALB/c mouse on day of 13.5 of gestation, washed with PBS, and filtered to make for single cell suspension, then cultured in DMEM/F12 medium supplemented with EGF (20ng/mL), bFGF (20ng/mL) and B27 (2%). NSCs of the third generation were used in the following experiments.⑵The establishment of the differentiation and culture model of NSCs infected by MCMV: The NSCs were infected by MCMV Smith strain with high, median and low multiplicity of infection(MOI) of 5, 1, and 0.1 respectively, then cultured in DMEM/F12 medium including two percent of fetal bovine serum (without EGF, bFGF and B27) to induce the differentiation. Normal NSCs of homochronous culture and differentiation without infection was as the negative control.⑶The influence of MCMV infection on the protein expression of the upstream-differentiation-related target genes(Wnt-3, Wnt-7a) of Wnt signaling pathway in NSCs : The dynamic protein expression of Wnt-3 and Wnt-7a in NSCs were measured by Western Blot assay at 0.5d, 1d, 2d, 3d, 4d and 5d after differentiation culture. All of the experiments were repeated thrice.Results⑴Murine NSCs were isolated and cultured in vitro successfully, and they could proliferate and duplicate with the appearance of neurosphere, and they also could be induced to differentiate when cultured in DMEM/F12 medium including two percent of fetal bovine serum (without bFGF, EGF and B27).⑵After MCMV infection, the adherence of NSCs were affected, the adherented nerosphere swelled and floated, and the marginal cells of the neurosphere dropped off, partial cells disintegrated. Such morphological change was more obvious with the MOI of virus increased.⑶The results of the Western blot assay: a. The expression of Wnt-3 protein of normal control showed the feature of two crest on day 2 and day 5 after differentiation, and the second crest was higher than the first. Although that of infection group also had a crest on 2d, only the subgroup of MOI=0.1 showed the second crest, which was obviously lower than the first. In MOI=1 and MOI=5 subgroup, the expression of Wnt-3 reduced gradually after 2d with absence of the second crest. The levels of Wnt-3 protein of the infection groups were obviously lower than those of normal control at all time points except 2d of the MOI=0.1 group (P<0.05); the levels of Wnt-3 of MOI=1 subgroup and MOI=5 subgroup were significantly lower than those of MOI=0.1 on 0.5d, 2d and 1d, 2d, 5d respectively(P<0.05); b. In normal control, the protein expression of Wnt-7a increased first, then decreased, and reached the peak on day 1 after differentiation. And that of the MOI=0.1 subgroup were similar. However, in MOI=1 and 5 subgroup, the level of Wnt-7a was obviously lower than that of normal control and MOI=0.1 subgroup on 0.5d and 1d (P<0.05), the peak of Wnt-7a expression delayed to day 3 after differentiation, and the peak of MOI=1 subgroup was significantly lower than that of normal control(P<0.05).Conclusions1) MCMV inhibited the protein expression of Wnt-3 in differentiated NSCs, which was more apparent with MOI increasing and differentiation proceeding. MCMV inhibited the protein expression of Wnt-7a in earlier period of differentiation (0.5d～1d), and delayed their peak, which also showed dose-dependence with MOI.2) MCMV could inhibit the protein expression of upstream-differentiation-related target genes (Wnt-3, Wnt-7a) of Wnt signaling pathway in NSCs, which may be one of the important mechanisms causing the impairment of fetal brain through interfering the proliferation and differentiation of NSCs.更多还原