MCMV感染对神经干细胞分化和细胞周期进程的影响及其机制研究
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摘要
【目的】
①在体外细胞水平研究巨细胞病毒感染对神经干细胞(NSCs)分化的影响及其机制;
②探讨巨细胞病毒感染对神经干细胞Wnt信号途径分化相关基因转录水平的影响;
③研究巨细胞病毒感染对神经干细胞细胞周期蛋白cyclinA、cyclinB1、cyclinD1、cyclinE和细胞周期进程的影响并探讨其机制。
【方法】
①小鼠神经干细胞的分离培养、鉴定和分化潜能的检测:剖宫取出孕13.5d的BALB/c胎鼠脑组织,制备单细胞悬液,过滤后用含EGF、bFGF和B27的DMEM/F12培养基培养;传4～5代后用nestin免疫荧光鉴定;用添加2%胎牛血清的DMEM/F12分化培养基(不含EGF、bFGF和B27)诱导NSCs分化,7天后采用免疫荧光法检测(nestin)、神经元特异性烯醇酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。
②MCMV感染NSCs分化培养模型的建立:用感染复数(MOI)分别为5、1和0.1的MCMV感染NSCs,并改用分化培养基进行分化培养。
③观察MCMV感染分化培养NSCs形态学的变化并示踪感染过程:采用免疫荧光法分别于分化培养后3d、6d、9d、12d、15d、18d和21d观察NSCs及其分化细胞标记物nestin、GFAP和NSE表达的变化,并用MCMV EA示踪感染过程。
④MCMV感染对NSCs分化细胞比率的影响:采用流式细胞术分别于分化培养后3d、6d和9d检测NSCs及其分化细胞比率的变化。
⑤MCMV感染对NSCs Wnt信号途径分化相关基因mRNA表达的影响:采用Real-time PCR法检测分化培养后0.5、1、2、3、4和5d时NSCs Wnt信号途径上游基因Wnt-3、Wnt-7a和下游基因Ngn-2、c-myc及CyclinD1 mRNA水平的动态变化。
⑥MCMV感染对NSCs cyclinA、cyclinB1、cyclinD1、cyclinE和细胞周期进程的影响:用感染复数(MOI)分别为5、1和0.1的MCMV感染NSCs,并用含EGF、bFGF和B27的DMEM/F12培养基继续培养,采用Cyclins/DNA双参数流式细胞术检测感染细胞cyclinA、cyclinB1、cyclinD1、cyclinE的表达和细胞周期时相的动态变化。
【结果】
①成功体外分离培养NSCs,NSCs呈球样生长,神经干细胞特异性标记nestin表达阳性,并可进一步分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞。
②分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,甚至崩解死亡;细胞nestin表达下调缓慢,显著高于对照组,GFAP和NSE表达显著低于对照组(P<0.05),分化细胞中可检测到MCMV EA(早期抗原)的阳性表达。
③分化培养后3～9d,感染组Nestin阳性细胞比率均显著高于对照组(P<0.05),而GFAP和NSE阳性细胞比率显著低于对照组(P<0.05),并随MOI增加差异越明显。
④分化培养后,感染组Wnt-3 mRNA的表达水平在第1～2d显著低于正常对照组(P<0.05),其后逐渐升高但未超过正常水平;感染组Wnt–7a和Ngn-2 mRNA水平分别在第0.5～2d和第1d明显低于正常组(P<0.05),均至第3d开始升高,但其总体峰值水平均低于正常组;感染组c-myc mRNA水平在第1～4d显著低于正常组(P<0.05);感染组CyclinD1 mRNA水平在第0.5～1d明显低于正常组,在第2d(MOI=5)和第3d(MOI=5、1、0.1)高于正常组(P<0.05);这五种基因表达变化的幅度随MOI的增加而增大。
⑤感染后,NSCs cyclinA、cyclinB1、cyclinD1和cyclinE的表达均上调,其中MOI=0.1表达逐渐上升,在第6d达峰值,MOI=1组表达高峰在第4d,MOI=5组表达高峰在第3d;感染组G0/G1期细胞比率减少,S期和G2/M期细胞比率显著增加,出现S期和G2/M期阻滞,并随MOI的增加变化越明显。
【结论】
①MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少,分化发育过程中的神经元和星形胶质细胞对MCMV易感。
②MCMV可下调NSCs Wnt信号途径分化相关基因Wnt-3、Wnt-7a、Ngn-2、c-myc的表达并干扰CyclinD1基因表达,这可能是MCMV感染抑制NSCs分化的重要作用机制。
③MCMV抑制NSCs分化的效应及对其Wnt信号途径分化相关基因表达的影响与MOI大小存在一定量效依赖关系,MCMV可通过干扰分化相关基因的表达来抑制NSCs分化,可能与CMV感染致脑发育异常密切相关。
④MCMV可上调cyclinA、cyclinB1、cyclinD1、cyclinE的表达,诱导NSCs从G0/G1期进入S期,出现S期和G2/M期偏移和阻滞,影响NSCs细胞周期进程,并与MOI存在一定量效依赖关系,这可能是CMV抑制NSCs增殖的主要原因。
⑤CMV感染对NSCs细胞周期进程和分化的抑制作用可能是先天性CMV感染致脑发育异常的重要机制。
Objectives:The purposes of the present study are:①to study the influence of cytomegalovirus infection on differentiation of neural stem cells (NSCs) in vitro.②to explore the effect of cytomegalovirus infection on the transcription of differentiation related genes in Wnt signal passway of NSCs.③to investigate the influence of cytomegalovirus infection on cyclins expression and cell cycle progression of NSC.
Methods:
①NSCs were isolated, cultured and identified, and it’s differentiation potency were observed. NSCs were isolated from fetal brain of BALB/c mouse on day 13.5d of gestation and cultured in DMEM/F12 medium including EGF, bFGF and B27. Subcultured NSCs were identified by detecting nestin. NSCs were induced to differentiate in DMEM/F12 medium including 2% fetal bovine serum. Nestin, GFAP and NSE of cells were detected by immunofluorescence.
②A differentiation culture model of infected NSCs was established. The NSCs infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively, were cultured in differentiation medium.
③Morphological changes of infected NSCs and infection process were observed. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence (MOI=1) at 3d, 6d, 9d, 12d, 15d, 18d and 21d. The EA expression of MCMV was detected to observe the infection process.
④The differentiated cells rate of infected NSCs was studied. Flow cytometry was employed to measure the rate changes of NSCs and its differentiated cells at 3d, 6d and 9d.
⑤The influence of MCMV on transcription of differentiation related genes in Wnt signal passway of NSCs in vitro were investigated. Real-time RT-PCR method was employed to measure the expression levels of the upperstream gene Wnt-3、Wnt-7a and the downstream gene Ngn-2、c-myc and CyclinD1 in Wnt signal pathway of NSCs at 0.5d, 1d, 2d, 3d, 4d and 5d.
⑥The influence of MCMV infection on cyclins expression and cell cycle progression of NSCs in vitro were investigated. The NSCs were infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively and cultured in DMEM/F12 medium including EGF, bFGF and B27. The dynamic changes of cyclinA, cyclinB1, cyclinD1, cyclinE and cell cycle progression of infected NSCs were detected at 1d, 2d, 3d, 4d, 5d and 6d.
Results
①NSCs were successfully isolated and cultured in vitro. NSCs could proliferate to form neurosphere and strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NeuN positive neurons or GFAP positive astrocytes.
②The infected NSCs couldn’t adhere to the wall and appear differentiation growth, but showed swelling gradually and disintegrated after differentiation culture. The nestin expression of the infected NSCs downregulated slowly and was higher than the control group (P<0.05). The expression of GFAP and NSE were lower than the control (P<0.05). The early antigen (EA) of MCMV could be always detected in the differentiated cells.
③The rate of nestin positive cells was higher than the control group and the rate of GFAP and NSE positive cells were lower than it from 3d to 9d during differentiation culture(P<0.05).The changes of the infected groups were obvious following the MOI increased
④The levels of Wnt-3 mRNA of infected groups decreased from 1d to 2d, obviously lower than that of normal control group (P<0.05), then increased but still not higher than the control. The levels of Wnt-7a and Ngn-2 mRNA were markedly lower than the control from 0.5d to 2d and at 1d, respectively (P<0.05) and both increased at 3d, but the general peak value were significantly lower than the control. The levels of c-myc mRNA were lower than the control from 1d to 4d (P<0.05). The levels of CyclinD1 mRNA were obviously lower than the control from 0.5d to 1d (P<0.05), and higher than it at 2d (MOI=5) and at 3d (MOI=5, 1, 0.1) (P<0.05). The expression change range of these genes increased following the MOI increase of MCMV.
⑤The expression of cyclinA, cyclinB1, cyclinD1 and cyclinE of infected NSCs upregulated. The expression of cyclins of the infected NSCs with MOI=0.1 gradually increased and reached to the peak at 6d. The peak expression of cyclins of the infected NSCs with MOI=1 and 5 appeared at 4d and 3d post infection. The cell rate of G0/G1 phase decreased and the rate of S phase and G2/M phase increased. The infected NSCs migrated to and blocked at S phase and G2/M phase. These changes were more obvious following the MOI increased.
Conclusions
①MCMV could inhibit significantly NSCs differentiate into neurons and astrocytes and lead to the decrease of differentiated cells. The neurons and astrocytes during differentiation development period were both susceptible to MCMV.
②MCMV could downregulate or interfere the expression of differentiation related genes Wnt-3, Wnt-7a, Ngn-2, c-myc and CyclinD1 in Wnt signal pathway of NSCs, which may be involved in the inhibitory effect of MCMV on NSCs differentiation.
③The inhibitory effect of MCMV on NSCs differentiation and the influence of MCMV on the transcription of differentiation related gene of NSCs showed dose-dependent with MOI. MCMV could inhibit differentiation of NSCs by inhibiting the transcription of differentiation related gene, which may be one of primary causes of brain development disorders caused by congenital CMV infection.
④MCMV could influence the cell cycle processes of NSCs by upregulating expression of cyclinA, cyclinB1, cyclinD1, cyclinE and inducing NSCs enter into S phase from G0/G1 phase and appear S phase arrest and G2/M phase arrest. The effect showed certain dose dependence relationship with MOI, which may be major causes of MCMV inhibit the proliferation of NSCs.
⑤The inhibitory effect of MCMV infection on differentiation and cell cycle progression of nerve stem cells maybe the important mechanisms of congenital encephalodysplasia induced by CMV.
①在体外细胞水平研究巨细胞病毒感染对神经干细胞(NSCs)分化的影响及其机制;
②探讨巨细胞病毒感染对神经干细胞Wnt信号途径分化相关基因转录水平的影响;
③研究巨细胞病毒感染对神经干细胞细胞周期蛋白cyclinA、cyclinB1、cyclinD1、cyclinE和细胞周期进程的影响并探讨其机制。
【方法】
①小鼠神经干细胞的分离培养、鉴定和分化潜能的检测:剖宫取出孕13.5d的BALB/c胎鼠脑组织,制备单细胞悬液,过滤后用含EGF、bFGF和B27的DMEM/F12培养基培养;传4～5代后用nestin免疫荧光鉴定;用添加2%胎牛血清的DMEM/F12分化培养基(不含EGF、bFGF和B27)诱导NSCs分化,7天后采用免疫荧光法检测(nestin)、神经元特异性烯醇酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。
②MCMV感染NSCs分化培养模型的建立:用感染复数(MOI)分别为5、1和0.1的MCMV感染NSCs,并改用分化培养基进行分化培养。
③观察MCMV感染分化培养NSCs形态学的变化并示踪感染过程:采用免疫荧光法分别于分化培养后3d、6d、9d、12d、15d、18d和21d观察NSCs及其分化细胞标记物nestin、GFAP和NSE表达的变化,并用MCMV EA示踪感染过程。
④MCMV感染对NSCs分化细胞比率的影响:采用流式细胞术分别于分化培养后3d、6d和9d检测NSCs及其分化细胞比率的变化。
⑤MCMV感染对NSCs Wnt信号途径分化相关基因mRNA表达的影响:采用Real-time PCR法检测分化培养后0.5、1、2、3、4和5d时NSCs Wnt信号途径上游基因Wnt-3、Wnt-7a和下游基因Ngn-2、c-myc及CyclinD1 mRNA水平的动态变化。
⑥MCMV感染对NSCs cyclinA、cyclinB1、cyclinD1、cyclinE和细胞周期进程的影响:用感染复数(MOI)分别为5、1和0.1的MCMV感染NSCs,并用含EGF、bFGF和B27的DMEM/F12培养基继续培养,采用Cyclins/DNA双参数流式细胞术检测感染细胞cyclinA、cyclinB1、cyclinD1、cyclinE的表达和细胞周期时相的动态变化。
【结果】
①成功体外分离培养NSCs,NSCs呈球样生长,神经干细胞特异性标记nestin表达阳性,并可进一步分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞。
②分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,甚至崩解死亡;细胞nestin表达下调缓慢,显著高于对照组,GFAP和NSE表达显著低于对照组(P<0.05),分化细胞中可检测到MCMV EA(早期抗原)的阳性表达。
③分化培养后3～9d,感染组Nestin阳性细胞比率均显著高于对照组(P<0.05),而GFAP和NSE阳性细胞比率显著低于对照组(P<0.05),并随MOI增加差异越明显。
④分化培养后,感染组Wnt-3 mRNA的表达水平在第1～2d显著低于正常对照组(P<0.05),其后逐渐升高但未超过正常水平;感染组Wnt–7a和Ngn-2 mRNA水平分别在第0.5～2d和第1d明显低于正常组(P<0.05),均至第3d开始升高,但其总体峰值水平均低于正常组;感染组c-myc mRNA水平在第1～4d显著低于正常组(P<0.05);感染组CyclinD1 mRNA水平在第0.5～1d明显低于正常组,在第2d(MOI=5)和第3d(MOI=5、1、0.1)高于正常组(P<0.05);这五种基因表达变化的幅度随MOI的增加而增大。
⑤感染后,NSCs cyclinA、cyclinB1、cyclinD1和cyclinE的表达均上调,其中MOI=0.1表达逐渐上升,在第6d达峰值,MOI=1组表达高峰在第4d,MOI=5组表达高峰在第3d;感染组G0/G1期细胞比率减少,S期和G2/M期细胞比率显著增加,出现S期和G2/M期阻滞,并随MOI的增加变化越明显。
【结论】
①MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少,分化发育过程中的神经元和星形胶质细胞对MCMV易感。
②MCMV可下调NSCs Wnt信号途径分化相关基因Wnt-3、Wnt-7a、Ngn-2、c-myc的表达并干扰CyclinD1基因表达,这可能是MCMV感染抑制NSCs分化的重要作用机制。
③MCMV抑制NSCs分化的效应及对其Wnt信号途径分化相关基因表达的影响与MOI大小存在一定量效依赖关系,MCMV可通过干扰分化相关基因的表达来抑制NSCs分化,可能与CMV感染致脑发育异常密切相关。
④MCMV可上调cyclinA、cyclinB1、cyclinD1、cyclinE的表达,诱导NSCs从G0/G1期进入S期,出现S期和G2/M期偏移和阻滞,影响NSCs细胞周期进程,并与MOI存在一定量效依赖关系,这可能是CMV抑制NSCs增殖的主要原因。
⑤CMV感染对NSCs细胞周期进程和分化的抑制作用可能是先天性CMV感染致脑发育异常的重要机制。
Objectives:The purposes of the present study are:①to study the influence of cytomegalovirus infection on differentiation of neural stem cells (NSCs) in vitro.②to explore the effect of cytomegalovirus infection on the transcription of differentiation related genes in Wnt signal passway of NSCs.③to investigate the influence of cytomegalovirus infection on cyclins expression and cell cycle progression of NSC.
Methods:
①NSCs were isolated, cultured and identified, and it’s differentiation potency were observed. NSCs were isolated from fetal brain of BALB/c mouse on day 13.5d of gestation and cultured in DMEM/F12 medium including EGF, bFGF and B27. Subcultured NSCs were identified by detecting nestin. NSCs were induced to differentiate in DMEM/F12 medium including 2% fetal bovine serum. Nestin, GFAP and NSE of cells were detected by immunofluorescence.
②A differentiation culture model of infected NSCs was established. The NSCs infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively, were cultured in differentiation medium.
③Morphological changes of infected NSCs and infection process were observed. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence (MOI=1) at 3d, 6d, 9d, 12d, 15d, 18d and 21d. The EA expression of MCMV was detected to observe the infection process.
④The differentiated cells rate of infected NSCs was studied. Flow cytometry was employed to measure the rate changes of NSCs and its differentiated cells at 3d, 6d and 9d.
⑤The influence of MCMV on transcription of differentiation related genes in Wnt signal passway of NSCs in vitro were investigated. Real-time RT-PCR method was employed to measure the expression levels of the upperstream gene Wnt-3、Wnt-7a and the downstream gene Ngn-2、c-myc and CyclinD1 in Wnt signal pathway of NSCs at 0.5d, 1d, 2d, 3d, 4d and 5d.
⑥The influence of MCMV infection on cyclins expression and cell cycle progression of NSCs in vitro were investigated. The NSCs were infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively and cultured in DMEM/F12 medium including EGF, bFGF and B27. The dynamic changes of cyclinA, cyclinB1, cyclinD1, cyclinE and cell cycle progression of infected NSCs were detected at 1d, 2d, 3d, 4d, 5d and 6d.
Results
①NSCs were successfully isolated and cultured in vitro. NSCs could proliferate to form neurosphere and strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NeuN positive neurons or GFAP positive astrocytes.
②The infected NSCs couldn’t adhere to the wall and appear differentiation growth, but showed swelling gradually and disintegrated after differentiation culture. The nestin expression of the infected NSCs downregulated slowly and was higher than the control group (P<0.05). The expression of GFAP and NSE were lower than the control (P<0.05). The early antigen (EA) of MCMV could be always detected in the differentiated cells.
③The rate of nestin positive cells was higher than the control group and the rate of GFAP and NSE positive cells were lower than it from 3d to 9d during differentiation culture(P<0.05).The changes of the infected groups were obvious following the MOI increased
④The levels of Wnt-3 mRNA of infected groups decreased from 1d to 2d, obviously lower than that of normal control group (P<0.05), then increased but still not higher than the control. The levels of Wnt-7a and Ngn-2 mRNA were markedly lower than the control from 0.5d to 2d and at 1d, respectively (P<0.05) and both increased at 3d, but the general peak value were significantly lower than the control. The levels of c-myc mRNA were lower than the control from 1d to 4d (P<0.05). The levels of CyclinD1 mRNA were obviously lower than the control from 0.5d to 1d (P<0.05), and higher than it at 2d (MOI=5) and at 3d (MOI=5, 1, 0.1) (P<0.05). The expression change range of these genes increased following the MOI increase of MCMV.
⑤The expression of cyclinA, cyclinB1, cyclinD1 and cyclinE of infected NSCs upregulated. The expression of cyclins of the infected NSCs with MOI=0.1 gradually increased and reached to the peak at 6d. The peak expression of cyclins of the infected NSCs with MOI=1 and 5 appeared at 4d and 3d post infection. The cell rate of G0/G1 phase decreased and the rate of S phase and G2/M phase increased. The infected NSCs migrated to and blocked at S phase and G2/M phase. These changes were more obvious following the MOI increased.
Conclusions
①MCMV could inhibit significantly NSCs differentiate into neurons and astrocytes and lead to the decrease of differentiated cells. The neurons and astrocytes during differentiation development period were both susceptible to MCMV.
②MCMV could downregulate or interfere the expression of differentiation related genes Wnt-3, Wnt-7a, Ngn-2, c-myc and CyclinD1 in Wnt signal pathway of NSCs, which may be involved in the inhibitory effect of MCMV on NSCs differentiation.
③The inhibitory effect of MCMV on NSCs differentiation and the influence of MCMV on the transcription of differentiation related gene of NSCs showed dose-dependent with MOI. MCMV could inhibit differentiation of NSCs by inhibiting the transcription of differentiation related gene, which may be one of primary causes of brain development disorders caused by congenital CMV infection.
④MCMV could influence the cell cycle processes of NSCs by upregulating expression of cyclinA, cyclinB1, cyclinD1, cyclinE and inducing NSCs enter into S phase from G0/G1 phase and appear S phase arrest and G2/M phase arrest. The effect showed certain dose dependence relationship with MOI, which may be major causes of MCMV inhibit the proliferation of NSCs.
⑤The inhibitory effect of MCMV infection on differentiation and cell cycle progression of nerve stem cells maybe the important mechanisms of congenital encephalodysplasia induced by CMV.
引文
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