【作者】 陈娟娟；

【导师】 陈素华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2011， 博士

【摘要】 第一部分MCMV感染对乳鼠海马突触数目及突触相关蛋白的影响[目的]研究MCMV感染对乳鼠海马神经元突触数目及突触相关蛋白表达的影响。[方法]①体外细胞培养法传毒增殖MCMV Smith毒株,Reed-Muench法计算病毒毒力。②建立乳鼠脑组织MCMV感染模型：ELISA法筛选MCMV IgM和IgG均为阴性的BALB/C小鼠,雌雄小鼠同笼受孕。仔鼠出生当天,按窝随机分为实验组和对照组。实验组(n=90)颅内接种MCMV病毒悬液10μl,对照组(n=90)颅内接种等量的细胞维持液。无菌采集接种后3d、15d、30d脑组织各30只,应用PCR法检测其MCMV感染情况,实验组内MCMV DNA阳性以及对照组内MCMV DNA阴性脑组织用于后续试验。③应用免疫荧光标记SYP,并在激光共聚焦显微镜下观察各组乳鼠海马SYP荧光颗粒数目,以此代表突触数目。④应用Real-time qPCR、免疫组织化学方法及Western blot法检测各组乳鼠海马突触相关蛋白SYP、PSD-95及AMPA受体GluR2亚基的编码基因转录水平及蛋白质表达水平。[结果]①CMV Smith毒株的TCID50为10-4.5/0.1ml。②实验组小鼠脑组织中均可检见MCMV DNA,对照组小鼠脑组织中未检见MCMV DNA。③随日龄增加,两组乳鼠海马SYP荧光颗粒数目不断增加；与对照组比较,实验组各时相乳鼠海马SYP荧光颗粒数目均减少,其中第15d和第30d减少更为明显,差异具有统计学意义。④随日龄增加,两组乳鼠海马SYP、PSD-95及AMPA受体GluR2亚基编码基因转录水平及蛋白质表达水平均不断增高；与对照组比较,实验组各时相三种蛋白的编码基因转录水平及蛋白质表达水平均降低,其中,第15d和第30d下降更明显,差异具有统计学意义。[结论]①CMV感染可导致发育中的乳鼠海马突触形成数目减少,并且随着日龄的增加,其影响更为明显。②MCMV可导致发育过程中的乳鼠突触相关蛋白SYP、PSD-95及AMPA受体GluR2亚基的编码基因转录水平及蛋白质表达水平下降。第二部分MCMV感染后星形胶质细胞分泌产物对神经元突触的影响[目的]研究MCMV感染后星形胶质细胞分泌产物对神经元突触数目及突触相关蛋白表达的影响。[方法]①建立星形胶质细胞MCMV感染模型：原代及传代培养乳鼠海马星形胶质细胞；应用免疫细胞化学方法检测星形胶质细胞特异性标记物GFAP的表达情况,鉴定并检测星形胶质细胞的纯度；以100TCID50MCMV攻击星形胶质细胞后,通过PCR检测MCMV DNA、观察细胞培养液对NIH 3T3细胞的细胞毒作用、CCK-8法检测MCMV对星形胶质细胞存活率的影响,以及绘制MCMV在星形胶质细胞内的生长曲线等方法,判定星形胶质细胞对MCMV的易感性、感染状态以及MCMV在细胞内的生长情况。②建立星形胶质细胞培养液(Astrocyte conditioned medium, ACM)与神经元共培养体系：原代培养乳鼠海马神经元；应用免疫细胞化学方法检测神经元特异性标记物NSE的表达情况,鉴定并检测神经元的纯度；以100TCID50MCMV攻击传代培养的星形胶质细胞,收集MCMV感染后6、12、24、48、72h以及相同时间点的对照组ACM,过滤及离心后用紫外灯照射15min灭活CMV；应用CCK-8法检测不同浓度ACM对神经元存活的影响,以确定最佳的ACM浓度应用于后续实验；根据神经元细胞培养液各组分不同,分为实验组(含最佳浓度的各不同时间点感染ACM、ACM对照组(含最佳浓度的各不同时间点正常ACM)及空白对照组(不含ACM)。③应用免疫荧光标记SYP,并在荧光显微镜下观察各组神经元突触数目,以此代表突触数目。④应用Real-time qPCR及免疫细胞化学方法检测各组神经元突触相关蛋白SYP、PSD-95及AMPA受体GluR2亚基的编码基因转录水平及蛋白质表达水平。[结果]①体外培养星形胶质细胞(纯度达95%以上)感染MCMV后发生典型细胞病变；在MCMV感染48h的星形胶质细胞内可以检测到MCMV IE DNA的表达；感染后3d-4d的ACM可使NIH 3T3细胞发生典型的CMV病毒蚀斑；星形胶质细胞在感染MCMV后24h开始死亡,感染后72h大部分细胞死亡,至感染后96h细胞几乎全部死亡;MCMV在星形胶质细胞内的复制于感染后12h开始增高,72h达到高峰。②体外培养神经元的纯度可达98%以上,培养基含50%ACM时神经元存活率最高。③与空白对照组相比,ACM对照组和实验组12h-72h SYP荧光颗粒数目均明显增多(P<0.01)；与ACM对照组比较,实验组12h-72h SYP荧光颗粒数目均明显减少(P<0.01)。组内比较发现,ACM对照组12h、24h、48h SYP荧光颗粒数目均比前一时相明显升高(P<0.01),72h变化不明显(P>0.05)；实验组仅12h SYP荧光颗粒数比前一时相明显升高(P<0.01),24h、48h、72h变化不明显(P>0.05)。④与空白对照组比较,ACM对照组和实验组12-72h三种蛋白的编码基因转录水平及蛋白质表达水平均显著升高(P<0.01)。与ACM对照组比较,实验组12-72h三种蛋白的编码基因转录水平及蛋白质表达水平均显著降低(P<0.01)。组内比较发现,ACM对照组12h、24h、48h三种蛋白的编码基因转录水平及蛋白质表达水平均比前一时相明显升高(P<0.01),72h变化不明显(P>0.05)；实验组仅12h三种蛋白的编码基因转录水平及蛋白质表达水平均比前一时相明显升高(P<0.0.1),24h、48h、72h变化不明显(P>0.05)。[结论]①MCMV可在传代培养的星形胶质细胞内复制、产毒并产生致细胞病变效应。②星形胶质细胞分泌物可促进突触形成,MCMV可能通过影响星形胶质细胞的合成及分泌功能而使突触形成减少。③星形胶质细胞分泌物可促进神经元突触相关蛋白的编码基因转录及蛋白质表达,MCMV可能通过影响星形胶质细胞分泌功能而干扰神经元突触相关蛋白的编码基因转录及蛋白质表达。第三部分MCMV感染对星形胶质细胞合成分泌TSP-1及TNF-a的影响[目的]研究MCMV对星形胶质细胞合成分泌TSP-1及TNF-a的影响。[方法]传代培养星形胶质细胞,分为实验组和对照组。实验组加入100 TCID50 MCMV及细胞维持液,对照组加入细胞维持液。收集6、12、24、48、72h的细胞和ACM, ACM过滤及离心后用紫外灯照射15min灭活CMV。①应用Real-time qPCR及免疫细胞化学方法检测MCMV感染对星形胶质细胞TSP-1、TNF-αmRNA及蛋白质表达水平的影响。②应用ELISA法检测MCMV感染对星形胶质细胞分泌TSP-1及TNF-α的影响。[结果]①与对照组比较,实验组各时相TSP-1及TNF-αmRNA及蛋白质表达水平均降低(P均<0.01)；感染时间越长,表达水平越低,差异越大。组内比较发现,感染组12h-72h表达水平均低于前一时相(P均<0.05)。②星形胶质细胞培养12h后才能在ACM中检测到TSP-1及TNF-α;对照组TSP-1及TNF-α含量在48h内迅速升高(与前一时相比较,P均<0.05),72h升高速度减慢；与对照组比较,实验组各时相TSP-1及TNF-α含量均减少(P均<0.01),且12h-72h含量基本保持不变(与前一时相比较,P均>0.05)。[结论]①CMV感染后星形胶质细胞TSP-1及TNF-αmRNA表达水平及蛋白质合成能力降低,且其降低程度随感染时间延长而加剧,感染72h后几乎无mRNA表达水平及蛋白质合成。②正常星形胶质细胞体外培养时TSP-1及TNF-α分泌量不断增加,而MCMV感染抑制了星形胶质细胞的分泌功能,TSP-1及TNF-α分泌量始终维持在较低水平。更多还原

【Abstract】 Part 1 Effect of MCMV infection on the number of hippocampus synapse and the expression of related proteins in neonate miceObjective To study the effect of MCMV infection on the number of hippocampus synapse and the expression of related proteins in neonate mice.Methods①MCMV smith strain was duplicated by cell culture in vitro, and the virulence was measured with Reed-Muench method.②The model of MCMV infection on neonate mice was built as follows:the BALB/C mice with negative MCMV IgG and IgM were screened and mated. The fetal mice were randomly divided into experimental group(n=90) and control group(n=90) on the day of delivery. The fetal mice in experimental group were inoculated via intracalvarium with 10μl of MCMV suspension, the mice in control were inoculated with identity volume of cell maintenance medium. On the 3rd,15th,30th days post-inoculation, the brain tissues of 30 mice in each group were obtained asepticly. The mice with MCMV infection were detected by PCR amplification. The brain tissues of MCMV DNA positive in experimental group and MCMV DNA negative in control group were reserved for further research.③The numbers of hippocampus synapse in fetal mice were detected by immunofluorescence.④The transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor were detected with RT-PCR, immunohistochemistry and western blotting.Results①The TCID50of MCMV Smith strain was 10-4.5 per 0.1ml.②MCMV DNA in brain tissue was positive in experimental group, while that was negative in control group.③The fluorescence granules of SYP increased as days going on in both groups. But the fluorescence granules of SYP in experimental group were less than that in control, the decrease was more obvious on days 15th and 30th.④The transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor were increasing as days going on. But the transcription and protein expression of SYP PSD-95 and GluR2 subunit of AMPA receptor in experimental group were lower than that in control group, the decrease was more obvious on days 15th and 30th.Conclusions①MCMV infection could result in the decrease of hippocampus synapse in fetal mice, this effect increased as the days going on.②MCMV infection could induce the decrease of the transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor in developing fetal mice. Objective To investigate the effect of secretory products of MCMV infected astrocytes on synapse quantity and expression of synapse-related protein.Methods①Establish MCMV infected astrocyte model:primarily culture and subculture neonatal rat hippocampal astrocytes; identification and purity of astrocytes were observed by using Immunohistochemistry to detect specific marker GFAP expression; Infect astrocytes with 100TCID50 MCMV, and then use the following methods to observe infection susceptibility and status of astrocyte to MCMV, growth situation of MCMV was also detected:PCR to detect MCMV DNA, CCK-8 to observe the survival rate of MCMV infected astrocytes, observing the cytotoxic effect of culture media on NIH 3T3 cell, drawing a growth curve of MCMV ect.②Establish the co-culture system of ACM(astrocyte conditioned medium) and neural cells in vitro:primarily culture neonatal rat hippocampal neurons; NSE(neuron specific enolase) expression and the purity of neurons were observed by Immunohistochemistry; collect ACM in the six-well plate after MCMV infection within 6h,12h,24h,48h,72h and that of control groups at the same time points, after filtration and centrifugation MCMV in the ACM was inactivated by ultraviolet light irradiating 15min; CCK-8 method was used to dentify the optimal concentration of ACM used in follow-up studies; the study is grouped according to various components of ACM:experiment group (including optimal concentration at different time points of infected ACM), ACM controls (including optimal concentration at different time points of noninfected ACM) and control group (without ACM).③Immunofluorescence was applied to detection the number of synapses.④Real-time qPCR and immunocytochemistry were used to detect the gene transcription and protein expression levels of synaptic associated protein SYP, PSD-95 and AMPA receptor GluR2 subunit.Results①Typical cytopathy occured after MCMV infection in cultured astrocytes (>95%purity); expression of MCMV IE DNA can be detected in infected cells at the 48h time point; ACM of 3d-4d after infection induced typical CMV virus plaque in NIH 3T3 cells; astrocytes began to die 24h after infecton, most died 72h after infection, at 96h time point there were no survivers; MCMV replication began to increase 12h after infection and peaked at 72h.②Purity of cultured neurons can be more than 98%, the neuron survival rate reach summit when medium containing 50%ACM.③Compared with the control group, ACM control and experiment groups, the number of 12h-72h SYP fluorescent particles significantly increased (P<0.01); with ACM control group, experimental group 12h-72h number of SYP fluorescent particles significantly reduced (P<0.01). Comparison group within-sample found, ACM control group 12h,24h,48h SYP fluorescent particles was significantly higher than the prior temporary phase (P<0.01),72h did not change significantly (P>0.05); experiment group only 12h the number of SYP fluorescent particles significantly increased than the prior temporary phase (P<0.01),24h,48h,72h did not change significantly (P>0.05).④ompared with the control group, ACM control and experiment groups 12-72h of three proteins’ coding gene transcription and protein expression levels were significantly higher (P<0.01). Compared with ACM control group, the experiment group 12-72h of three proteins’ coding gene transcription and protein expression levels were significantly lower (P<0.01). Comparison group within-sample found, ACM control group 12h,24h,48h three proteins’ coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.01), 72h did not change significantly (P>0.05); experiment group, only 12h coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.0.1),24h,48h,72h did not change significantly (P> 0.05).Compared with the control group, ACM control and experiment groups 12-72h of coding gene transcription and protein expression levels were significantly higher (P<0.01). Compared with ACM control group, the experiment group 12-72h of coding gene transcription and protein expression levels were significantly lower (P<O.01). Comparison group within-sample found, ACM control group 12h,24h,48h three proteins’ coding genes transcription and protein expression levels significantly higher than the prior temporary phase (P<0.01),72h did not change significantly (P>0.05); experiment group, only 12h coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.0.1),24h,48h,72h did not change significantly (P> 0.05).Conclusion①MCMV can replicate and induce cytopathic effects in astrocyte.②Astrocyte secretion can promote the formation of synapse, and MCMV may reduce the formation by making an effect on the synthesis and secretion function of astrocyte.③Astrocyte secretion can promote the coding gene transcription and protein expression of synaptic associated proteins, MCMV may influence the gene transcription and protein expression by disturb the secretory function of astrocyte. Objective To study the effect of MCMV infection on TSP-1 and TNF-a synthesized and excreted by astrocyte.Methods The astrocyte cells were cultured and then divided into experimental and control groups.100 TCID50MCMV and cell maintenance medium were added into the medium of experimental group, while in control, only cell maintenance medium was added. After 6,12, 24,48 and 72h, the cultured cells and astrocyte conditioned medium (ACM) were collected respectively. The ACM was exposed to ultraviolet rays for 15 minutes to inactivate CMV, after filtration and centrifugalization.①The mRNA and protein expression of TSP-1 and TNF-a were detected by RT-PCR, immunohistochemistry.②TSP-1 and TNF-a excreted by astrocyte were detected with ELISA method.Results①Comparing to control group, the mRNA and protein expression of TSP-1 and TNF-a were lower in experimental group at each time point. The diversity increased as the time going on. Furtherly, the mRNA and protein expression of TSP-1 and TNF-a decreased during 12h-72h post-culturing.②By ELISA, TSP-1 and TNF-a in ACM can be detected from 12h post-culturing. TSP-1 and TNF-a in control showed a sharp increasing during 0-48h, and then increased mildly during 48-72h. While in experimental group, they were invariable during 12-72h. At each time point, TSP-1 and TNF-a in experimental group were lower than that in control.Conclusions①TSP-1 and TNF-a synthesized and excreted by astrocyte decreased after CMV infection. As the time going on, the decrease was more distinguished.②TSP-1 and TNF-a excreted by astrocyte increased in normal, while it was inhibited by CMV infection. The excretion of TSP-1 and TNF-a maintained in low level with CMV infection.更多还原