HCMV先天性感染老年小鼠大脑皮质细胞凋亡与AD样病理改变相关性研究

发布时间：2018-05-19 18:12

本文选题：人巨细胞病毒 + 先天性潜伏感染　；参考：《安徽医科大学》2006年硕士论文

【摘要】： 目的在已建立的人巨细胞病毒(human cytomegalovirus ,HCMV)先天性潜伏感染老年小鼠模型基础上,观察了HCMV先天性潜伏感染以及潜伏后再激活感染对大脑皮质细胞凋亡的影响和小鼠大脑皮质AD样病理改变;探求HCMV先天性潜伏感染以及潜伏后再激活感染与大脑皮质凋亡、AD之间的内在联系,以期为AD病因学研究提供新的思路和合适的动物模型。方法将已知亲代感染HCMV所生子代小鼠经测试确定有感染后,于屏障级鼠笼内继续饲养。试验分为三组: HCMV先天性潜伏感染组、HCMV先天性潜伏感染再激活组(腹腔注射免疫抑制剂环磷酰胺以激活体内潜伏感染的病毒,环磷酰胺剂量为150mg/kg,每6天1次,共3次)、正常对照组。在各组小鼠达18~20月龄时,分别在各组随机选取小鼠5只。按照实验设计处死小鼠,无菌取小鼠大脑皮质并进行下列各项检测。(1)采用细胞共培养方法进行病毒分离、PCR和RT-PCR法检测病毒基因和基因转录的有无确定模型小鼠的感染状态。(2)采用透射电镜检测模型小鼠大脑皮质凋亡形态学改变;取大脑皮质石蜡切片用TUNEL法检测小鼠大脑皮质细胞凋亡;取新鲜大脑皮质组织用机械碾磨加物理吹撒法制成单细胞悬液,流式细胞仪通过检测细胞周期DNA含量来检测小鼠大脑皮质凋亡改变。(3)检测各组老年小鼠AD样病理改变:HE染色检测小鼠大脑皮质组织构筑、病理改变和生物学特性;刚果红染色检测大脑皮质组织中的淀粉样斑块;镀银染色检查大脑皮质组织中的神经原纤维缠结和淀粉样斑块。结果(1)模型小鼠感染状态检测:HCMV先天性潜伏感染组小鼠大脑皮质可以用PCR检测出HCMV IE和UL83的DNA,但RT-PCR检测不出相应的mRNA;HCMV先天性潜伏感染再激活组用PCR、RT-PCR均可检测出相应产物;而正常对照组PCR、RT-PCR结果均为阴性。细胞共培养病毒分离实验发现HCMV先天性潜伏感染组和病毒潜伏感染再激活组老年小鼠大脑皮质神经元病毒在HF细胞内增殖产生特征性细胞病变,而对照组阴性。以上结果说明模型小鼠感染状态与试验设计相符合。 (2)凋亡检测结果:透射电镜检测发现HCMV先天性潜伏感染组和HCMV先天性潜伏感染再激活组部分神经元细胞核染色体发生固缩,核型不规则,核膜表面凹凸不平甚至消失,核物质碎片化。正常对照组未见这些特征性凋亡改变。透射电镜检测同时还发现HCMV先天性潜伏感染组和HCMV先天性潜伏感染再激活组神经元胞浆中的细胞器数量减少,线粒体的嵴消失。 TUNEL法采用高倍光镜下计10个视野平均阳性细胞数,正常对照组TUNEL阳性细胞数为2.0±0.7(mean±SD),HCMV先天性潜伏感染组TUNEL阳性细胞数为5.2±1.3,HCMV先天性潜伏感染再激活组TUNEL阳性细胞数为18.1±3.5。检测结果经T检验发现HCMV先天性潜伏感染组、HCMV先天性潜伏感染再激活组与正常对照组各组间均有明显差异(P0.05)。数流式细胞仪PI染色检测发现正常对照组大脑皮质凋亡率为1.75±0.3%,HCMV先天性潜伏感染组大脑皮质凋亡率为4.83±0.96%,HCMV先天性潜伏感染再激活组大脑皮质凋亡率为10.09±1.18%。检测结果经T检验发现HCMV先天性潜伏感染组、HCMV先天性潜伏感染再激活组与正常对照组各组均有明显差异(P0.05)。说明病毒潜伏感染能促进大脑皮质凋亡,病毒再激活后更进一步促进大脑皮质凋亡,造成大脑皮质神经元功能丧失。 (3)病理检测结果:HE染色发现HCMV先天性潜伏感染组大脑皮质神经元细胞核着色较正常对照组浅,神经细胞数大为减少,残有的细胞出现空泡化,突起减少,细胞层数减少。HCMV先天性潜伏感染再激活组小鼠大脑皮质神经元细胞核着色非常浅,细胞核固缩甚至消失。刚果红染色发现HCMV先天性潜伏感染组和HCMV先天性潜伏感染再激活组小鼠大脑皮质中细胞外有淀粉样斑块,正常对照组未见淀粉样斑块。镀银染色试验发现HCMV先天性潜伏感染组和HCMV先天性潜伏感染再激活组的大脑皮质中找到淀粉样斑块,而对照组未发现淀粉样斑块;正常对照组小鼠大脑皮质中仅发现个别的神经原纤维缠结,HCMV先天性潜伏感染组和HCMV先天性潜伏感染再激活组小鼠大脑皮质发现有神经原纤维缠结。结论 (1)在成功建立的HCMV先天性老年小鼠中枢神经系统潜伏感染模型基础上,发现HCMV先天性感染可引起AD样病理改变,提示HCMV感染是AD病的重要病原之一。从而为AD病因学研究及其发生机制提供新的思路和新的技术平台。 (2)HCMV先天性感染促进老年小鼠大脑皮质细胞凋亡,使中枢神经系统细胞功能逐渐丧失,从而继发一系列AD样病理改变。这一发现提示先天性HCMV感染诱发AD病的重要途径之一就是促进神经细胞凋亡,为HCMV感染诱发AD病的机制研究提供了理论和实验依据。
[Abstract]:Objective on the basis of the established human cytomegalovirus (HCMV) congenital latent infection model of senile mice, the effects of HCMV congenital latent infection and latent infection on cerebral cortex cell apoptosis and the change of AD like disease in cerebral cortex of mice were observed, and the congenital latent infection and latent infection of HCMV were explored. After reactivation, the intrinsic relationship between infection and cerebral cortex apoptosis, AD, in order to provide new ideas and suitable animal models for AD etiology research.
Methods the offspring of the offspring of HCMV were tested and kept in the barrier rat cage. The test was divided into three groups: HCMV congenital latent infection group, HCMV congenital latent infection reactivation group (intraperitoneal injection of immunosuppressive cyclophosphamide to activate the latent infection virus, and the dose of cyclophosphamide 150m G/kg, every 6 days 1 times, a total of 3 times), in the normal control group, 5 mice were selected randomly in each group at the age of 18~20 months. The mice were sacrificed according to the experimental design, the cerebral cortex of the mice was aseptic and the following tests were carried out. (1) the cell co culture method was used to isolate the virus and the PCR and RT-PCR methods were used to detect the gene and gene transcription of the virus. (2) the morphological changes in the cerebral cortex of the model mice were detected by transmission electron microscopy, and the paraffin section of the cerebral cortex was used to detect the apoptosis of the cerebral cortex cells of the mice by TUNEL method, and the fresh cerebral cortex tissue was made by mechanical grinding and physical blowing into single cell suspension, and the flow cytometry was detected. Cell cycle DNA content was used to detect the changes in the apoptosis of cerebral cortex in mice. (3) AD like pathological changes in the aged mice were detected: HE staining was used to detect the tissue construction, pathological changes and biological characteristics in the cerebral cortex of mice; the amyloid plaque in the cerebral cortex was detected by Congo red staining; and the silver plating was used to examine the nerve fibrils in the cerebral cortex. Vascular tangles and amyloid plaques.
Results (1) detection of infection state in model mice: the cerebral cortex of HCMV congenital latent infection group can detect DNA of HCMV IE and UL83 in PCR, but RT-PCR can not detect the corresponding mRNA; HCMV congenital latent infection reactivation group can detect the corresponding product with PCR and RT-PCR, while the normal control group is negative. Cells are all negative. The culture virus isolation experiment found that the HCMV congenital latent infection group and the virus latent infection reactivation group proliferated in the cerebral cortex neuron of the aged mice to produce the characteristic cytopathic in the HF cell, but the control group was negative. The results showed that the infection state of the model mice was in accordance with the test set.
(2) the results of apoptosis detection: transmission electron microscopy showed that the nuclei of HCMV congenital latent infection group and HCMV congenital latent infection reactivation group had chromosomal contraction, irregular karyotype, uneven and even disappearance of nuclear membrane surface, and nuclear material fragmentation. No characteristic apoptosis changes were found in normal group. Transmission electron microscope detection At the same time, it was also found that the number of organelles in the cytoplasm of HCMV latent infection group and HCMV congenital latent infection reactivation group decreased, and the cristae of mitochondria disappeared.
In the TUNEL method, the average number of positive cells in 10 visual fields was measured by a high optical microscope, and the number of TUNEL positive cells in the normal control group was 2 + 0.7 (mean + SD). The number of TUNEL positive cells in the HCMV congenital latent infection group was 5.2 + 1.3. The number of TUNEL positive cells in the HCMV congenital latent infection reactivation group was 18.1 + 3.5. detection results, and the HCMV congenital latency was found by T test. Infection group, HCMV congenital latent infection reactivation group and normal control group were significantly different (P0.05).
The apoptosis rate of cerebral cortex in the normal control group was 1.75 + 0.3%, the apoptosis rate of cerebral cortex in the HCMV congenital latent infection group was 4.83 + 0.96%, the cerebral cortex apoptosis rate of the HCMV congenital latent infection group was 10.09 + 1.18%., and the T test found the HCMV congenital latent infection group, and the HCMV congenital latent infection was found in the HCMV congenital latent infection group. The infection reactivation group was significantly different from that of the normal control group (P0.05). It indicated that the latent infection of the virus could promote the apoptosis of the cerebral cortex. After the reactivation of the virus, the apoptosis of the cerebral cortex was further promoted, resulting in the loss of neuron function in the cerebral cortex.
(3) pathological examination results: HE staining showed that the nucleus coloration of cerebral cortical neurons in the HCMV congenital latent infection group was lighter than that of the normal control group, the number of nerve cells decreased, the residual cell appeared vacuolization, the protuberance decreased, and the number of cell layers reduced.HCMV congenital latent infection, and the nucleus coloring of the cerebral cortex neuron of the mice was very good. Congo red staining found that there were amyloid plaques in the cerebral cortex of the HCMV congenital latent infection group and the HCMV congenital latent infection group, and no amyloid plaques were found in the normal control group. The silver plating staining test found the HCMV congenital latent infection group and the HCMV congenital latent infection reactivation group. Amyloid plaques were found in the cerebral cortex, but no amyloid plaques were found in the control group; only a few neurofibrillary tangles were found in the cerebral cortex of the normal control group, and the cerebral cortex of the HCMV congenital latent infection group and the HCMV congenital latent infection reactivation group found the neurofibrillary tangles in the cerebral cortex.
conclusion
(1) on the basis of the latent infection model of the central nervous system in HCMV congenital senile mice, it is found that HCMV congenital infection can cause AD like pathological changes, suggesting that HCMV infection is one of the important pathogens of AD disease, thus providing new ideas and new technical platform for the etiology and pathogenesis of AD.
(2) HCMV congenital infection promotes the apoptosis of cerebral cortex cells in the aged mice, which leads to the gradual loss of the function of the central nervous system, and secondary to a series of AD like pathological changes. This discovery suggests that one of the important ways to induce AD disease induced by congenital HCMV infection is to promote the withering of the nerve cells and provide the mechanism for the AD disease induced by HCMV infection. Theoretical and experimental basis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373.9;R361

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