本文選題：人巨細胞病毒 + 先天性潛伏感染��； 參考：《安徽醫(yī)科大學》2006年碩士論文

【摘要】： 目的在已建立的人巨細胞病毒(human cytomegalovirus ,HCMV)先天性潛伏感染老年小鼠模型基礎(chǔ)上,觀察了HCMV先天性潛伏感染以及潛伏后再激活感染對大腦皮質(zhì)細胞凋亡的影響和小鼠大腦皮質(zhì)AD樣病理改變;探求HCMV先天性潛伏感染以及潛伏后再激活感染與大腦皮質(zhì)凋亡、AD之間的內(nèi)在聯(lián)系,以期為AD病因?qū)W研究提供新的思路和合適的動物模型。 方法將已知親代感染HCMV所生子代小鼠經(jīng)測試確定有感染后,于屏障級鼠籠內(nèi)繼續(xù)飼養(yǎng)。試驗分為三組: HCMV先天性潛伏感染組、HCMV先天性潛伏感染再激活組(腹腔注射免疫抑制劑環(huán)磷酰胺以激活體內(nèi)潛伏感染的病毒,環(huán)磷酰胺劑量為150mg/kg,每6天1次,共3次)、正常對照組。在各組小鼠達18~20月齡時,分別在各組隨機選取小鼠5只。按照實驗設(shè)計處死小鼠,無菌取小鼠大腦皮質(zhì)并進行下列各項檢測。(1)采用細胞共培養(yǎng)方法進行病毒分離、PCR和RT-PCR法檢測病毒基因和基因轉(zhuǎn)錄的有無確定模型小鼠的感染狀態(tài)。(2)采用透射電鏡檢測模型小鼠大腦皮質(zhì)凋亡形態(tài)學改變;取大腦皮質(zhì)石蠟切片用TUNEL法檢測小鼠大腦皮質(zhì)細胞凋亡;取新鮮大腦皮質(zhì)組織用機械碾磨加物理吹撒法制成單細胞懸液,流式細胞儀通過檢測細胞周期DNA含量來檢測小鼠大腦皮質(zhì)凋亡改變。(3)檢測各組老年小鼠AD樣病理改變:HE染色檢測小鼠大腦皮質(zhì)組織構(gòu)筑、病理改變和生物學特性;剛果紅染色檢測大腦皮質(zhì)組織中的淀粉樣斑塊;鍍銀染色檢查大腦皮質(zhì)組織中的神經(jīng)原纖維纏結(jié)和淀粉樣斑塊。 結(jié)果(1)模型小鼠感染狀態(tài)檢測:HCMV先天性潛伏感染組小鼠大腦皮質(zhì)可以用PCR檢測出HCMV IE和UL83的DNA,但RT-PCR檢測不出相應(yīng)的mRNA;HCMV先天性潛伏感染再激活組用PCR、RT-PCR均可檢測出相應(yīng)產(chǎn)物;而正常對照組PCR、RT-PCR結(jié)果均為陰性。細胞共培養(yǎng)病毒分離實驗發(fā)現(xiàn)HCMV先天性潛伏感染組和病毒潛伏感染再激活組老年小鼠大腦皮質(zhì)神經(jīng)元病毒在HF細胞內(nèi)增殖產(chǎn)生特征性細胞病變,而對照組陰性。以上結(jié)果說明模型小鼠感染狀態(tài)與試驗設(shè)計相符合。 (2)凋亡檢測結(jié)果:透射電鏡檢測發(fā)現(xiàn)HCMV先天性潛伏感染組和HCMV先天性潛伏感染再激活組部分神經(jīng)元細胞核染色體發(fā)生固縮,核型不規(guī)則,核膜表面凹凸不平甚至消失,核物質(zhì)碎片化。正常對照組未見這些特征性凋亡改變。透射電鏡檢測同時還發(fā)現(xiàn)HCMV先天性潛伏感染組和HCMV先天性潛伏感染再激活組神經(jīng)元胞漿中的細胞器數(shù)量減少,線粒體的嵴消失。 TUNEL法采用高倍光鏡下計10個視野平均陽性細胞數(shù),正常對照組TUNEL陽性細胞數(shù)為2.0±0.7(mean±SD),HCMV先天性潛伏感染組TUNEL陽性細胞數(shù)為5.2±1.3,HCMV先天性潛伏感染再激活組TUNEL陽性細胞數(shù)為18.1±3.5。檢測結(jié)果經(jīng)T檢驗發(fā)現(xiàn)HCMV先天性潛伏感染組、HCMV先天性潛伏感染再激活組與正常對照組各組間均有明顯差異(P0.05)。數(shù) 流式細胞儀PI染色檢測發(fā)現(xiàn)正常對照組大腦皮質(zhì)凋亡率為1.75±0.3%,HCMV先天性潛伏感染組大腦皮質(zhì)凋亡率為4.83±0.96%,HCMV先天性潛伏感染再激活組大腦皮質(zhì)凋亡率為10.09±1.18%。檢測結(jié)果經(jīng)T檢驗發(fā)現(xiàn)HCMV先天性潛伏感染組、HCMV先天性潛伏感染再激活組與正常對照組各組均有明顯差異(P0.05)。說明病毒潛伏感染能促進大腦皮質(zhì)凋亡,病毒再激活后更進一步促進大腦皮質(zhì)凋亡,造成大腦皮質(zhì)神經(jīng)元功能喪失。 (3)病理檢測結(jié)果:HE染色發(fā)現(xiàn)HCMV先天性潛伏感染組大腦皮質(zhì)神經(jīng)元細胞核著色較正常對照組淺,神經(jīng)細胞數(shù)大為減少,殘有的細胞出現(xiàn)空泡化,突起減少,細胞層數(shù)減少。HCMV先天性潛伏感染再激活組小鼠大腦皮質(zhì)神經(jīng)元細胞核著色非常淺,細胞核固縮甚至消失。剛果紅染色發(fā)現(xiàn)HCMV先天性潛伏感染組和HCMV先天性潛伏感染再激活組小鼠大腦皮質(zhì)中細胞外有淀粉樣斑塊,正常對照組未見淀粉樣斑塊。鍍銀染色試驗發(fā)現(xiàn)HCMV先天性潛伏感染組和HCMV先天性潛伏感染再激活組的大腦皮質(zhì)中找到淀粉樣斑塊,而對照組未發(fā)現(xiàn)淀粉樣斑塊;正常對照組小鼠大腦皮質(zhì)中僅發(fā)現(xiàn)個別的神經(jīng)原纖維纏結(jié),HCMV先天性潛伏感染組和HCMV先天性潛伏感染再激活組小鼠大腦皮質(zhì)發(fā)現(xiàn)有神經(jīng)原纖維纏結(jié)。 結(jié)論 (1)在成功建立的HCMV先天性老年小鼠中樞神經(jīng)系統(tǒng)潛伏感染模型基礎(chǔ)上,發(fā)現(xiàn)HCMV先天性感染可引起AD樣病理改變,提示HCMV感染是AD病的重要病原之一。從而為AD病因?qū)W研究及其發(fā)生機制提供新的思路和新的技術(shù)平臺。 (2)HCMV先天性感染促進老年小鼠大腦皮質(zhì)細胞凋亡,使中樞神經(jīng)系統(tǒng)細胞功能逐漸喪失,從而繼發(fā)一系列AD樣病理改變。這一發(fā)現(xiàn)提示先天性HCMV感染誘發(fā)AD病的重要途徑之一就是促進神經(jīng)細胞凋亡,為HCMV感染誘發(fā)AD病的機制研究提供了理論和實驗依據(jù)。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R373.9;R361

【參考文獻】

相關(guān)期刊論文 前1條

1 王明麗,唐久來,胡聞,史百芬,胡勇,畢克菊,李京培;人巨細胞病毒先天性中樞神經(jīng)系統(tǒng)感染小鼠模型的建立[J];動物學報;2000年02期

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本文編號：1911127