本文選題：人巨細胞病毒 + 增殖性感染�。� 參考：《安徽醫(yī)科大學》2007年碩士論文

【摘要】： 目的研究人巨細胞病毒(human cytomegalovirus，HCMV)體外增殖性感染胎鼠大腦皮質(zhì)細胞，初步探討HCMV跨種屬感染的分子機制。為臨床研究HCMV中樞神經(jīng)系統(tǒng)感染提供實驗依據(jù)。 方法將本實驗室純化后的HCMV AD169株通過空斑形成試驗(plaque forming unit，PFU)測定病毒感染性。以4×10~5 PFU／ml病毒感染量接種至Balb／c胎鼠的大腦皮質(zhì)細胞和人胚肺成纖維細胞(human embryo lung cell，HEL)，在感染后的第1、3、5和7天分別收集細胞培養(yǎng)物及其上清，用不同的方法檢測如下指標：(1)HCMV接種后，通過倒置顯微鏡逐日觀察HCMV致胎鼠大腦皮質(zhì)細胞病變效應(yīng)(cytopathic effect，CPE)，并觀察其病變特征；(2)通過PCR和RT-PCR方法檢測胎鼠大腦皮質(zhì)細胞中與HCMV復制增殖的重要基因IE、UL83和UL54 DNA和mRNA；同時，以β-actin為內(nèi)參，研究其mRNA含量的變化特征及趨勢；(3)通過空斑形成試驗檢測HCMV感染胎鼠大腦皮質(zhì)細胞病變效應(yīng)達到+++～++++時感染性病毒數(shù)量；(4)通過透射電鏡觀察HCMV感染后胎鼠大腦皮質(zhì)細胞胞內(nèi)的病毒顆粒及其超微結(jié)構(gòu)的變化。 結(jié)果(1)將HCMV AD169毒株以4×10~5 PFU／ml接種至胎鼠大腦皮質(zhì)細胞2天后，出現(xiàn)病毒致細胞病變效應(yīng)(CPE)，可見少數(shù)神經(jīng)元和膠質(zhì)細胞細胞腫脹、變圓，胞內(nèi)顆粒增多，折光性增強等病變。在第3、5和7天時，細胞病變數(shù)量逐漸增加，病變程度逐步加重，表現(xiàn)為軸突斷裂、細胞溶解甚至脫落等情況。而接種病毒的HEL則在第1天就出現(xiàn)CPE，且隨著時間的延長，細胞病變數(shù)目增多，病變加重，，表現(xiàn)為細胞腫脹、變圓、折光性增強、脫落等情況。相對來說，HCMV對HEL更為敏感、CPE出現(xiàn)早、細胞病變程度重；(2)HCMV接種至胎鼠大腦皮質(zhì)細胞的第1、3、5和7天，PCR檢測到細胞培養(yǎng)物HCMV IE、UL83和UL54 DNA均呈陽性；同時，以β-actin為內(nèi)參，RT—PCR檢測各時間點mRNA，結(jié)果表明：各時間點均可檢測到HCMV IE、UL83和UL54 mRNA，產(chǎn)物大小分別為872bp、842bp和784bp，經(jīng)Bioscience image anlysis system進行灰度值分析顯示，隨著HCMV感染胎鼠大腦皮質(zhì)細胞時間的延長，HCMV IE、UL83和UL54 mRNA表達量均明顯增加，證實HCMV在胎鼠大腦皮質(zhì)細胞內(nèi)為增殖性感染；(3)采用空斑形成實驗結(jié)果表明：約在第7天，接種病毒懸液的HEL細胞層出現(xiàn)圓形空斑，隨著感染天數(shù)的增加，空斑變大，數(shù)目增多。約14天空斑基本不在發(fā)生變化，根據(jù)公式計算得到病毒感染量為3.8×10~5PFU／ml。(4)透射電鏡可見感染后的胎鼠大腦皮質(zhì)細胞構(gòu)筑破壞嚴重，細胞器均有不同程度損傷，并在胞核內(nèi)出現(xiàn)病毒核酸、胞質(zhì)內(nèi)出現(xiàn)皰疹樣病毒顆粒。 結(jié)論HCMV AD169株具有感染體外培養(yǎng)的胎鼠大腦皮質(zhì)細胞的能力，證實了HCMV可以跨種屬感染，為臨床研究HCMV中樞神經(jīng)系統(tǒng)感染提供了實踐依據(jù)。
[Abstract]:Objective to study the proliferative infection of human cytomegalovirus (HCMV) in fetal rat cerebral cortex cells in vitro and to explore the molecular mechanism of HCMV transspecies infection. To provide experimental basis for clinical study of HCMV central nervous system infection. Methods the purified HCMV AD169 strain was detected for viral infection by plaque forming unitu test. 4 脳 10 ~ (5) PFU/ml virus was inoculated into the cerebral cortex cells and human embryonic lung fibroblasts of Balb/c fetal mice. The cell cultures and their supernatants were collected on the 1st day after infection and the supernatants were collected on the 7th day after inoculation, and the following indexes were detected by different methods. To observe the cytopathic effect of HCMV on fetal rat cerebral cortex cytopathic cytopathic cytopathic effect and its pathological characteristics by inverted microscope. To detect the replication and proliferation of UL54 DNA, UL54 DNA and mRNAs in fetal rat cerebral cortex cells by PCR and RT-PCR. Taking 尾 -actin as internal parameter, Study on the change characteristics and trend of mRNA content: (3) using plaque formation test to detect the number of infective viruses when the effect of HCMV infection on fetal cortical cytopathic effect reaches ~ ~ ~) observing the infection of HCMV by transmission electron microscope (TEM). Changes of viral particles and ultrastructure in fetal rat cerebral cortex cells. Results 1) 2 days after inoculating HCMV AD169 strain with 4 脳 10 ~ 5 PFU/ml to fetal rat cerebral cortex cells, the cytopathic effect of virus was found. A few neurons and glial cells were swollen, round, intracellular granules increased and refractive index increased. On the 5th and 7th day, the number of cytopathic lesions increased gradually, and the degree of cytopathic lesions became more serious, such as axonal breakage, cell dissolution and even exfoliation. However, the HEL inoculated with the virus appeared on the first day, and with the prolongation of time, the number of cytopathic lesions increased and the pathological changes became more serious, which showed cell swelling, roundness, enhanced refraction and shedding. Relatively speaking, HEL was more sensitive to HEL and the cytopathic degree was higher. Both HCMV IEMUL83 and UL54 DNA were detected by PCR on the 5th and 7th day after inoculation of HCMV with fetal rat cerebral cortex cells. HCMV IEUL83 and UL54 mRNAs were detected at each time point by RT-PCR with 尾 -actin as the internal reference. The product sizes were 872 BP and 784 BP, respectively. The results of Bioscience image anlysis system showed that the mRNAs were 872 BP and 784 BP, respectively. With the prolongation of the time of HCMV infection in fetal rat cerebral cortex cells, the expression of HCMV-IEMV-UL83 and UL54 mRNA increased significantly. It was confirmed that HCMV was proliferative infection in fetal rat cerebral cortex cells. The results of plaque formation test showed that: about the 7th day, the expression of HCMV-IEMV-UL83 and UL54 mRNA in fetal rat cerebral cortex cells was increased. Round plaque appeared in the HEL cell layer inoculated with virus suspension. With the increase of infection days, the plaque became larger and the number increased. According to the formula, the virus infection amount was 3.8 脳 10 ~ 5 PFU / ml. 4) transmission electron microscope showed that the damage of cerebral cortex cell architecture was serious and the organelles were damaged in different degrees. Virus nucleic acid appeared in the nucleus and herpesvirus particles appeared in the cytoplasm. Conclusion HCMV AD169 strain has the ability of infecting fetal rat cerebral cortex cells cultured in vitro, which proves that HCMV can transspecies infection and provides a practical basis for clinical study of HCMV central nervous system infection.
【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】：2007
【分類號】：R373

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