體外共培養(yǎng)潛伏感染的老年小鼠大腦皮質(zhì)及肺組織內(nèi)HCMV研究
發(fā)布時(shí)間：2019-04-19 22:51
【摘要】：目的 取HCMV先天性潛伏感染小鼠腦、肺組織并制成細(xì)胞懸液,體外與HF細(xì)胞進(jìn)行共培養(yǎng),觀察細(xì)胞中潛伏的病毒再激活現(xiàn)象,為研究HCMV潛伏再激活及闡明機(jī)制提供證據(jù)和新線索。 方法 依據(jù)隨機(jī)抽樣原則,取HCMV先天性潛伏感染組小鼠及DMEM對(duì)照組小鼠各6只,分別將其大腦皮質(zhì)和肺組織制成細(xì)胞懸液。染色計(jì)數(shù)活細(xì)胞,細(xì)胞終濃度為1×10~6/ml,接種至放有蓋玻片的細(xì)胞培養(yǎng)板中。隨即加入1×10~6/ml的人胚成纖維細(xì)胞(HF)懸液,建立共培養(yǎng)體系。同時(shí)設(shè)立HCMV陽性對(duì)照和HF陰性對(duì)照。逐同觀察細(xì)胞生長(zhǎng)情況并記錄。分別在共培養(yǎng)后的第1、3、7、14、28、35d收獲細(xì)胞,進(jìn)行如下檢測(cè):(1)觀察共培養(yǎng)物上HCMV特征性CPE;(2)PCR、RT-PCR分別檢測(cè)HCMV IE、UL83 DNA和相應(yīng)的mRNA;(3)取共培養(yǎng)細(xì)胞爬片做間接免疫熒光試驗(yàn),分別檢測(cè)HCMV IE和pp65抗原的表達(dá);(4)透射電鏡觀察皰疹病毒樣顆粒和感染的HF細(xì)胞超微結(jié)構(gòu)變化。 結(jié)果 HCMV先天性潛伏感染的老年小鼠大腦皮質(zhì)和肺組織細(xì)胞體外分別與HF共培養(yǎng)組:(1)HCMV潛伏感染的老年小鼠大腦皮質(zhì)和肺組織細(xì)胞經(jīng)體外與HF共培養(yǎng),于第7d開始出現(xiàn)HCMV特征性CPE,病變呈灶性并逐漸擴(kuò)展至整個(gè)單層,病變特征同病毒陽性對(duì)照。潛伏病毒再激活率為100%。(2)PCR擴(kuò)增出HCMVIE、UL83 DNA且序列測(cè)定與Genbank上登錄的基因序列比較,一致率分別為99%和100%。RT-PCR檢測(cè)見HCMVIE、UL83特異性條帶,HCMV mRNA在第3d開始表達(dá)。HCMV基因的復(fù)制具有明顯的時(shí)序性,HCMV IE基因的表達(dá)及轉(zhuǎn)錄均早于UL83,隨著時(shí)間的延長(zhǎng),表達(dá)水平逐漸升高。(3)間接免疫熒光試驗(yàn)于共培養(yǎng)后第7d檢出IE、pp65蛋白。(4)透射電鏡下觀察到HF細(xì)胞超微結(jié)構(gòu)明顯病變,發(fā)現(xiàn)典型的皰疹病毒樣顆粒。DMEM對(duì)照組及HF陰性對(duì)照組均末檢測(cè)到所設(shè)置
[Abstract]:Objective to obtain the brain and lung tissue of HCMV congenital latent infection mice and make cell suspension, and to co-culture with HF cells in vitro to observe the reactivation of latent virus in the cells. It provides evidence and new clues for studying the mechanism of latent reactivation and elucidation of HCMV. Methods according to the principle of random sampling, 6 mice in HCMV congenital latent infection group and 6 mice in DMEM control group were used to make cell suspension from cerebral cortex and lung tissue respectively. The cells were stained and counted, and the final cell concentration was 1 脳 10? 6? ml. The cells were inoculated into the cell culture plate with cover glass. The co-culture system was established by adding 1 脳 10 ~ 6/ml human embryonic fibroblast (HF) suspension. At the same time, HCMV positive control and HF negative control were established. Cell growth was observed and recorded. The cells were harvested at 1,3,7,14,28,35 days after co-culture, respectively. The results were as follows: (1) the characteristic CPE; (2) PCR,RT-PCR of HCMV on the co-culture medium was observed to detect HCMV IE,UL83 DNA and corresponding mRNA;, respectively. (3) the expression of HCMV IE and pp65 antigen were detected by indirect immunofluorescence assay, and (4) the ultrastructural changes of herpes virus-like granules and infected HF cells were observed by transmission electron microscopy. Results the cells of cerebral cortex and lung of aged mice with congenital latent infection of HCMV were co-cultured with HF in vitro. (1) the cells of cerebral cortex and lung of aged mice infected with HCMV latent infection were co-cultured with HF in vitro. On the 7th day, the characteristic CPE, lesions of HCMV appeared focal and gradually extended to the whole monolayer, and the pathological features of the lesions were compared with those of the virus positive controls. The reactivation rate of latent virus was 100%. (2) HCMVIE,UL83 DNA was amplified by PCR and compared with the gene sequence logged on Genbank, the coincidence rate was 99% and the specific band of HCMVIE,UL83 was detected by 100%.RT-PCR, respectively. HCMV mRNA began to express on the 3rd day. The replication of HCMV gene had obvious time sequence, HCMV IE gene expression and transcription earlier than that of UL83, with the prolongation of time. (3) IE,pp65 protein was detected by indirect immunofluorescence assay on the 7th day after co-culture. (4) obvious ultrastructural changes of HF cells were observed under transmission electron microscope (TEM). Typical herpesvirus-like granules were found in both the DMEM control group and the HF-negative control group.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
本文編號(hào)：2461362
[Abstract]:Objective to obtain the brain and lung tissue of HCMV congenital latent infection mice and make cell suspension, and to co-culture with HF cells in vitro to observe the reactivation of latent virus in the cells. It provides evidence and new clues for studying the mechanism of latent reactivation and elucidation of HCMV. Methods according to the principle of random sampling, 6 mice in HCMV congenital latent infection group and 6 mice in DMEM control group were used to make cell suspension from cerebral cortex and lung tissue respectively. The cells were stained and counted, and the final cell concentration was 1 脳 10? 6? ml. The cells were inoculated into the cell culture plate with cover glass. The co-culture system was established by adding 1 脳 10 ~ 6/ml human embryonic fibroblast (HF) suspension. At the same time, HCMV positive control and HF negative control were established. Cell growth was observed and recorded. The cells were harvested at 1,3,7,14,28,35 days after co-culture, respectively. The results were as follows: (1) the characteristic CPE; (2) PCR,RT-PCR of HCMV on the co-culture medium was observed to detect HCMV IE,UL83 DNA and corresponding mRNA;, respectively. (3) the expression of HCMV IE and pp65 antigen were detected by indirect immunofluorescence assay, and (4) the ultrastructural changes of herpes virus-like granules and infected HF cells were observed by transmission electron microscopy. Results the cells of cerebral cortex and lung of aged mice with congenital latent infection of HCMV were co-cultured with HF in vitro. (1) the cells of cerebral cortex and lung of aged mice infected with HCMV latent infection were co-cultured with HF in vitro. On the 7th day, the characteristic CPE, lesions of HCMV appeared focal and gradually extended to the whole monolayer, and the pathological features of the lesions were compared with those of the virus positive controls. The reactivation rate of latent virus was 100%. (2) HCMVIE,UL83 DNA was amplified by PCR and compared with the gene sequence logged on Genbank, the coincidence rate was 99% and the specific band of HCMVIE,UL83 was detected by 100%.RT-PCR, respectively. HCMV mRNA began to express on the 3rd day. The replication of HCMV gene had obvious time sequence, HCMV IE gene expression and transcription earlier than that of UL83, with the prolongation of time. (3) IE,pp65 protein was detected by indirect immunofluorescence assay on the 7th day after co-culture. (4) obvious ultrastructural changes of HF cells were observed under transmission electron microscope (TEM). Typical herpesvirus-like granules were found in both the DMEM control group and the HF-negative control group.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
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