人巨细胞病毒感染对U251胶质瘤干细胞增殖分化及凋亡的影响

发布时间：2018-03-28 10:54

本文选题：人巨细胞病毒　切入点：胶质瘤干细胞　出处：《青岛大学》2017年硕士论文

【摘要】：目的:1.探讨不同感染复数(MOI)的人巨细胞病毒(HCMV)在体外对U251胶质瘤干细胞(GSCS)增殖、分化及凋亡的影响。2.用MOI为1的HCMV AD169毒株感染U251GSCS,观察HCMV对U251GSCS在裸鼠大脑内成瘤及血管生成的影响。方法:1.用HCMV AD169毒株感染人胚肺成纤维细胞进行病毒扩增,收集上清并用空斑定量法检测病毒滴度。2.用单克隆培养法从U251胶质瘤细胞中分离出GSCS,用流式细胞仪检测U251GSCS中表达CD133的细胞数。3.用MOI分别为0.2、1、5的HCMV感染U251GSCS。分别在感染后12h、24h、36h、48h时间点,用MTT法检测U251GSCS增殖情况。4.在HCMV感染后48h,用RT-PCR检测U251GSCS中IE-1 m RNA、Bcl-2 m RNA及Bax m RNA的表达。用Western blot检测U251GSCS中IE-1、Bcl-2、Bax和CD133蛋白的表达。5.将MOI为1的HCMV感染U251GSCS,然后接种到10只裸鼠颅内,设为实验组;对照组接种未感染HCMV的U251GSCS。观察裸鼠生长情况及外观变化。6.处死濒死裸鼠,取出脑组织并分理出肿瘤组织做HE染色。用免疫组化法检测肿瘤组织中IE-1、CD34蛋白的表达。7.采用weidner血管计数法计算出实验组和对照组肿瘤微血管密度(MVD)。结果:1.U251胶质瘤细胞在无血清培养基中悬浮生长。流式细胞仪结果显示,U251GSCS中CD133阳性细胞比例达98.00%。2.MTT结果显示,用MOI为1和5的HCMV感染U251GSCS 12h、24h、36h、48h后,与对照组相比,细胞增殖活性显著增加(P0.05)。而用MOI为0.2的HCMV感染U251GSCS12h、24h、36h、48h后,与对照组比较,细胞增殖活性差异不明显(P0.05)。3.RT-PCR结果显示,HCMV(MOI=1,5)感染U251GSCS 48h后,细胞IE-1 m RNA、Bcl-2 m RNA的表达上调,Bax m RNA表达下调,与对照组比较,差异显著(P0.05)。而用MOI为0.2的HCMV感染U251GSCS 48h后,细胞IE-1 m RNA、Bcl-2m RNA及Bax m RNA表达与对照组相比无明显差异(P0.05)。4.Western blot检测结果显示,与对照组相比,HCMV(MOI=1,5)感染U251GSCS48h后,细胞IE-1、Bcl-2及CD133蛋白表达增多,而Bax蛋白表达下降(P0.05)。5.接种感染HCMV(MOI=1)的U251GSCS3周后,大部分裸鼠出现活动减少、皮肤皱缩和一些神经症状。6.移植瘤HE染色显示,肿瘤细胞核异形明显。免疫组化结果显示,与对照组相比,HCMV(MOI=1)感染的U251GSCS移植瘤IE-1表达阳性。7.weidner血管计数结果显示,HCMV(MOI=1)感染的U251GSCS移植瘤微血管密度显著高于对照组(P0.05)。结论:1.一定滴度的HCMV感染U251GSCS可促进其增殖,使其维持干性特征。2.HCMV感染U251GSCS后,细胞表达IE-1并通过下调Bax蛋白的表达和上调Bcl-2蛋白的表达来抑制U251GSCS凋亡。3.HCMV感染可通过促进CD133阳性U251GSCS分化为CD34血管内皮细胞来促进U251GSCS移植瘤的生长,从而增加其恶性程度。
[Abstract]:Objective: 1. To investigate the proliferation of human cytomegalovirus (HCMV), a human cytomegalovirus virus (HCMV) infected with multiple moi, on U251 glioma stem cells (GSCS) in vitro. Effect of differentiation and apoptosis. U251GSCS was infected with HCMV AD169 strain with MOI 1 to observe the effect of HCMV on U251GSCS tumorigenesis and angiogenesis in the brain of nude mice. Methods: 1. Human embryonic lung fibroblasts were infected with HCMV AD169 strain for virus amplification. GSCSs were isolated from U251 glioma cells by monoclonal culture method, and the number of cells expressing CD133 in U251GSCS was detected by flow cytometry. The U251G SCSs were infected with HCMV with MOI of 0.2GSCS0.The cells were infected with U251GSCSs at 12h, 24h, 36hs, 48h after infection, respectively, and the results showed that GSCSs were isolated from U251 glioma cells by Monoclonal culture, and the cells expressing CD133 in U251 glioma cells were detected by flow cytometry. The proliferation of U251GSCS was detected by MTT. At 48h after HCMV infection, the expression of IE-1 m RNA-Bcl-2 m RNA and Bax m RNA in U251GSCS was detected by RT-PCR, and the expression of IE-1bcl-2Bcl-2Bax and CD133 protein in U251GSCS was detected by Western blot. The MOI was infected with U251GSCSand then inoculated into 10 nude mice. The control group was inoculated with U251 GSCS. the growth and appearance changes of nude mice were observed. Take out brain tissue and divide it into tumor tissue for HE staining. Immunohistochemical method was used to detect the expression of IE-1pCD34 protein in tumor tissue .7.The microvessel density of tumor in experimental group and control group was calculated by weidner vessel count method. Results: 1. U251 glioma. The percentage of CD133 positive cells in U251 GSCS was higher than that in 98.00%.2.MTT. Compared with the control group, the proliferation activity of the cells infected with 1 and 5 HCMV (1 and 5 HCMV) increased significantly compared with the control group for 48 h, and the proliferation activity of the cells infected with U251 GSCS 12 h and 24 h HCMV with MOI 0. 2 was not significantly different from that of the control group (P 0 05. 3. RT-PCR results showed that the cell proliferation activity was not significantly different from that of the control group (P 0. 05. 3. RT-PCR results showed that the cells were infected with U251GSCS 48 h after infection with MOI 0. 2 GSCS 12 h or 24 h ~ 12 h) and the results of RT-PCR showed that there was no significant difference in cell proliferation activity between the control group and the control group. The expression of Bcl-2 m RNA in IE-1 m RNAs up-regulated the expression of Bax m RNA, and the difference was significant compared with the control group. However, the expression of IE-1 m RNAs Bcl-2m RNA and Bax m RNA was not significantly different from that of the control group after the U251GSCS was infected with MOI 0.2 for 48 h. The results of Western blot analysis showed that the expression of Bcl-2m RNA and Bax m RNA in the cells were not significantly different from those in the control group. Compared with the control group, the expression of IE-1Bcl 2 and CD133 protein increased after U251GSCS48h infection, while the expression of Bax protein decreased (P0.05. 5). After inoculation with HCMV / MOI1), the activity of most nude mice was decreased, skin shrinkage and some neurologic symptoms. The nuclei of the tumor were abnormal. The immunohistochemical results showed that, Compared with the control group, the microvessel density of U251GSCS transplanted tumor infected with HCMV / moi 1) was significantly higher than that of the control group (P 0.05). Conclusion: 1. A certain titer of HCMV infection with U251GSCS can promote the proliferation of U251GSCS transplant tumor. 7. Weidner blood vessel count showed that the microvessel density of U251GSCS transplanted tumor infected with HCMV was significantly higher than that of the control group (P 0.05). Conclusion: 1. A certain titer of HCMV infection with U251GSCS can promote the proliferation of U251GSCS transplant tumor. After HCMV was infected with U251GSCS, The cells expressed IE-1 and down-regulated the expression of Bax protein and up-regulated the expression of Bcl-2 protein to inhibit U251GSCS apoptosis. 3. HCMV infection could promote the growth of U251GSCS transplanted tumor by promoting the differentiation of CD133 positive U251GSCS into CD34 vascular endothelial cells, thus increasing the malignant degree of U251GSCS transplanted tumor.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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