轮状病毒感染动物模型建立及其发病机制研究

发布时间：2018-03-16 20:10

本文选题：轮状病毒　切入点：感染　出处：《重庆医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的轮状病毒(Rotavirus, RV)是引起婴幼儿腹泻的最主要病原,同时也可引起肠外脏器损害,致病率及病死率均较高,但目前对其发病机制仍未完全清楚,也未能研制出很满意的疫苗。本实验通过猴RV SA-11感染生后7天昆明乳鼠,建立RV感染的乳鼠动物模型,并用该动物模型研究轮状病毒腹泻及肠外脏器损伤发病机制,为RV疫苗研制及更好地防治RV感染提供理论依据。方法以恒河猴胚肾细胞(MA-104)培养猴RV SA-11,扩增,空斑实验滴定病毒滴度,分装,-80℃保存备用。实验动物选用生后7天清洁级昆明乳鼠,实验组每只乳鼠经口接种0.1 ml病毒培养细胞冻融液(RV含量为3.75×10~7 PFU/ml),而对照组每只乳鼠经口接种0.1 ml正常无病毒对照培养细胞冻融液。感染后1～7天(Day Post Inoculation,DPI),每日观察乳鼠活动情况、毛色光泽、大便变化、体重变化,并收集大便经ELISA检测其中RV抗原,空斑实验滴定其中RV滴度,建立轮状病毒感染动物模型。在1、2、3、4、7、10、15 DPI各时间点取小肠组织,苏木精-伊红染色(hematoxylin-eosin,HE)染色光镜下观察形态学改变,并用图像分析软件(Image pro plus 5.1, IPP5.1)测定空肠绒毛高度和隐窝深度;取3 DPI空肠组织2.5%戊二醛固定后送电镜切片观察肠上皮细胞超形结构改变;取3 DPI空肠石蜡切片用免疫组化染色观察乳鼠空肠RV抗原分布;取1、2、3、4、10、15 DPI空肠石蜡切片用Phalloidine-TRITC染色观察小肠绒毛丝状肌动蛋白分布并用IPP5.1测量、SAS软件统计分析;取3 DPI空肠石蜡切片用Tunnel法检测肠上皮细胞原位凋亡,以研究RV肠炎腹泻发病机制。取3 DPI乳鼠脑、肺、心、胰、肝、肾组织,石蜡切片HE染色光镜和电镜观察病理改变;用免疫荧光法检测RV抗原在各脏器分布;以Phalloidine-TRITC染色观察1、2、3、4 DPI乳鼠心脏丝状肌动蛋白分布并用IPP5.1测量、SAS软件统计分析;用Tunnel法检测3 DPI乳鼠肝脏和心脏原位细胞凋亡;以巢式RT-PCR(nested reverse transcription polymerase reaction ,nRT-PCR)法检测组织中RV RNA分布,以研究RV感染肠外脏器损伤机制。结果RV SA-11在MA-104细胞内增殖良好,感染后1天即可出现病变,表现为拉网、细胞圆缩、脱落,4天病变可达到+++～++++。空斑实验滴定所收集的病毒液,滴度为3.75×10~7噬斑形成单位(Plaque Forming Unit,PFU)/ml。 RV感染后乳鼠出现腹泻,活动减少,皮毛光泽差,肛周可见粪便污染。腹泻潜伏期为1～5天,持续约1～5天,感染后第3～4天腹泻最重,感染后第8天所有乳鼠停止腹泻。感染后乳鼠大便RV抗原阳性且便中有病毒排出,1～7 DPI大便RV滴定结果依次为3.75×103、6.25×10~3、1.25×10~4、5.00×10~4、6.25×10~3、8.75×10~3、8.75×10~2 PFU/ml,乳鼠腹泻越严重,其大便RV滴度也越高。RV腹泻对乳鼠体重未见明显影响。乳鼠感染RV后光镜下见小肠肠壁和绒毛轻度充血水肿,绒毛萎缩,绒毛上皮细胞广泛空泡状变性,未见有明显炎性细胞浸润及肠壁坏死等表现。电镜下见绒毛上皮细胞空泡变性部位呈大量脂滴样结构,微绒毛排列紊乱或脱落,而细胞间连结未见明显结构改变。1、2、3、4、7、10、15DPI空肠石蜡切片HE染色并经IPP5.1测量及SAS软件统计分析显示:RV感染后实验组和对照组空肠绒毛高度比较1、2 DPI有统计学意义(P0.05),提示绒毛有萎缩;2、3、4 DPI可见两组肠隐窝深度有差异(P0.05),实验组肠隐窝增生明显;1、2、3、4 DPI肠绒毛高度和肠隐窝深度比值有差异(P0.05),表明其后绒毛上皮增生趋于正常。免疫组化检测提示RV抗原分布主要在小肠绒毛的上部。Palloidine丝状肌动蛋白染色显示1、2 DPI时实验组乳鼠空肠组织丝状肌动蛋白含量有明显减少,但其后则两组无显著差异。原位细胞凋亡检测显示RV感染后空肠上皮细胞凋亡增加。 3 DPI时取乳鼠脑、肺、心、肝、胰、肾组织,光镜下显示轻度心肌间质性水肿、轻度心肌细胞浊肿,肝组织轻度充血、肝细胞空泡样变性,肾小球和肾间质轻度充血、肾小管轻度浊肿,而脑、肺、胰未见明显改变。电镜下见心肌细胞肌丝溶解,滑面内质网扩张,线粒体内室肿胀,核周间隙增宽。免疫组化显示除脑组织外,肺、心、肝、胰、肾组织中均检测到RV抗原。Phalloidine丝状肌动蛋白染色显示1、2、3、4 DPI心肌丝状肌动蛋白含量改变不明显。原位细胞凋亡检测显示肝细胞凋亡明显,而心肌细胞未见明显凋亡。nRT-PCR检测显示脑、肺、心、肝、胰、肾组织中均检测到RV核酸。结论RV感染昆明乳鼠动物模型建立成功。RV主要感染小肠成熟绒毛上皮细胞。腹泻的发生与小肠绒毛上皮细胞骨架损害、微绒毛损害、细胞凋亡脱落和绒毛萎缩相关,但与肠上皮细胞间连接损害关系不大。RV可由肠道向全身播散,并造成肠外多脏器不同程度损害。造成肠外脏器损害的机制不除外病毒局部增殖破坏组织的可能,也可能由于病毒结构蛋白或非结构蛋白作用造成,如影响丝状肌动蛋白合成,破坏细胞骨架等。病毒感染引起细胞凋亡增加也是造成肠外脏器损害的机制之一。
[Abstract]:The purpose of rotavirus (Rotavirus, RV) is caused by the main pathogen of infantile diarrhea, also can cause extraintestinal organ damage, the morbidity and mortality are high, but its pathogenesis is not fully understood, also failed to develop satisfactory vaccine. This experiment through the monkey RV SA-11 infection after birth Kunming 7 days of suckling mice, establish rat animal model of RV infection, and the pathogenesis of the animal model for the study of rotavirus diarrhea and extraintestinal organ damage, and provide a theoretical basis for the development of RV vaccine and better prevention and treatment of RV infection.
Taking Ganges RIver monkey kidney cell (MA-104) cultured monkey RV SA-11 amplification, packed plaque assay titration of virus titer, -80 C, save spare. Choosing of the experimental animal 7 days after birth and clean Kunming rat, each experimental group rats were orally inoculated with 0.1 ml virus cell lysate was (RV content 3.75 * 10~7 PFU/ml), while the control group of each rat by oral inoculation of 0.1 ml normal virus control cell lysate was cultured. 1~7 days after infection (Day Post, Inoculation, DPI), the daily observation of rat activity, color gloss, stool change, weight change, and stool collection by ELISA the detection of RV antigen, plaque assay titration and RV titer, establish the animal model of rotavirus infection. Small intestine in 1,2,3,4,7,10,15 DPI at different time points, hematoxylin eosin staining (hematoxylin-eosin, HE) staining to observe the morphological changes under light microscope and image analysis software (Image Pro plu S 5.1, IPP5.1) determination of jejunal villus height and crypt depth; 3 DPI jejunal tissue fixed in 2.5% glutaraldehyde after electron microscopic observation of intestinal epithelial cells changes in ultra structure; 3 DPI jejunum paraffin sections by immunohistochemical staining of rat jejunum RV antigen distribution; 1,2,3,4,10,15 DPI jejunum paraffin sections using Phalloidine-TRITC staining to observe the intestinal villi the distribution of filamentous actin and IPP5.1 measurement, analysis of SAS statistical software; 3 DPI jejunum paraffin detection of intestinal epithelial cell apoptosis by Tunnel method, to study the pathogenesis of RV diarrhea enteritis. 3 DPI rat brain, lung, heart, pancreas, liver, kidney tissue, paraffin section and HE staining, light microscopy and change electron microscopic observation of Pathology; distribution by immunofluorescence detection of RV antigen in organs was observed with Phalloidine-TRITC staining; 1,2,3,4 DPI rat cardiac actin filament distribution and measured by IPP5.1, SAS statistical software The apoptosis of liver and heart in 3 DPI suckling mice was detected by Tunnel method. The RV RNA distribution in tissues was detected by nested reverse transcription polymerase reaction (nRT-PCR), so as to study the mechanism of the infection of intestinal organs in rats infected by RT-PCR.
The results of RV SA-11 in MA-104 cell proliferation, infection 1 days after lesion, manifested as net of rounded cells, 4 days off, lesions can be reached + + + ~ ++++. virus by plaque assay titration collected, titer of 3.75 * 10~7 plaque forming unit (Plaque Forming Unit, PFU /ml.)
After RV infection of suckling mice diarrhea, reduced activity, shiny coat, perianal visible fecal contamination. Diarrhea incubation period is 1~5 days, lasted about 1~5 days, from third to 4 days after infection diarrhea most, eighth days after infection. All rats stop diarrhea of newborn mice infected by stool RV antigen positive and virus at from 1~7 DPI stool RV titration results were 3.75 * 103,6.25 * 10~3,1.25 * 10~4,5.00 * 10~4,6.25 * 10~3,8.75 * 10~3,8.75 * 10~2 PFU/ml, neonatal rat diarrhea is more serious, affecting the titer of RV was also higher.RV stool diarrhea on rat body weight showed no obvious.
RV infection of suckling mice under the light microscope to see the intestinal wall and villi mild hyperemia and edema, atrophy of the villi and villous epithelial cells widely vacuolar degeneration, no obvious infiltration of inflammatory cells and intestinal necrosis. Under electron microscope, villus epithelial cells showed vacuolar degeneration of large lipid droplets like structure, microvilli arranged in disorder or loss the links between the cell, no obvious structural changes.1,2,3,4,7,10,15DPI jejunum paraffin section and HE staining and display analysis by IPP5.1 measurement and SAS statistical software: RV after infection of the experimental group and the control group of jejunum was statistically significant compared with 1,2 DPI (P0.05), suggesting that there are different villous atrophy; 2,3,4 DPI showed two groups of intestinal crypt depth (P0.05), the experimental group of intestinal crypt hyperplasia; 1,2,3,4 DPI ratio of villus height and crypt depth difference (P0.05), showed that villus epithelial hyperplasia subsequently became normal. Immunohistochemical detection showed RV antigen mainly distributed at the top of the villi.Palloidine filamentous actin staining showed that 1,2 DPI experimental content of filamentous actin in rat jejunum tissue were significantly decreased, but was no significant difference between the two groups. TUNEL showed increased jejunal epithelial cell apoptosis after RV infection.
3 DPI from rat brain, lung, heart, liver, pancreas, kidney, light microscope showed mild myocardial interstitial edema, mild myocardial cell swelling, liver tissue hyperemia, hepatocyte vacuolation, glomerular and interstitial hyperemia, slight tubular swelling, and the brain. Lung and pancreas had no obvious change. Under electron microscope, myofilament dissolved myocardial cells, smooth endoplasmic reticulum expansion, vacuolated mitochondria swelling, enlarged perinuclear space. Immunohistochemistry showed that except for brain, lung, heart, liver, pancreas, kidney tissues were detected by RV antigen.Phalloidine filamentous actin staining showed that 1,2,3,4 myocardial DPI filamentous actin content did not change significantly. TUNEL showed that hepatic cell apoptosis significantly, and no obvious myocardial cell apoptosis in.NRT-PCR assay showed that the brain, lung, heart, liver, pancreas, kidney tissues were detected by RV nucleic acid.
Kunming rat animal model of.RV infection mainly mature villous epithelial cells infected with RV. Conclusion the incidence of diarrhea and intestinal villus epithelial cytoskeleton damage, microvilli damage, apoptosis and loss of villous atrophy, but with intestinal epithelial cell junction damage has little relation to.RV from the gut to spread all over the body, and caused many parenteral different organs damage. The mechanism causing extraintestinal organ damage except for local tissue destruction may be virus proliferation, may also be due to a virus structural protein or non structural proteins, such as the influence of filamentous actin cytoskeleton synthesis, damage caused by virus infection. One of the mechanisms of cell apoptosis is caused by intestinal damage of organs.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R-332;R725.1

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