本文選題：輪狀病毒　切入點(diǎn)：感染　出處：《重慶醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 目的輪狀病毒(Rotavirus, RV)是引起嬰幼兒腹瀉的最主要病原,同時(shí)也可引起腸外臟器損害,致病率及病死率均較高,但目前對(duì)其發(fā)病機(jī)制仍未完全清楚,也未能研制出很滿意的疫苗。本實(shí)驗(yàn)通過(guò)猴RV SA-11感染生后7天昆明乳鼠,建立RV感染的乳鼠動(dòng)物模型,并用該動(dòng)物模型研究輪狀病毒腹瀉及腸外臟器損傷發(fā)病機(jī)制,為RV疫苗研制及更好地防治RV感染提供理論依據(jù)。 方法以恒河猴胚腎細(xì)胞(MA-104)培養(yǎng)猴RV SA-11,擴(kuò)增,空斑實(shí)驗(yàn)滴定病毒滴度,分裝,-80℃保存?zhèn)溆�。�?shí)驗(yàn)動(dòng)物選用生后7天清潔級(jí)昆明乳鼠,實(shí)驗(yàn)組每只乳鼠經(jīng)口接種0.1 ml病毒培養(yǎng)細(xì)胞凍融液(RV含量為3.75×10~7 PFU/ml),而對(duì)照組每只乳鼠經(jīng)口接種0.1 ml正常無(wú)病毒對(duì)照培養(yǎng)細(xì)胞凍融液。感染后1～7天(Day Post Inoculation,DPI),每日觀察乳鼠活動(dòng)情況、毛色光澤、大便變化、體重變化,并收集大便經(jīng)ELISA檢測(cè)其中RV抗原,空斑實(shí)驗(yàn)滴定其中RV滴度,建立輪狀病毒感染動(dòng)物模型。在1、2、3、4、7、10、15 DPI各時(shí)間點(diǎn)取小腸組織,蘇木精-伊紅染色(hematoxylin-eosin,HE)染色光鏡下觀察形態(tài)學(xué)改變,并用圖像分析軟件(Image pro plus 5.1, IPP5.1)測(cè)定空腸絨毛高度和隱窩深度;取3 DPI空腸組織2.5%戊二醛固定后送電鏡切片觀察腸上皮細(xì)胞超形結(jié)構(gòu)改變;取3 DPI空腸石蠟切片用免疫組化染色觀察乳鼠空腸RV抗原分布;取1、2、3、4、10、15 DPI空腸石蠟切片用Phalloidine-TRITC染色觀察小腸絨毛絲狀肌動(dòng)蛋白分布并用IPP5.1測(cè)量、SAS軟件統(tǒng)計(jì)分析;取3 DPI空腸石蠟切片用Tunnel法檢測(cè)腸上皮細(xì)胞原位凋亡,以研究RV腸炎腹瀉發(fā)病機(jī)制。取3 DPI乳鼠腦、肺、心、胰、肝、腎組織,石蠟切片HE染色光鏡和電鏡觀察病理改變;用免疫熒光法檢測(cè)RV抗原在各臟器分布;以Phalloidine-TRITC染色觀察1、2、3、4 DPI乳鼠心臟絲狀肌動(dòng)蛋白分布并用IPP5.1測(cè)量、SAS軟件統(tǒng)計(jì)分析;用Tunnel法檢測(cè)3 DPI乳鼠肝臟和心臟原位細(xì)胞凋亡;以巢式RT-PCR(nested reverse transcription polymerase reaction ,nRT-PCR)法檢測(cè)組織中RV RNA分布,以研究RV感染腸外臟器損傷機(jī)制。 結(jié)果RV SA-11在MA-104細(xì)胞內(nèi)增殖良好,感染后1天即可出現(xiàn)病變,表現(xiàn)為拉網(wǎng)、細(xì)胞圓縮、脫落,4天病變可達(dá)到+++～++++。空斑實(shí)驗(yàn)滴定所收集的病毒液,滴度為3.75×10~7噬斑形成單位(Plaque Forming Unit,PFU)/ml。 RV感染后乳鼠出現(xiàn)腹瀉,活動(dòng)減少,皮毛光澤差,肛周可見(jiàn)糞便污染。腹瀉潛伏期為1～5天,持續(xù)約1～5天,感染后第3～4天腹瀉最重,感染后第8天所有乳鼠停止腹瀉。感染后乳鼠大便RV抗原陽(yáng)性且便中有病毒排出,1～7 DPI大便RV滴定結(jié)果依次為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,乳鼠腹瀉越嚴(yán)重,其大便RV滴度也越高。RV腹瀉對(duì)乳鼠體重未見(jiàn)明顯影響。 乳鼠感染RV后光鏡下見(jiàn)小腸腸壁和絨毛輕度充血水腫,絨毛萎縮,絨毛上皮細(xì)胞廣泛空泡狀變性,未見(jiàn)有明顯炎性細(xì)胞浸潤(rùn)及腸壁壞死等表現(xiàn)。電鏡下見(jiàn)絨毛上皮細(xì)胞空泡變性部位呈大量脂滴樣結(jié)構(gòu),微絨毛排列紊亂或脫落,而細(xì)胞間連結(jié)未見(jiàn)明顯結(jié)構(gòu)改變。1、2、3、4、7、10、15DPI空腸石蠟切片HE染色并經(jīng)IPP5.1測(cè)量及SAS軟件統(tǒng)計(jì)分析顯示:RV感染后實(shí)驗(yàn)組和對(duì)照組空腸絨毛高度比較1、2 DPI有統(tǒng)計(jì)學(xué)意義(P0.05),提示絨毛有萎縮;2、3、4 DPI可見(jiàn)兩組腸隱窩深度有差異(P0.05),實(shí)驗(yàn)組腸隱窩增生明顯;1、2、3、4 DPI腸絨毛高度和腸隱窩深度比值有差異(P0.05),表明其后絨毛上皮增生趨于正常。免疫組化檢測(cè)提示RV抗原分布主要在小腸絨毛的上部。Palloidine絲狀肌動(dòng)蛋白染色顯示1、2 DPI時(shí)實(shí)驗(yàn)組乳鼠空腸組織絲狀肌動(dòng)蛋白含量有明顯減少,但其后則兩組無(wú)顯著差異。原位細(xì)胞凋亡檢測(cè)顯示RV感染后空腸上皮細(xì)胞凋亡增加。 3 DPI時(shí)取乳鼠腦、肺、心、肝、胰、腎組織,光鏡下顯示輕度心肌間質(zhì)性水腫、輕度心肌細(xì)胞濁腫,肝組織輕度充血、肝細(xì)胞空泡樣變性,腎小球和腎間質(zhì)輕度充血、腎小管輕度濁腫,而腦、肺、胰未見(jiàn)明顯改變。電鏡下見(jiàn)心肌細(xì)胞肌絲溶解,滑面內(nèi)質(zhì)網(wǎng)擴(kuò)張,線粒體內(nèi)室腫脹,核周間隙增寬。免疫組化顯示除腦組織外,肺、心、肝、胰、腎組織中均檢測(cè)到RV抗原。Phalloidine絲狀肌動(dòng)蛋白染色顯示1、2、3、4 DPI心肌絲狀肌動(dòng)蛋白含量改變不明顯。原位細(xì)胞凋亡檢測(cè)顯示肝細(xì)胞凋亡明顯,而心肌細(xì)胞未見(jiàn)明顯凋亡。nRT-PCR檢測(cè)顯示腦、肺、心、肝、胰、腎組織中均檢測(cè)到RV核酸。 結(jié)論RV感染昆明乳鼠動(dòng)物模型建立成功。RV主要感染小腸成熟絨毛上皮細(xì)胞。腹瀉的發(fā)生與小腸絨毛上皮細(xì)胞骨架損害、微絨毛損害、細(xì)胞凋亡脫落和絨毛萎縮相關(guān),但與腸上皮細(xì)胞間連接損害關(guān)系不大。RV可由腸道向全身播散,并造成腸外多臟器不同程度損害。造成腸外臟器損害的機(jī)制不除外病毒局部增殖破壞組織的可能,也可能由于病毒結(jié)構(gòu)蛋白或非結(jié)構(gòu)蛋白作用造成,如影響絲狀肌動(dòng)蛋白合成,破壞細(xì)胞骨架等。病毒感染引起細(xì)胞凋亡增加也是造成腸外臟器損害的機(jī)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R-332;R725.1

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