發(fā)布時(shí)間：2018-06-10 18:57

  本文選題：CMV + 圍生期��； 參考：《復(fù)旦大學(xué)》2010年博士論文

【摘要】： 研究背景與目的：膽道閉鎖是發(fā)生于嬰幼兒的進(jìn)行性炎癥性膽管病,最終導(dǎo)致肝外、肝內(nèi)膽管的梗阻及肝硬化。膽道閉鎖的病因和發(fā)病機(jī)制仍不清楚。來自臨床和動(dòng)物實(shí)驗(yàn)的證據(jù)提示圍生期的某種病毒感染誘發(fā)的宿主炎性反應(yīng)很可能是致病原因之一。其中,巨細(xì)胞病毒和輪狀病毒是膽道閉鎖研究中最受關(guān)注的兩種病毒。盡管研究發(fā)現(xiàn)膽道閉鎖患兒有很高的巨細(xì)胞病毒感染率,但是臨床上仍缺乏足夠的證據(jù)表明這樣的感染與肝外膽道損傷繼而閉鎖存在直接的因果聯(lián)系。因此我們的研究目的之一是在原有豚鼠先天性感染模型的基礎(chǔ)上,根據(jù)國內(nèi)高致病力毒株無法通過近交系傳代獲得的現(xiàn)狀,摸索圍產(chǎn)期遠(yuǎn)交系豚鼠巨細(xì)胞病毒(Guinea pig cytomegalovirus, gpCMV)感染并導(dǎo)致幼鼠肝膽損傷的合適病毒滴度,建立感染模型,并在此基礎(chǔ)上分析圍產(chǎn)期CMV感染后肝臟實(shí)質(zhì)和膽道系統(tǒng)的病變情況,進(jìn)一步了解CMV在膽道閉鎖發(fā)病中的作用；恒河猴輪狀病毒感染(Rhesus Rotavirus, RRV)的膽道閉鎖動(dòng)物模型是目前膽道閉鎖研究中最為常用的工具。由于輪狀病毒對(duì)反轉(zhuǎn)錄基因基因技術(shù)的抵抗,使得對(duì)該病毒致病分子生物學(xué)機(jī)制研究受到極大限制。本研究的第二部分?jǐn)M利用同組輪狀病毒混合感染后可發(fā)生基因重排的特性,篩選單基因重排病毒株,通過病毒株的表現(xiàn)型分析確定RRV致膽道損傷的關(guān)鍵致病基因節(jié)段及其致病機(jī)制。 材料與方法：一,豚鼠圍生期巨細(xì)胞病毒感染模型的建立及肝膽損傷機(jī)制研究：gpCMV于豚鼠胚胎肺成纖維細(xì)胞上傳代適應(yīng)。按病毒接種時(shí)間將動(dòng)物分組為：1,孕晚期組,孕晚期豚鼠于孕40-43天腹腔接種1×109TCID50病毒懸液,同時(shí)設(shè)生理鹽水或空白對(duì)照,活產(chǎn)子豚鼠出生后不同時(shí)間點(diǎn)處死取標(biāo)本。2,新生鼠組,隨機(jī)取新生豚鼠23只,于生后12-24小時(shí)腹腔注射1×108TCID50病毒懸液,于接種后不同時(shí)間點(diǎn)處死取標(biāo)本。3,幼鼠組,隨機(jī)取新生豚鼠16只,于生后10天腹腔注射1×108TCID50病毒懸液,于接種后不同時(shí)間點(diǎn)處死取標(biāo)本。觀察生長情況及黃疸癥狀。所有血液標(biāo)本常規(guī)肝功能檢測。肝臟及肝外膽道標(biāo)本石蠟切片常規(guī)HE染色,原位檢測細(xì)胞凋亡情況。部分標(biāo)本冰凍切片原位雜交檢測gpCMVmRNA在肝膽系統(tǒng)的表達(dá)。膽汁標(biāo)本雙抗酶標(biāo)ELISA法檢測膽汁肝細(xì)胞生長因子(Hepatocyte growth factor, HGF)和白介素6(Interleukin 6, IL-6)表達(dá)情況。免疫組化檢測CD8+細(xì)胞的表達(dá)。二,輪狀病毒致小鼠膽管損傷關(guān)鍵基因的研究：將毒性病毒株RRV與非毒性病毒株EDIM(Epizootic Diarrhea ofInfant Mice)(體內(nèi))或TUCH(Tulane University and Cincinnati Childen's Hospital,新分離出的猴輪狀病毒株)(體外)共感染。所得部分雙基因或三基因重排子代與親代,或其他子代進(jìn)行回交或雜交以施加選擇性壓力。將培養(yǎng)所得病毒懸液接種MA104細(xì)胞系后瓊脂糖凝膠鋪板,挑選病毒斑。病毒純化后抽提RNA,聚丙烯酰胺凝膠電泳進(jìn)行基因型分析。將所篩選出的單基因重排病毒株接種新生BALB/C小鼠,進(jìn)行病毒致病性分析,部分小鼠接種后7天殺死取肝臟及肝外膽道標(biāo)本進(jìn)行組織病理分析。此外,將單基因重排各株接種膽管細(xì)胞系,4℃共孵育1小時(shí)后計(jì)算結(jié)合病毒量與總接種量的比例進(jìn)行病毒細(xì)胞結(jié)合力分析；重排病毒株接種膽管上皮細(xì)胞系,感染劑量MOI (Multiplicity Of Infection)為1。48小時(shí)后對(duì)病毒進(jìn)行滴定,了解增殖情況。各重排克隆接種新生鼠后7天后取肝外膽道標(biāo)本制成勻漿,于MA104細(xì)胞系上進(jìn)行滴定,了解病毒負(fù)荷量。 結(jié)果： 一,圍生期豚鼠巨細(xì)胞病毒感染模型建立及分析： 孕晚期及出生時(shí)腹腔接種巨細(xì)胞病毒可引起子代豚鼠肝膽系統(tǒng)損傷,表現(xiàn)為： 1,孕晚期感染豚鼠出生體重明顯低于鹽水及空白對(duì)照(P=0.0029,P=0027)。生后20天,體重增長也明顯落后于鹽水及空白對(duì)照。新生鼠及幼鼠組病毒接種后豚鼠體重增長較對(duì)照組相比無明顯差異。 2,孕晚期感染子豚鼠與空白及鹽水對(duì)照組相比,出生時(shí)及生后10天TB、DB、ALT、AST水平均明顯高于對(duì)照組(P＜0.05),出生時(shí)及生后10天兩組比較各指標(biāo)無明顯差異(P0.05),生后20天各指標(biāo)均明顯低于前兩組,較對(duì)照無明顯差別(P0.05)。部分子豚鼠肉眼可見大便變白,伴TB和DB水平的升高。新生鼠組病毒接種后10天AST水平較20天組和空白對(duì)照升高(P=0.027,P=0.043)。 3,63.6%的孕晚期感染子豚鼠肝臟存在單核淋巴細(xì)胞浸潤為主的炎癥／病理改變,炎性細(xì)胞浸潤以匯管區(qū)為主。可見小葉間匯管區(qū)的破壞和纖維化改變,合并小膽管增生。伴有肝細(xì)胞氣球樣變性和壞死,肝內(nèi)膽汁淤積。偶可見肝內(nèi)微膿腫形成。新生鼠接種后10天出現(xiàn)肝臟病變(3／8),但病變較孕晚期組明顯減輕。幼鼠組未見明顯肝臟病變。 4,有肝膽損傷者肝內(nèi)細(xì)胞凋亡信號(hào)密度分布基本與炎癥浸潤程度一致,主要表達(dá)于肝臟實(shí)質(zhì)細(xì)胞和匯管區(qū)間質(zhì)細(xì)胞,血管和膽管上皮偶見信號(hào)表達(dá)。孕晚期組出生時(shí)肝臟細(xì)胞凋亡指數(shù)均值6.7±1.322,匯管區(qū)細(xì)胞凋亡指數(shù)均值5.13±2.112,與對(duì)照相比有明顯增加(P0.05),與新生鼠組肝損傷者凋亡指數(shù)無明顯差別。 5,病毒gpCMV陽性雜交信號(hào)表達(dá)于膽管上皮及血管內(nèi)皮細(xì)胞及匯管區(qū)基質(zhì)內(nèi),但肝細(xì)胞內(nèi)未見表達(dá)。孕晚期組豚鼠gpCMV陽性率17.27%,而新生鼠及幼鼠組豚鼠肝膽系gpCMV-mRNA陽性率(55%,50%)均明顯高于孕晚期組(P=0.011,0.0180.05)。 6,新生鼠感染后10天,20天膽汁中可見HGF表達(dá)增加(P=0.0292,P=0.0461)。各組膽汁標(biāo)本中未見IL-6的表達(dá)。 7,孕晚期組(8／14)和新生鼠組(3／3)肝膽損傷者有CD8+細(xì)胞表達(dá),主要分布于肝臟實(shí)質(zhì)和匯管區(qū)間質(zhì)。 二,輪狀病毒致小鼠膽管損傷關(guān)鍵基因的研究： 1,在RRV對(duì)EDIM的體內(nèi)雜交中,得到單基因重排株4株,將EDIM背景單基因重排株D6／2接種新生鼠后(第4基因節(jié)段來自RRV),誘導(dǎo)出與RRV類似的膽管損傷癥狀。RRV對(duì)TUCH的體外混合感染中(包括回交及子代雜交),分離出所有22個(gè)RRV或TUCH背景下單基因重排病毒株。分別命名為RTn(RRV背景重排株,第n個(gè)基因節(jié)段來自TUCH)或TRn(TUCH背景重排株,第n個(gè)基因節(jié)段來自RRV)。n代表第1至11個(gè)基因節(jié)段。 2,致病性分析發(fā)現(xiàn)：重排株RT4沒有致病性。相反,重排株TR4誘導(dǎo)出與RRV相似的黃疸癥狀,致病率(94.2%vs.100%,P=1.000)及致死率(88.24%vs.80.96%,P=1.000)無明顯差別。58.33%的RT3感染小鼠表現(xiàn)膽道梗阻癥狀,其致病率和致死率明顯低于RRV(p=0.001,0.0000.05)。相對(duì)應(yīng)的TR3盡管誘導(dǎo)出與RRV相似的致病率(88.89%,P=0.218),77.78%的小鼠最終黃疸癥狀消退并存活。其余RRV背景重排病毒株疾病的表現(xiàn)型與RRV類似。TR7、TR1和TR2一樣未誘導(dǎo)出任何肝膽損傷癥狀。其余大多數(shù)TUCH背景單基因重排株誘導(dǎo)—過性肝膽損傷癥狀,幾乎所有小鼠最終存活。 3,病理分析證實(shí)各重排株的疾病表現(xiàn)型與匯管區(qū)及肝外膽道病理改變密切相關(guān)。 4,細(xì)胞結(jié)合能力：重排株RT4的細(xì)胞結(jié)合率(3.97%±0.93%)明顯低于RRV(16.03%±1.31%,P=0.003＜0.05)及其他所有RRV背景重排株(P＜0.05)。其余RRV背景重排株細(xì)胞結(jié)合率與RRV無明顯差別(P＞0.05)。相反,重排株TR4(12.43%±2.25%)表現(xiàn)出明顯高于TUCH：5.33%±1.27%,p=0.001)及所有其他TUCH背景重排株的細(xì)胞結(jié)合率(P0.05)。除了TR8和TR2外,其它TUCH背景重排株細(xì)胞結(jié)合率與TUCH相似(P0.05)。 5,感染性分析：克隆RT4在膽管上皮細(xì)胞系和肝外膽道的滴度均明顯低于RRV(P=0.013,0.000)。相對(duì)應(yīng)的克隆TR4的滴度無論在膽管細(xì)胞系還是小鼠肝外膽道均明顯高于TUCH(P=0.004,0.0446)。在膽管細(xì)胞系上,所有其它RRV背景重排株的滴度與RRV類似(P＞0.05),在肝外膽道大部分低于RRV(P＜0.05)。TR7、TR1和TR2在膽管細(xì)胞系及肝外膽道滴度均低于TUCH(P=0.001,0.012,0.0069)。其余引起癥狀TUCH背景病重排株在膽管細(xì)胞系的滴度均高于TUCH(P＜0.05)。等級(jí)相關(guān)分析提示TUCH背景重排株在肝外膽道平均滴度與病毒致病率相關(guān)。 結(jié)論： 一,圍生期豚鼠巨細(xì)胞病毒感染模型建立及分析： 1,孕晚期和出生后即刻接種gpCMV病毒可導(dǎo)致子代豚鼠的感染和肝膽系統(tǒng)炎癥損傷。 2,肝內(nèi)和匯管區(qū)浸潤炎性細(xì)胞以單核淋巴細(xì)胞為主,伴有CD8+T細(xì)胞的表達(dá),提示Thl抗病毒免疫在此病理過程中發(fā)揮重要作用。 3,病毒mRNA主要表達(dá)于匯管區(qū)及內(nèi)皮系統(tǒng),證實(shí)了CMV病毒對(duì)膽管上皮的親和性。其攻擊膽管上皮標(biāo)本并引起炎癥損傷的機(jī)制待進(jìn)一步研究。 4,損傷主要發(fā)生于圍產(chǎn)期,與接種時(shí)間直接相關(guān),表明宿主免疫狀態(tài)不成熟可能是致病的重要背景因素。 二,輪狀病毒致小鼠膽管損傷關(guān)鍵基因的研究： 1,基因節(jié)段4是決定小鼠膽道模型RRV對(duì)膽管上皮致病性的關(guān)鍵基因,也決定了RRV對(duì)膽管上皮感染的特異性,其機(jī)制在于影響病毒與膽管上皮細(xì)胞的結(jié)合能力,繼而影響病毒在細(xì)胞內(nèi)復(fù)制,其蛋白產(chǎn)物VP4與膽管上皮相關(guān)受體的相互作用很可能是決定膽管細(xì)胞對(duì)病毒易感性的關(guān)鍵環(huán)節(jié)。 2,基因節(jié)段7被替代后,病毒的致病性、細(xì)胞結(jié)合能力和膽管上皮復(fù)制均未受影響,其蛋白產(chǎn)物VP7相關(guān)細(xì)胞表面受體的表達(dá)可能對(duì)膽管上皮的易感性影響不大。 3,基因節(jié)段3對(duì)RRV致病性有重要影響,其被替代所帶來的“中間型”可能是包括宿主因素在內(nèi)的多因素相互作用的結(jié)果。 4,基因節(jié)段1,2對(duì)病毒致病性無明顯影響,但兩者之間可能在病毒復(fù)制中存在協(xié)同作用。
[Abstract]:Background and objective: biliary atresia is an progressive inflammatory cholangiosis occurring in infants and infants, which eventually leads to extrahepatic, intrahepatic bile duct obstruction and cirrhosis. The etiology and pathogenesis of biliary atresia are still unclear. Evidence from clinical and animal experiments suggests that the host inflammatory response induced by a certain viral infection perinatal may be very likely. It is one of the causes of disease. Among them, cytomegalovirus and rotavirus are two of the most concerned viruses in the study of biliary atresia. Although the study found that children with biliary atresia have high cytomegalovirus infection rates, there is still lack of sufficient evidence to show that such infection and extrahepatic bile duct injury and subsequent atresia have direct cause and effect. Therefore, one of the objectives of our study is to explore the appropriate viral titer of the Guinea pig cytomegalovirus (gpCMV) infection of the perinatal guinea pig and the liver and gallbladder injury of the perinatal system, based on the existing congenital infection model of the guinea pig. The infection model was established and the pathological changes of liver parenchyma and biliary tract after perinatal CMV infection were analyzed, and the role of CMV in the pathogenesis of biliary atresia was further understood. The biliary atresia model of Ganges RIver monkey rotavirus infection (Rhesus Rotavirus, RRV) was the most commonly used tool in the current study of biliary atresia. The resistance of rotavirus to the reverse transcriptional gene technology makes the study of the molecular biological mechanism of the virus pathogenic very limited. The second part of this study intends to use the characteristics of gene rearrangement after the mixed infection of the same group of rotavirus, screening the single gene rearrangement virus strain, and determining the RRV gallbladder through the phenotype analysis of the virus strain. The key gene segment and pathogenicity mechanism of road injury.
Materials and methods: first, the establishment of a perinatal giant cytomegalovirus infection model in guinea pigs and the mechanism of hepatobiliary injury: gpCMV in guinea pig embryonic lung fibroblasts. The animals were grouped into 1, late trimester, late trimester, 40-43 days of pregnancy and intraperitoneal inoculation of 1 * 109TCID50 virus suspension at the same time of pregnancy, and physiological salts were set up at the same time. Water or blank control, the living producing guinea pigs were killed at different time points after birth of.2, the newborn rat group, 23 newborn guinea pigs were randomly taken, 12-24 hours after birth, intraperitoneal injection of 1 x 108TCID50 virus suspension, after inoculation at different time points were killed.3, young rats, randomly taken 16 new born guinea pigs, intraperitoneal injection of 1 x 108TCID50 virus 10 days after birth, 10 days after birth, 1 * 108TCID50 virus intraperitoneal Suspension, specimens were killed at different time points after inoculation. Observe growth and jaundice symptoms. Routine examination of liver function in all blood specimens. Routine HE staining of paraffin section in liver and extrahepatic bile duct specimens, in situ detection of cell apoptosis. Partial specimens of frozen section in situ hybridization were used to detect the expression of gpCMVmRNA in the hepatobiliary system. Detection of the expression of bile hepatocyte growth factor (Hepatocyte growth factor, HGF) and interleukin 6 (Interleukin 6, IL-6) by double anti enzyme ELISA. Immunohistochemical detection of the expression of CD8+ cells. Two, study on key genes of bile duct injury in mice induced by rotavirus: toxic virus strain RRV and non toxic virus strain EDIM (Epizootic Diarrhea) Nfant Mice) (in vivo) or TUCH (Tulane University and Cincinnati Childen's Hospital, newly isolated monkey rotavirus strain) Co infection. A partial double gene or three gene rearrangement progeny is recrossed or hybridized with the parent or other progeny to apply selective pressure. The cultured virus suspension is inoculated to the MA104 cell line. The virus plaques were selected by agarose gel pads. The virus was purified by RNA and polyacrylamide gel electrophoresis for genotype analysis. The screened single gene rearrangement virus strain was inoculated to the new BALB/C mice and the virus pathogenicity was analyzed. The liver and extrahepatic bile duct specimens were killed 7 days after the inoculation, and the histopathological analysis was carried out. The single gene rearrangement of each strain was inoculated to the bile duct cell line, and after 1 hours of incubation at 4 C, the virus cell binding force was calculated combined with the proportion of virus and total inoculation. The virus strain was inoculated with the bile duct epithelial cell line. The infection dose MOI (Multiplicity Of Infection) was titrated to the virus after 1.48 hours to understand the proliferation. After 7 days inoculation, the extrahepatic bile duct specimens were homogenized and the MA104 cells were titrated to understand the viral load.
Result錛，

本文編號(hào)：2004257