胆道闭锁与病毒感染的动物实验研究

发布时间：2018-06-10 18:57

  本文选题：CMV + 围生期　； 参考：《复旦大学》2010年博士论文

【摘要】： 研究背景与目的：胆道闭锁是发生于婴幼儿的进行性炎症性胆管病,最终导致肝外、肝内胆管的梗阻及肝硬化。胆道闭锁的病因和发病机制仍不清楚。来自临床和动物实验的证据提示围生期的某种病毒感染诱发的宿主炎性反应很可能是致病原因之一。其中,巨细胞病毒和轮状病毒是胆道闭锁研究中最受关注的两种病毒。尽管研究发现胆道闭锁患儿有很高的巨细胞病毒感染率,但是临床上仍缺乏足够的证据表明这样的感染与肝外胆道损伤继而闭锁存在直接的因果联系。因此我们的研究目的之一是在原有豚鼠先天性感染模型的基础上,根据国内高致病力毒株无法通过近交系传代获得的现状,摸索围产期远交系豚鼠巨细胞病毒(Guinea pig cytomegalovirus, gpCMV)感染并导致幼鼠肝胆损伤的合适病毒滴度,建立感染模型,并在此基础上分析围产期CMV感染后肝脏实质和胆道系统的病变情况,进一步了解CMV在胆道闭锁发病中的作用；恒河猴轮状病毒感染(Rhesus Rotavirus, RRV)的胆道闭锁动物模型是目前胆道闭锁研究中最为常用的工具。由于轮状病毒对反转录基因基因技术的抵抗,使得对该病毒致病分子生物学机制研究受到极大限制。本研究的第二部分拟利用同组轮状病毒混合感染后可发生基因重排的特性,筛选单基因重排病毒株,通过病毒株的表现型分析确定RRV致胆道损伤的关键致病基因节段及其致病机制。 材料与方法：一,豚鼠围生期巨细胞病毒感染模型的建立及肝胆损伤机制研究：gpCMV于豚鼠胚胎肺成纤维细胞上传代适应。按病毒接种时间将动物分组为：1,孕晚期组,孕晚期豚鼠于孕40-43天腹腔接种1×109TCID50病毒悬液,同时设生理盐水或空白对照,活产子豚鼠出生后不同时间点处死取标本。2,新生鼠组,随机取新生豚鼠23只,于生后12-24小时腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。3,幼鼠组,随机取新生豚鼠16只,于生后10天腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。观察生长情况及黄疸症状。所有血液标本常规肝功能检测。肝脏及肝外胆道标本石蜡切片常规HE染色,原位检测细胞凋亡情况。部分标本冰冻切片原位杂交检测gpCMVmRNA在肝胆系统的表达。胆汁标本双抗酶标ELISA法检测胆汁肝细胞生长因子(Hepatocyte growth factor, HGF)和白介素6(Interleukin 6, IL-6)表达情况。免疫组化检测CD8+细胞的表达。二,轮状病毒致小鼠胆管损伤关键基因的研究：将毒性病毒株RRV与非毒性病毒株EDIM(Epizootic Diarrhea ofInfant Mice)(体内)或TUCH(Tulane University and Cincinnati Childen's Hospital,新分离出的猴轮状病毒株)(体外)共感染。所得部分双基因或三基因重排子代与亲代,或其他子代进行回交或杂交以施加选择性压力。将培养所得病毒悬液接种MA104细胞系后琼脂糖凝胶铺板,挑选病毒斑。病毒纯化后抽提RNA,聚丙烯酰胺凝胶电泳进行基因型分析。将所筛选出的单基因重排病毒株接种新生BALB/C小鼠,进行病毒致病性分析,部分小鼠接种后7天杀死取肝脏及肝外胆道标本进行组织病理分析。此外,将单基因重排各株接种胆管细胞系,4℃共孵育1小时后计算结合病毒量与总接种量的比例进行病毒细胞结合力分析；重排病毒株接种胆管上皮细胞系,感染剂量MOI (Multiplicity Of Infection)为1。48小时后对病毒进行滴定,了解增殖情况。各重排克隆接种新生鼠后7天后取肝外胆道标本制成匀浆,于MA104细胞系上进行滴定,了解病毒负荷量。 结果： 一,围生期豚鼠巨细胞病毒感染模型建立及分析： 孕晚期及出生时腹腔接种巨细胞病毒可引起子代豚鼠肝胆系统损伤,表现为： 1,孕晚期感染豚鼠出生体重明显低于盐水及空白对照(P=0.0029,P=0027)。生后20天,体重增长也明显落后于盐水及空白对照。新生鼠及幼鼠组病毒接种后豚鼠体重增长较对照组相比无明显差异。 2,孕晚期感染子豚鼠与空白及盐水对照组相比,出生时及生后10天TB、DB、ALT、AST水平均明显高于对照组(P＜0.05),出生时及生后10天两组比较各指标无明显差异(P0.05),生后20天各指标均明显低于前两组,较对照无明显差别(P0.05)。部分子豚鼠肉眼可见大便变白,伴TB和DB水平的升高。新生鼠组病毒接种后10天AST水平较20天组和空白对照升高(P=0.027,P=0.043)。 3,63.6%的孕晚期感染子豚鼠肝脏存在单核淋巴细胞浸润为主的炎症／病理改变,炎性细胞浸润以汇管区为主。可见小叶间汇管区的破坏和纤维化改变,合并小胆管增生。伴有肝细胞气球样变性和坏死,肝内胆汁淤积。偶可见肝内微脓肿形成。新生鼠接种后10天出现肝脏病变(3／8),但病变较孕晚期组明显减轻。幼鼠组未见明显肝脏病变。 4,有肝胆损伤者肝内细胞凋亡信号密度分布基本与炎症浸润程度一致,主要表达于肝脏实质细胞和汇管区间质细胞,血管和胆管上皮偶见信号表达。孕晚期组出生时肝脏细胞凋亡指数均值6.7±1.322,汇管区细胞凋亡指数均值5.13±2.112,与对照相比有明显增加(P0.05),与新生鼠组肝损伤者凋亡指数无明显差别。 5,病毒gpCMV阳性杂交信号表达于胆管上皮及血管内皮细胞及汇管区基质内,但肝细胞内未见表达。孕晚期组豚鼠gpCMV阳性率17.27%,而新生鼠及幼鼠组豚鼠肝胆系gpCMV-mRNA阳性率(55%,50%)均明显高于孕晚期组(P=0.011,0.0180.05)。 6,新生鼠感染后10天,20天胆汁中可见HGF表达增加(P=0.0292,P=0.0461)。各组胆汁标本中未见IL-6的表达。 7,孕晚期组(8／14)和新生鼠组(3／3)肝胆损伤者有CD8+细胞表达,主要分布于肝脏实质和汇管区间质。 二,轮状病毒致小鼠胆管损伤关键基因的研究： 1,在RRV对EDIM的体内杂交中,得到单基因重排株4株,将EDIM背景单基因重排株D6／2接种新生鼠后(第4基因节段来自RRV),诱导出与RRV类似的胆管损伤症状。RRV对TUCH的体外混合感染中(包括回交及子代杂交),分离出所有22个RRV或TUCH背景下单基因重排病毒株。分别命名为RTn(RRV背景重排株,第n个基因节段来自TUCH)或TRn(TUCH背景重排株,第n个基因节段来自RRV)。n代表第1至11个基因节段。 2,致病性分析发现：重排株RT4没有致病性。相反,重排株TR4诱导出与RRV相似的黄疸症状,致病率(94.2%vs.100%,P=1.000)及致死率(88.24%vs.80.96%,P=1.000)无明显差别。58.33%的RT3感染小鼠表现胆道梗阻症状,其致病率和致死率明显低于RRV(p=0.001,0.0000.05)。相对应的TR3尽管诱导出与RRV相似的致病率(88.89%,P=0.218),77.78%的小鼠最终黄疸症状消退并存活。其余RRV背景重排病毒株疾病的表现型与RRV类似。TR7、TR1和TR2一样未诱导出任何肝胆损伤症状。其余大多数TUCH背景单基因重排株诱导—过性肝胆损伤症状,几乎所有小鼠最终存活。 3,病理分析证实各重排株的疾病表现型与汇管区及肝外胆道病理改变密切相关。 4,细胞结合能力：重排株RT4的细胞结合率(3.97%±0.93%)明显低于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背景重排株细胞结合率与RRV无明显差别(P＞0.05)。相反,重排株TR4(12.43%±2.25%)表现出明显高于TUCH：5.33%±1.27%,p=0.001)及所有其他TUCH背景重排株的细胞结合率(P0.05)。除了TR8和TR2外,其它TUCH背景重排株细胞结合率与TUCH相似(P0.05)。 5,感染性分析：克隆RT4在胆管上皮细胞系和肝外胆道的滴度均明显低于RRV(P=0.013,0.000)。相对应的克隆TR4的滴度无论在胆管细胞系还是小鼠肝外胆道均明显高于TUCH(P=0.004,0.0446)。在胆管细胞系上,所有其它RRV背景重排株的滴度与RRV类似(P＞0.05),在肝外胆道大部分低于RRV(P＜0.05)。TR7、TR1和TR2在胆管细胞系及肝外胆道滴度均低于TUCH(P=0.001,0.012,0.0069)。其余引起症状TUCH背景病重排株在胆管细胞系的滴度均高于TUCH(P＜0.05)。等级相关分析提示TUCH背景重排株在肝外胆道平均滴度与病毒致病率相关。 结论： 一,围生期豚鼠巨细胞病毒感染模型建立及分析： 1,孕晚期和出生后即刻接种gpCMV病毒可导致子代豚鼠的感染和肝胆系统炎症损伤。 2,肝内和汇管区浸润炎性细胞以单核淋巴细胞为主,伴有CD8+T细胞的表达,提示Thl抗病毒免疫在此病理过程中发挥重要作用。 3,病毒mRNA主要表达于汇管区及内皮系统,证实了CMV病毒对胆管上皮的亲和性。其攻击胆管上皮标本并引起炎症损伤的机制待进一步研究。 4,损伤主要发生于围产期,与接种时间直接相关,表明宿主免疫状态不成熟可能是致病的重要背景因素。 二,轮状病毒致小鼠胆管损伤关键基因的研究： 1,基因节段4是决定小鼠胆道模型RRV对胆管上皮致病性的关键基因,也决定了RRV对胆管上皮感染的特异性,其机制在于影响病毒与胆管上皮细胞的结合能力,继而影响病毒在细胞内复制,其蛋白产物VP4与胆管上皮相关受体的相互作用很可能是决定胆管细胞对病毒易感性的关键环节。 2,基因节段7被替代后,病毒的致病性、细胞结合能力和胆管上皮复制均未受影响,其蛋白产物VP7相关细胞表面受体的表达可能对胆管上皮的易感性影响不大。 3,基因节段3对RRV致病性有重要影响,其被替代所带来的“中间型”可能是包括宿主因素在内的多因素相互作用的结果。 4,基因节段1,2对病毒致病性无明显影响,但两者之间可能在病毒复制中存在协同作用。
[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.
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