發(fā)布時間：2018-03-29 13:15

  本文選題：昆明地區(qū)　切入點：戊型肝炎　出處：《揚州大學(xué)》2014年博士論文

【摘要】：戊型肝炎(Hepatitis E, HE)是由戊型肝炎病毒(Hepatitis E virus, HEV)感染引起的一種人畜共患病,主要暴發(fā)和流行于亞洲和非洲一些衛(wèi)生條件相對較差的發(fā)展中國家。人對HEV普遍易感,易感人群以大齡兒童和青壯年為主。在孕婦中可導(dǎo)致流產(chǎn)或引起死亡,病死率高達到15%-30%。2002-2004年衛(wèi)生部傳染病疫情報告顯示我國是戊型肝炎高發(fā)的國家,呈現(xiàn)持續(xù)增長的態(tài)勢,HE已經(jīng)成為一個重要的公共衛(wèi)生問題。越來越多的研究證據(jù)表明HEV可感染豬、犬、牛、山羊、綿羊、鹿、靈長類、雞、貝類、嚙齒類等多種動物,在這些動物宿主中感染率最高是豬,而且從豬中分離到的病毒基因序列和人源毒株有較高的同源性。加之HEV與其它嗜肝性病毒相似,易降解、不穩(wěn)定,很難從糞便、膽汁等中分離到較大量的純HEV,缺乏合適的細胞培養(yǎng)系統(tǒng),在很大程度上限制了HEV深入的研究。為了解昆明地區(qū)戊型肝炎流行情況和探索建立豬HEV感染動物模型,我們開展了昆明地區(qū)人、豬、犬HE的血清流行病學(xué)調(diào)查,并以長爪沙鼠和樹嗣建立豬HEV感染模型。本研究為進一步研究豬HEV的感染機制、研制有效的防控手段奠定了基礎(chǔ)。具體研究內(nèi)容如下：1.昆明地區(qū)戊型肝炎血清流行病學(xué)調(diào)查研究。為了解昆明地區(qū)人、豬、犬戊型肝炎的血清流行病學(xué)特征,應(yīng)用雙夾心抗原ELISA對采集自昆明地區(qū)人血清樣品456份、豬血清樣品835份、犬血清樣品285份進行HEV IgG抗體檢測。結(jié)果表明昆明地區(qū)人、豬、犬血清樣品中HEV IgG抗體陽性分別為43.20%、55.45%、20.35%。人群中男性和女性HEV IgG抗體陽性率分別為43.25%和43.11%,男性和女性二者之間無顯著差異(尸0.05)；21-30歲(48.82%)、 31-40歲(50.86%)、41-50歲(42.86%)人群的HEV抗體陽性率顯著高于10-20歲(23.08%)、51歲以上(32.89%)人群(P0.05)。豬群中仔豬(1月齡-2月齡)、育肥豬(2月齡-6月齡)、種豬(6月齡以上)HEV IgG抗體陽性率分別為46.85%、57.06%、65.42%。不同年齡組豬群抗HEV IgG陽性率存在差異且差異顯著(P(0.05)。犬群中城市流浪犬、家庭散養(yǎng)犬和養(yǎng)殖場犬HEV IgG抗體陽性率分別為59.18%、13.16%和10.71%,家庭散養(yǎng)犬、養(yǎng)殖場犬HEV與流浪犬HEV IgG抗體陽性率差異顯著(P0.05)。德國牧羊犬、昆明犬與雪橇犬、其他品種之間及雪橇犬與其他品種之間HEV IgG抗體陽性率顯著差異(P0.05)。1歲以下與1-5歲、5-10歲、10歲以上犬HEV IgG抗體陽性率顯著差異(P0.05)。研究表明昆明地區(qū)人、豬、犬中存在戊型肝炎的流行,人群-豬群和犬HEV IgG陽性率與年齡有關(guān),21-50歲人群HEV IgG抗體陽性率相對10～20歲、51歲以上較高；豬群HEV IgG陽性率隨年齡的增長而逐步升高；犬HEV IgG抗體陽性率隨年齡的增長而逐步下降。2.昆明及周邊縣豬戊型肝炎病毒分子流行病學(xué)調(diào)查研究。為了解昆明地區(qū)規(guī)�；i場中仔豬HEV的分子背景和流行特征,針對GenBank中HEV的ORF2保守區(qū)域序列,設(shè)計合成兩對巢式PCR引物。分別對采集自昆明及周邊縣的187份仔豬糞便樣品進行擴增,PCR擴增出348bp左右的片段為陽性,陽性率為6.95%。祿勸縣、江川縣、富民縣陽性率分別為4.54%、8.00%、9.52%。對擴增片段進行核苷酸同源性比較和遺傳進化分析,9株豬HEV 348bp擴增片段的核苷酸同源性為87.1%-99.4%,與HEVⅠ、Ⅱ、Ⅲ型同源性分別為77.0%-81.9%、52.2%～53.6%、77.0%～88.2%,與HEV Ⅳ型的同源性為77.9%～96.8%。通過遺傳進化樹分析得出所檢測到的9株豬HEV毒株均為HEV基因Ⅳ型。研究表明昆明地區(qū)規(guī)模化豬場中仔豬群中存在HEV感染并且都為基因Ⅳ型。3.戊型肝炎病毒感染豬肝臟的組織病理學(xué)觀察。為了解豬戊型肝炎病毒感染肝的組織病理學(xué)變化,應(yīng)用免疫組織化學(xué)染色和蘇木素一伊紅染色對采集自昆明地區(qū)農(nóng)貿(mào)市場上的待售豬肝樣品176份進行了組織病理學(xué)觀察。免疫組織化學(xué)染色觀察得出,市售豬肝呈HEV抗原陽性率為65.34%。蘇木素一伊紅染色觀察得出,51.30%的豬肝組織出現(xiàn)炎性細胞浸潤,28.70%的豬肝組織出現(xiàn)纖維結(jié)締組織增生,20.87%的豬肝出現(xiàn)肝細胞萎縮,19.13%的豬肝出現(xiàn)肝細胞變性、壞死。研究表明昆明地區(qū)市售豬肝中普遍存在HEV感染并出現(xiàn)相應(yīng)的病理學(xué)變化。4.豬戊型肝炎病毒感染長爪沙鼠的實驗研究。為了探討建立豬戊型肝炎病毒感染長爪沙鼠模型的可能性,應(yīng)用HEV陽性豬糞便上清液腹腔注射接種長爪沙鼠,定期檢測長爪沙鼠血清中丙氨酸轉(zhuǎn)氨酶(ALT)和天門冬氨酸轉(zhuǎn)氨酶(AST)水平的變化及肝臟、糞便、血液、小腸中HEV RNA的產(chǎn)生,病理組織學(xué)變化及HEV在肝臟中的分布情況。結(jié)果表明長爪沙鼠接種豬HEV后7天血清中ALT和AST均同步上升,此后開始降低。但ALT值到接種后35天還是沒有降低到對照組水平,攻毒組比陰性對照組數(shù)值高出2倍左右。AST到接種后35天基本回落到正常范圍內(nèi)(根據(jù)陰性對照)。糞便和肝臟中均能檢測到HEV RNA,血液和小腸偶能檢測HEV RNA。肝臟組織觀察到肝小葉間淋巴細胞浸潤、肝細胞顆粒變性,局灶性淋巴細胞浸潤、肝細胞索排列紊亂、膽管增生等。后期表現(xiàn)為多發(fā)性淋巴細胞浸潤、肝細胞壞死,枯否細胞增多,匯管區(qū)纖維結(jié)締組織增生。小腸、腎、腦、脾臟等組織也有不同程度病理變化。免疫組化檢測結(jié)果可見到肝細胞漿中有強陽性信號。說明成功建立了豬戊型肝炎病毒感染長爪沙鼠模型。5.豬戊型肝炎病毒感染樹晌的實驗研究。為了探討建立豬戊型肝炎病毒感染樹(?)模型的可能性,應(yīng)用HEV RNA陽性豬糞便上清液腹腔注射接種樹(?),定期檢測樹(?)血清中丙氨酸轉(zhuǎn)氨酶(ALT)和天門冬氨酸轉(zhuǎn)氨酶(AST)水平的變化及肝臟、糞便、血液、小腸、腎臟、脾臟中HEV RNA的產(chǎn)生,病理組織學(xué)變化。結(jié)果表明樹(?)接種豬HEV后7天血清中ALT和AST均同步升高,此后開始降低。但ALT和AST到接種第28天還是沒有降低到對照組水平,攻毒組比陰性對照組數(shù)值高。樹(?)接種豬HEV 7天后肝臟、糞便、血液、小腸中能檢測到HEVRNA,腎臟和脾臟中偶能檢測HEV RNA。肝臟可觀察到靜脈竇淤血、肝細胞空泡變性、多發(fā)性淋巴細胞浸潤、枯否細胞增多、匯管區(qū)纖維結(jié)締組織增生等病變。小腸、腎等組織也有不同程度病理變化。說明建立豬戊型肝炎病毒感染樹昀模型是可行的,可為進一步研究HEV跨物種傳播和致病機制等奠定基礎(chǔ)。
[Abstract]:Hepatitis E (Hepatitis E, HE) is caused by hepatitis E virus (Hepatitis E, virus, HEV) infection is a zoonosis originated from, major outbreaks and epidemics in Asia and Africa some health conditions are relatively poor in developing countries. People of HEV generally susceptible, susceptible to older children and young adults. In pregnant women can cause miscarriage or cause death, high mortality rate reached epidemic reporting of infectious diseases 15%-30%.2002-2004 Ministry of health show that China is a country with high incidence of hepatitis E, showing sustained growth, HE has become an important public health problem. More and more evidence indicates that HEV can infect pigs, dogs and cattle. Goat, sheep, deer, primates, chicken, shellfish, rodents and other animal, in these animal hosts the highest infection rate of pigs, and isolated from swine virus gene sequence and human strains had high homology. In addition to HEV and other similar hepavirus, easy degradation and instability, it is difficult to separate from fecal bile, to a large number of pure HEV, the lack of suitable cell culture system, to a large extent limited the further study of HEV. To investigate the prevalence of hepatitis E in Kunming and explore the establishment of animal model of swine HEV we carried out infection in Kunming area, pig serum epidemiological investigation of canine HE, and Meriones unguiculatus and tree shrew establishment of pig model of HEV infection. This study for further study of the mechanism of infection of porcine HEV, laid the foundation for the development of effective means of prevention and control. The specific contents are as follows: sero epidemiological study on hepatitis E 1. Kunming area. In order to understand the Kunming region, pig, sero epidemiology of canine hepatitis E, application of double sandwich antigen ELISA in human serum samples collected from Kunming area of 456 copies, 835 copies of blood proof products, blood of dogs 285 proofs were tested for HEV IgG antibody. The results show that the Kunming region, pig, HEV IgG antibody positive canine serum samples were 43.20%, 55.45%, 20.35%. crowd of men and women HEV IgG antibody positive rates were 43.25% and 43.11%, between male and female two have no significant difference (P 0.05); at the age of 21-30 (48.82%), 31-40 (50.86%), 41-50 (42.86%) HEV antibody positive rate was significantly higher than that of 10-20 people (23.08%), 51 years old (32.89%) group (P0.05). Pigs piglets (1 month old -2 months), pigs (2 month old -6 months), pigs (6 month old above) the positive rate of HEV IgG antibody were 46.85%, 57.06%, the positive rate of 65.42%. among different age groups of anti HEV IgG differences and the difference was significant (P (0.05). The dogs in the city of stray dogs, family backyard dogs and farm dogs HEV IgG antibody positive rates were 59.18%, 13.16% and 10.71%, dog family farms scattered The difference in dogs HEV and stray dogs HEV IgG antibody positive rate significantly (P0.05). The German shepherd dog, Kunming dog and sled dogs, among other varieties and between the sled dogs and other varieties of HEV IgG antibody positive rate was significant difference (P0.05).1 years of age and 1-5 years old, 5-10 years old, 10 years old of canine HEV IgG antibody positive the rate of significant differences (P0.05). The results show that the Kunming area people, pigs, dogs have hepatitis E epidemic, population - pigs and dogs HEV positive rate of IgG is related with age, people aged 21-50 HEV IgG antibody positive rate relative to 10~20 years old, 51 years old is higher; the positive rate of swine HEV IgG with age gradually the positive rate of HEV increased; canine IgG antibodies with age gradually decreased the investigation of molecular epidemiology of hepatitis E virus.2. in Kunming and the surrounding counties. In order to understand the molecular background of pig farms in Kunming area HEV piglets and the epidemic characteristic, according to the GenBank ORF2 conserved region sequence of HEV, designed and synthesized two pairs of nested PCR primers. 187 fecal samples of piglets were collected from Kunming and the surrounding counties were amplified, amplified PCR fragment about 348bp was positive. The positive rate of 6.95%. in Luquan County, Jiangchuan County, the positive rate of Fumin County were 4.54%, 8%, 9.52%. the amplified fragment of the nucleotide homology and phylogenetic analysis, 9 strains of porcine HEV 348bp amplified fragment nucleotide homology was 87.1%-99.4%, and HEV I, II, III type homology were 77.0%-81.9%, 52.2% ~ 53.6%, 77% ~ 88.2%, the homology with HEV type IV was 77.9% ~ 96.8%. by phylogenetic analysis the detected 9 strains of Porcine HEV strains were HEV genotype. The results show that the scale of pig farms in Kunming province were pigs are infected by HEV and.3. for the genotype of hepatitis E virus infection of porcine liver tissue pathology Observation. In order to understand the swine hepatitis E virus infection of liver tissue pathological changes, immunohistochemical staining and hematoxylin eosin staining of collected from Kunming area farmers market on the sale of 176 samples of liver histopathology were observed. Immunohistochemical staining was used to observe the liver showed a positive rate of HEV the 65.34%. antigen for hematoxylin eosin staining, 51.30% liver tissue inflammatory cell infiltration, 28.70% liver tissue fiber connective tissue hyperplasia, 20.87% liver liver cell atrophy, 19.13% liver the liver cell degeneration, necrosis of liver. The results show that sold in Kunming in the prevalence of HEV infection and experimental study on the corresponding pathological changes of.4. swine hepatitis E virus infection in Mongolian gerbils. In order to explore the possibility of swine hepatitis E virus infection in Mongolian gerbil model, Application of HEV positive swine fecal supernatant by intraperitoneal injection inoculation of gerbil, Meriones unguiculatus regular detection of alanine transaminase in serum (ALT) and aspartate aminotransferase (AST) levels and changes of liver, feces, blood, intestine HEV RNA, histopathological changes and distribution of HEV in the liver. The results showed that gerbil pigs inoculated with ALT and AST HEV after 7 days in serum were increased, then began to decrease. But the value of ALT to 35 days after inoculation was not reduced to the level of control group, infection group and control group values as high as 2 times.AST to 35 days after inoculation the basic back to normal range than negative (according to the negative control). Feces and liver were detected in HEV RNA, blood and intestine I can detect HEV RNA. in liver tissue was observed between hepatic lobules of lymphocytic infiltration, degeneration of liver cell granules, focal lymphocytic infiltration, liver cell cords Disorder of bile duct hyperplasia. The late show multiple lymphocytic infiltration, necrosis of liver cells and Kupffer cells increased, hyperplasia of connective tissue fibers. Hui Guan Qu small intestine, kidney, brain, spleen tissues have different degrees of pathological changes. The immunohistochemical results can be seen a strong positive signal in liver cytoplasm. Successfully established experimental Study on gerbil model of swine hepatitis E virus.5. infection of tree of swine hepatitis E virus infection. In order to explore the establishment of swine hepatitis E virus infection tree (?) the possibility of application of HEV model, RNA positive swine fecal supernatant intraperitoneally inoculated trees (?), regular testing tree (?) in serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and changes of liver, feces, blood, intestine, kidney, spleen HEV RNA, histopathological changes. The results showed that the tree (?) HEV 7 days after inoculation of porcine serum ALT and A ST increased, then began to decrease. But the ALT and AST to twenty-eighth days of inoculation or not reduced to the level of control group, infection group than negative control group. High value tree (?) HEV vaccinated pigs after 7 days of feces, blood, liver, small intestine HEVRNA could be detected in the kidney and spleen, I can detect HEV RNA. can be observed in the liver sinus congestion, liver cell degeneration, multiple lymphocytic infiltration, Kupffer cells increased, periportal fibrous connective tissue hyperplasia lesions. The small intestine, kidney and other tissues have different degrees of pathological changes. The establishment of a porcine model of hepatitis E virus infection tree Yun is feasible, laid the foundation for further study on HEV cross species transmission and pathogenesis.

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.63;S855.3

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