本文關(guān)鍵詞：樹鼩慢性感染乙肝病毒過程中肝組織病理學(xué)觀察及枯否細胞變化意義的探討，由筆耕文化傳播整理發(fā)布。

        目的乙型肝炎病毒(Hepatitis B virus,HBV)感染是全球面臨的重大公共衛(wèi)生問題,是引起急慢性肝炎、肝硬化及肝癌的主要病原。建立一種簡便、有效、穩(wěn)定的HBV感染動物模型對于探索其感染機制、尋找有效的防治方法、研制抗HBV藥物都具有重要的意義。本課題組前期研究證實,新生期接種HBV的樹鼩能夠長期感染HBV,并且HBV能夠在樹鼩體內(nèi)穩(wěn)定復(fù)制和長期存在。本研究擬在課題組以往研究的基礎(chǔ)上,一方面繼續(xù)觀察慢性感染HBV的樹鼩肝組織的病理組織學(xué)改變進展、分析和比較其與人類慢性感染HBV后的病理變化過程的異同,另一方面,為尋找樹鼩HBV感染率的影響因素,本課題還對慢性感染HBV的樹鼩肝組織的枯否細胞數(shù)量、功能及其可能的調(diào)節(jié)因子進行檢測,以探索枯否細胞在樹鼩感染HBV慢性化過程中的意義,為進一步優(yōu)化樹鼩模型提供線索。方法動物分為3組：A組,6只,為前期實驗已確定慢性感染HBV(接種后1-6年)的樹鼩；B組,3只,為前期實驗疑似慢性感染HBV的樹鼩(接種后3-4年)；C組,4只,為未接種HBV的正常對照樹鼩。全部動物定期抽血和進行肝活檢手術(shù),采集的血清和肝組織標(biāo)本分別作以下兩部分研究。第一部分,分析慢性感染HBV的樹鼩肝組織病理學(xué)變化,主要內(nèi)容有：1.檢測HBV感染指標(biāo),包括應(yīng)用ELISA/TRFIA方法定性/定量檢測HBV血清免疫學(xué)標(biāo)志(兩對半)、應(yīng)用FQ-PCR定量檢測血清和肝組織的HBVDNA水平、應(yīng)用免疫組織化學(xué)方法檢測肝組織中的HBsAg和HBcAg陽性肝細胞。2.觀察肝組織病變,包括對組織切片應(yīng)用HE染色,結(jié)合網(wǎng)狀纖維、Masson等特殊染色及透射電鏡等方法,觀察肝臟病理組織學(xué)及細胞超微結(jié)構(gòu)的改變,評價病變級別；同時,通過Ki67、P53和Cyclin D1免疫組化染色,評價肝細胞增殖水平。第二部分,分析慢性感染HBV的樹鼩肝內(nèi)枯否細胞的功能和數(shù)量變化,并分析其可能的影響因子。主要內(nèi)容有：1.檢測樹鼩肝組織中枯否細胞數(shù)量變化,即應(yīng)用流式細胞術(shù)檢測從樹鼩肝活檢組織分離的肝非實質(zhì)細胞中CD163+細胞的比例,以及用免疫組化染色方法原位檢測樹鼩肝組織中的枯否細胞數(shù)量。2.檢測樹鼩肝組織中枯否細胞的功能,即對手術(shù)切取的樹鼩肝組織進行枯否細胞的分離、鑒定和原代培養(yǎng),然后檢測枯否細胞的遷移功能、吞噬功能及合成促炎癥介質(zhì)TNF-a的功能。其中,遷移功能的檢測為以原代培養(yǎng)的枯否細胞開展細胞遷移實驗,以及用免疫熒光染色法檢測枯否細胞內(nèi)的與遷移能力有關(guān)的細胞骨架成分微絲蛋白及微管蛋白的表達水平；吞噬功能的檢測為應(yīng)用溶酶體熒光探針檢測原代培養(yǎng)的枯否細胞內(nèi)溶酶體數(shù)量,以及用免疫組化方法原位檢測肝組織中溶菌酶的表達情況；合成促炎癥介質(zhì)TNF-a功能的檢測為應(yīng)用VVestern blot及實時熒光定量RT-PCR方法,分別檢測肝組織TNF-a蛋白及原代培養(yǎng)的枯否細胞TNF-a mRNA表達水平。3.檢測樹鼩肝組織中可能影響枯否細胞功能的因子,即應(yīng)用實時熒光定量RT-PCR方法,檢測原代培養(yǎng)的枯否細胞TLR-2及TLR-4等基因mRNA表達水平。結(jié)果第一部分,1.A組樹鼩呈現(xiàn)HBV持續(xù)感染狀態(tài),6只動物于最后一次檢測(接種HBV后1-6年)仍全部顯示血清HBsAg和HBV DNA陽性、肝組織中有HBsAg陽性肝細胞；B組僅1只動物為血清HBsAg馬陽性,其余指標(biāo)陰性；C組全部動物的HBV感染標(biāo)志均為陰性。2.A組各動物肝組織均有不同程度的慢性肝炎改變,表現(xiàn)為散在或彌漫分布的肝細胞水腫、脂肪變性、嗜酸性變,以及匯管區(qū)炎癥——匯管區(qū)出現(xiàn)較明顯的以淋巴細胞為主的炎細胞浸潤,伴隨小膽管增生；其中1只感染時間最長的動物(A1,接種HBV后6年)還出現(xiàn)小葉內(nèi)多處壞死灶融合甚至橋接壞死及纖維化、大細胞性不典型增生等組織學(xué)改變。A組樹鼩的肝活檢組織學(xué)評分均值為5.33±3.93,顯著高于B組(1分,P=-0.018)和C組(0分,P=0.008)；秩相關(guān)分析結(jié)果表明,組織學(xué)評分與動物HBV感染時長及其血清HBV DNA拷貝數(shù)呈顯著的正相關(guān)關(guān)系(與感染時長的關(guān)系為r=0.808、P=0.000,與血清HBVDNA拷貝數(shù)的關(guān)系為r=0.494、P=0.014)。電鏡下,A組動物的肝細胞超微結(jié)構(gòu)變化表現(xiàn)為部分肝細胞腫大、表面微絨毛腫脹、溶酶體數(shù)量增多、胞質(zhì)中糖原顆粒多少不均、線粒體腫脹變大、內(nèi)質(zhì)網(wǎng)擴張或形成不規(guī)則囊泡。免疫組化檢測肝組織中的細胞增殖因子結(jié)果顯示,A組動物的Ki67、P53和Cyclin D1表達水平均顯著高于B組(分別為P=0.043、P=0.039和P=0.016)和C組(分別為P=0.050、P=0.021和P=0.007)；秩相關(guān)分析結(jié)果表明,肝組織的Ki67、P53及Cyclin D1的表達水平與肝組織學(xué)評分、血清HBV DNA考貝數(shù)及肝組織HBV DNA拷貝數(shù)均有顯著的正相關(guān)關(guān)系(與組織學(xué)評分的關(guān)系,分別為r=0.829和P=0.000、r=0.815和P=0.001、r=0.913和P=0.000；與血清HBV DNA拷貝數(shù)的關(guān)系,分別為r=0.868和P=0.000、r=0.919和P=0.000、r=0.874和P=0.000；與肝組織HBV DNA拷貝數(shù)的關(guān)系,分別為r=0.744和P=0.004、r=0.846和P=0.000、r=0.876和P=0.000)。第二部分,樹鼩肝組織分離枯否細胞的產(chǎn)量約為1.2±0.2x106個細胞/g肝臟,細胞活力為90%,純度為85%。對原代培養(yǎng)的枯否細胞以及對肝組織中的枯否細胞原位檢測結(jié)果顯示：1.A組樹鼩肝內(nèi)枯否細胞數(shù)量增加——經(jīng)流式細胞術(shù)測定,A、B、C三組CD163+細胞占肝非實質(zhì)細胞的比例分別為89.80±0.36%、77.92±1.22%及77.97±1.13%,A組顯著高于B組(P=0.034)和C組(P=0.021)；免疫組化檢測結(jié)果顯示,A、B、C三組肝組織內(nèi)CD163陽性細胞計數(shù)分別為39.92±6.61、24.73±3.85和21.78±2.31個/高倍視野,A組顯著高于B組(P=0.028)和C組(P=0.010)；秩相關(guān)分析結(jié)果顯示,枯否細胞的數(shù)量與動物感染HBV時長及血清HBV DNA拷貝數(shù)存在正相關(guān)關(guān)系(與感染時長的關(guān)系為r=0.737、P=0.000,與血清HBV DNA拷貝數(shù)的關(guān)系為r=0.497、P=0.013)。2.A組樹鼩枯否細胞功能下降——經(jīng)細胞遷移實驗檢測,A、B、C三組的遷移細胞數(shù)均數(shù)分別為6.50±0.93、16.13±0.70和16.25±0.87,A組顯著低于B組(P=0.034)和C組(P=0.021)；A組枯否細胞的微絲蛋白及微管蛋白的熒光強度、細胞溶酶體熒光強度、溶菌酶免疫組化陽性表達的細胞計均低于B組和C組(微絲蛋白,P=0.034和P=0.021；微管蛋白,P=0.034和P=0.021；溶酶體熒光,P=0.034和P=0.021；溶菌酶,P=0.020和P=0.011)；A組枯否細胞TNF-a mRNA的表達水平低于B組和C組(P=0.034和P=0.021),肝組織的TNF-a蛋白質(zhì)表達水平也支持上述結(jié)果。秩相關(guān)分析結(jié)果顯示,溶菌酶陽性細胞計數(shù)與動物感染HBV時長及其血清HBV DNA拷貝數(shù)均存在顯著的負(fù)相關(guān)關(guān)系(與感染時長的關(guān)系為r=-0.890、P=0.000；與血清HBVDNA拷貝數(shù)的關(guān)系為r=-0.601、P=0.002),而TNF-a mRNA表達量與動物肝組織的HBV DNA拷貝數(shù)也呈負(fù)相關(guān)關(guān)系(r=-0.622、P=0.041)。3.檢測可能影響枯否細胞功能的因子的結(jié)果顯示,A組TLR-2mRNA及TLR-4mRNA表達水平均低于B組(P=0.034及P=0.021)和C組(P=0.034及P=0.021)。秩相關(guān)分析結(jié)果顯示,TLR-2mRNA及TLR-4mRNA表達水平均與動物的肝組織HBV DNA拷貝數(shù)呈負(fù)相關(guān)關(guān)系(分別為r=-0.622、P=0.041和r=-0.673、P=0.023)；而該二基因的mRNA表達水平與枯否細胞的其它指標(biāo)則均呈正相關(guān)關(guān)系,如與枯否細胞遷移數(shù)的關(guān)系(分別為r=0.809、P=0.003和r=0.845、P=0.001)、與溶酶體密度的關(guān)系(分別為r=0.745、P=0.008和r=0.609、P=0.047),以及與TNF-a mRNA表達水平的關(guān)系(分別為r=0.782、P=0.006和r=0.739、P=0.010)結(jié)論1..慢性感染HBV的樹鼩在血清病毒學(xué)指標(biāo)、組織病理學(xué)改變、超微結(jié)構(gòu)特點及病程發(fā)展等方面與人類慢性感染HBV后的改變有諸多相似之處,此為其他同類動物模型所不具備的特點,是動物感染HBV研究領(lǐng)域的首次報道,提示樹鼩感染HBV模型更適用于人類感染HBV相關(guān)的基礎(chǔ)和臨床研究。2.慢性感染HBV的樹鼩肝組織損傷、肝細胞增殖指數(shù)分別與其感染HBV病程的長短、血清HBV DNA拷貝數(shù)呈顯著的正相關(guān)關(guān)系,提示HBV在宿主體內(nèi)的持續(xù)感染及復(fù)制可促進肝組織慢性病變的發(fā)展；對這些慢性感染HBV的樹鼩繼續(xù)進行觀察,將有可能證明HBV是肝癌的獨立誘發(fā)因素之一。3.慢性感染HBV的樹鼩肝臟枯否細胞數(shù)量增加,但該細胞的遷移功能、吞噬功能及合成促炎癥介質(zhì)TNF-a等功能均下降,后者可能由該細胞表達TLR-2mRNA和TLR-4mRNA的水平下降所致；樹鼩感染HBV的病程與其肝內(nèi)枯否細胞的數(shù)量及功能變化顯著相關(guān)。此系列結(jié)果提示,枯否細胞在宿主感染HBV的慢性化過程中可能起一定的調(diào)節(jié)作用。

    ObjectiveHepatitis B virus (HBV) infection is a major public health problem in the world. It is the major cause of acute and chronic hepatitis, cirrhosis and liver cancer. A simple, effective and stable HBV-infection animal model is impotent for exploring the infection mechanism, finding the effective prevention and treatment, and developing anti-HBV drugs. Our preliminary studies confirmed that neonatal tree shrews inoculated with HBV can become chronically infected with HBV, and HBV can replicate and exist in vivo stably for long. On the basis of previous studies, one of the purposes of this study is to examine the histopathological changes in the liver tissues of the tree shrews infected chronically with HBV, to compare the similarities with the pathological changes in the human liver of chronic HBV infection. On the other hand, in order to explore the significance of Kupffer cells in the process of HBV infection, this study is going to investigate the changes of Kupffer cells in the liver of tree shrew infected chronically with HBV, including the changes in number, function and the possible regulatory factors of Kupffer cells.MethodsAnimals were divided into three groups. Group A had6tree shrews that were identified as infected with HBV chronically, group B had3tree shrews that were suspected as infected with HBV chronically, while group C had4tree shrews that did not inoculated with HBV and were as normal controls. The serum and liver biopsy were collected regularly from all animals, the samples were then applied for the two-part study as following.Part one is to analyze the histopathological changes in the liver, including:(1) To detect the markers of HBV infection, such as to qualitatively/quantitatively detect the HBV serum immunological markers by the methods of ELISA and TRFIA, to quantitatively detect the level of HBV DNA in serum and liver tissue by FQ-PCR, and to immunohistochemically detect HBsAg and HBcAg in liver tissues.(2) To observation the histopathologic changes in liver tissues, by HE-stained tissues-slides, with some special staining such as reticular fibers stain and Masson’s stain, followed by evaluating the changes. Meanwhile, transmission electron microscopy was used to observation the ultrastructural changes in liver, and immunohistochemical stains for Ki67, P53and Cyclin D1were used to evaluate status of cell proliferation in liver.Part two is to analyze the changes in function and number of the intrahepatic Kupffer cells in tree shrews infected chronically with HBV, followed the isolation, identification and primary culture of Kupffer cells from liver tissue. The tests including:(1) To test the number of Kupffer cells, by flow cytometry to detect the proportion of CD163+cell within the non-parenchymal cells isolated from the tree shrew liver biopsy, as well as by immunohistochemical staining to detect in situ the proportion of Kupffer cells within the liver tissue of the tree shrew.(2) To detect the functions Kupffer cells such as migration, phagocytosis and synthesis of pro-inflammatory mediator TNF-a. The migration function were tested by cell migration assay on primary cultured Kupffer cells, as well as by immunofluorescence staining for cytoskeletal components microfilament and microglobulin of Kupffer cells. The phagocytosis function were tested by lysosomal fluorescent probe to detect the number of lysosomes on primary cultured Kupffer cells, as well as by immunohistochemical staining to detect the lysosomes on liver tissue samples. While the ability for synthesizing the pro-inflammatory mediators were detected by Western blot on liver tissue sample for TNF-a protein, and by real-time RT-PCR on primary cultured Kupffer cells for TNF-a mRNA, respectively.(3) To detect the factors that might affect the functions of Kupffer cell, by applying real-time RT-PCR on primary cultured Kupffer cells for the expression of TLR-2and TLR-4at mRNA levels.ResultsThe results of part one showed:(1) Tree shrews in group A presented persistent HBV infection, by showing positive HBsAg and HBV DNA in their serum as well as in liver tissues, which had lasted longer than3years after HBV vaccination. Only one animal in group B occasionally showed weakly positive serum HBsAg, while the remaining markers were negative. All animals in group C group were negative for the all markers of HBV infection.(2) Animals in group A showed different levels of chronic hepatitis change, such as scattered or diffuse distribution of liver cell edema, fatty and eosinophilic degeneration, as well as inflammation of the portal area which appeared obvious lymphocyte infiltration, accompanied by small bile duct hyperplasia. One animal (number A1, had been infected with HBV longer than six years) showed multiple necrosis which even fused to form bridging necrosis and fibrosis, as well as large cell dysplasia and other histological changes. The hepatic histological score of the animals in group A was5.33±3.93, which was higher than that in group B (1score, P=0.018) and group C (0score, P=0.008).Correlation analysis showed that the histological score was positively correlated with the duration of infection (r=0.808and P=0.000), and with the HBV DNA level in serum (r=0.494and P=0.014). Under electron microscope, ultrastructure changes in the liver cells of animal in group A were cell swell, microvilli swelling, lysosomal increase, uneven number of glycogen granules in the cytoplasm, mitochondrial swelling, endoplasmic reticulum expansion or the formation of irregular vesicles. Immunohistochemical detection for cell proliferation factor showed that the expression levels of Ki67, P53and cyclin D1of group A group were all higher than those in group B (P=0.043, P=0.039and P=0.016, respectively) and group C (P=0.050, P=0.021and P=0.007, respectively). Correlation analysis showed that Ki67, P53and cyclin D1expression levels in liver tissue were significantly and positively correlated with the liver histological scores (r=0.829and P=0.000, r=0.815and P=0.001, r=0.913and P=0.000, respectively), serum HBV DNA copy number (r=0.868and P=0.027, r=0.919and P=0.000, r=0.874and P=0.000, respectively), and the HBV DNA copy number in liver tissues (r=0.744and P=0.004, r=0.846and P=0.000, r=0.876, P=0.000, respectively).The results of part two showed that the harvest of Kupffer cell from tree shrew liver tissue were1.2±0.2×106cells/g liver, the cell viability was90%and the purity was85%. The results of tests on Kupffer cells in primary culture, as well as of the in the liver tissue showed:(1) The number of Kupffer cells increased in the tree shrews of group A. Measured by flow cytometry, CD163+cells within the non-parenchyma cells of group A, B, C were89.80±0.36%、77.92±1.22%and77.97±1.13%, respectively. The number of group A was higher significantly than that of group B (P=0.034) and group C (P=0.021). Immunohistochemistry test results showed that the CD163-positive cells in group A, B and C were39.92±6.61,24.73±3.85and21.78±2.31/high power field, respectively. The number of group A was higher than that of group B (P=0.028) and group C (P=0.010) too. Correlation analysis showed that the number of Kupffer cells was positively correlated with the duration of animals infected with HBV (r=0.737and P=0.000), as well as with the level of HBV DNA in serum (r=0.479and P=0.013).(2) Function of Kupffer cells reduced in the animals of group A. The average numbers of migrating cells in group A, B and C were6.50±0.93,16.13±0.70and16.25±0.87, respectively. The number in group A was lower than that in group B (P=0.034) and C group (P=0.021). Meanwhile, group A showed lower levels than group B and C in the fluorescence intensities of microfilament protein (P=0.034and P=0.021respectively), of microtubule protein (P=0.034and P=0.021respectively), of lysosomal (P=0.034and P=0.021respectively), and in the number of lysozyme-positive cells detected by immunohistochemistry (P=0.020and P=0.011respectively). Also, the TNF-a mRNA expression level of Kupffer cell in group A was lower than that in group B (P=0.034) and group C (P=0.021), while the TNF-a protein expression levels in liver tissue tested by Western blot showed the similar tendency. Correlation analysis showed that the number of lysozyme-positive cells was significantly and negatively correlated with the duration of animal infected with HBV (r=-0.892and P=0.000), the level of HBV DNA in serum (r=-0.601and P=0.002). Meanwhile, expression level of TNF-a mRNA animal was negatively correlated with the levels of HBV DNA in liver tissues (r=-0.622and P=0.041).(3) Result of the detection on the factors that might affect the function of Kupffer cell showed expression levels of TLR-2mRNA and TLR-4mRNA in group A were lower than those in group B (P=0.034and P=0.021, respectively) and C group (P=0.034and P=0.021, respectively). Correlation analysis showed that the expression levels of TLR-2mRNA and TLR-4mRNA were negatively related with the level of HBV DNA in liver tissues (r=-0.622and P=0.041, r=-0.673and P=0.023, respectively). However, the expression levels of TLR-2mRNA and TLR-4mRNA were positively related with some other factors, such as the number of migrated Kupffer cells (r=0.809and P=0.003, r=0.845and P=0.001, respectively), the density of lysosomes relationship (r=0.745and P=0.008, r=0.609and P=0.047, respectively), and the expression level of TNF-a mRNA (r=0.782and P=0.006, r=0.739and P=0.010, respectively).Conclusions1. Tree shrew with chronic infection of HBV is similar to human beings based on the serum virological indicators, histopathological changes, ultrastructural characteristics and progression of infection. These characters are unique in tree shrews than other animals. Therefore, tree shrews can be used as a research model of chronic HBV infection.2. In tree shrews with chronic HBV infection, liver histology score, liver cell proliferation index, infection time and serum HBV DNA copy number were positively correlated. These results suggested that the liver histological changes in tree shrews with chronic HBV infection correlated with persistent HBV replication and infection 3. Kupffer cells number increase in tree shrews with chronic HBV infection; however, the cell migration, phagocytosis function and TNF-α synthesis function declined. It suggested that the course of the tree shrews chronic HBV infection correlated with Kupffer cell volume and function changes4. Decreased expression of TLR-2and TLR-4mRNA was observed in Kupffer cells from the tree shrew with chronic HBV infection. It is possible reason that Kupffer cells showed a decrease of migration function, phagocytosis and TNF-a synthesis function.

樹鼩慢性感染乙肝病毒過程中肝組織病理學(xué)觀察及枯否細胞變化意義的探討

中英文縮略詞4-5摘要5-10ABSTRACT10-16前言17-34材料與方法34-62結(jié)果與討論62-113    1、第一部分結(jié)果62-77    2、第一部分討論77-85    3、第二部分結(jié)果85-105    4、第二部分討論105-113結(jié)論113-114問題與展望114-115參考文獻115-125綜述：固有免疫在乙型肝炎病毒感染中的作用125-142    參考文獻136-142致謝142-143攻讀學(xué)位期間發(fā)表的學(xué)術(shù)論文143

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