【作者】 郑吉春；

【导师】 裴雪涛； 岳文；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目前,全球各种因遗传、肝炎病毒、药物、酒精等引起的急慢性肝炎、肝硬化、肝癌等患者数以千万,这些患者肝功能在不同程度上受到损害,并且可能累及到其它脏器和组织,并影响其功能,从而对患者身体健康造成损害,到晚期不可避免的发展为终末期肝衰竭(End-stage Liver Failure)。而当前对于肝功能衰竭患者的治疗仍然十分困难,传统的保守治疗生存率仅为15%-25%。自1985年进行紧急原位肝移植(emergency orthotropic total live transplantation, EOTLT)治疗以来,重症患者的术后一年的生存率已提高至60%-70%,5年实际存活率达到60%,远优于传统的保守治疗效果。但是EOTLT存在以下不足之处:(1)供体短缺、器官不能较长期地保存、手术操作复杂;(2)患者术后需终生使用免疫抑制剂;(3)部分可能通过自身肝再生而康复的患者失去免于肝移植而治愈的机会;(4)部分因一般情况差的患者不能在短期内接受EOTLT而丧失治疗机会;(5)当一些由于肝功能恶化迅速的病例紧急移植了ABO不符或供肝的条件欠佳时,往往出现移植肝无功能,不得不面临进行再次肝移植的危险状况。以上原因使紧急原位肝移植治疗肝功能衰竭的应用受到一定的限制。近年来,国内外学者为解决上述问题积极地寻找新的治疗方法治疗肝功能衰竭,例如:紧急辅助肝移植(emergency auxiliary liver transplantation, EALT)和肝细胞的紧急辅助移植等,以期达到替代原位肝移植或延缓肝衰竭的种种致命并发症并等待合适的供体的目的。而生物人工肝( bioartificial liver system, BAL )的研究进展为利用肝干细胞移植和肝组织工程治疗肝功能衰竭提供了诱人的前景。生物人工肝是专指人工培养的肝细胞为基础构体的体外生物反应系统。生物人工肝在体外与患者循环通路相连,由人工部分(生物反应器)和生物部分(肝细胞)组成。其基本原理是将体外培养的肝细胞置于体外循环装置(生物反应器)中,患者血液(血浆)流过生物反应器时,通过容器内的纤维素半透膜或直接与肝细胞进行物质交换,从而达到人工肝支持肝功能的目的。生物人工肝最初是为急性肝功能衰竭设计,利用它临时支持肝脏解毒功能,等待肝脏自身功能的恢复。现代的人工肝技术已发生巨大变化,长期支持肝脏多种功能包括解毒、合成、分泌及凝血等已成为可能,生物人工肝主要适用于肝功能衰竭,特别是急、慢性重型肝功能衰竭患者;其次还适用于肝脏移植术前支持,术后肝脏无功能,极量肝切除后肝功能不全等。临床研究发现,生物人工肝治疗能够改善患者意识状态和血生化指标。但生物人工肝设计中常常忽略了胆汁酸排除问题。胆固醇在肝中转化成胆汁酸是胆固醇在体内代谢的主要去路,分泌胆汁酸是肝细胞特有的生理功能,不仅对胆固醇的溶解和食物脂类的消化、吸收具有重要意义,而且是体内有毒代谢产物,药物,激素等的排除途径,如胆固醇、胆红素等。胆汁酸的生物合成有经典途径和替代途径两种合成途径,大部分胆汁酸由经典途径合成。胆固醇7α羟化酶(cholesterol 7αhydroxylase, CYP7A1)是肝细胞生物合成胆汁酸经典途径的起始酶,也是限速酶,其表达水平的高低反映了胆汁酸合成的快慢。生物人工肝中大量具有生物活性肝细胞的规模化培养,将不可避免的涉及胆汁酸的合成、分泌和排泄问题,由于没有正常的胆道供胆汁排出,随着时间的推移,肝细胞生长环境中会发生“胆汁淤积”,因此胆汁酸只能靠残留的受损肝脏或直接经过血液循环排出。生物人工肝内的肝细胞不断产生胆汁酸盐,它们多数具有与蛋白结合的毒性,并进一步增加了血中胆汁酸盐浓度。为了避免植入患者体内的生物人工肝中肝细胞分泌胆汁酸对肝细胞本身的毒害作用及可能造成的其他不良影响,本课题中我们应用RNAi技术获得低表达CYP7A1的肝细胞,课题研究的具体内容及实验结果如下:一、构建胆固醇7α羟化酶慢病毒RNA干涉载体已有的研究表明CYP7A1基因敲除的小鼠胆汁酸分泌明显减少。那么对于人CYP7A1基因,如果用RNAi技术,是否也能减少胆汁酸的分泌呢?我们首先利用生物信息学手段从GenBank中查找人CYP7A1的mRNA序列(NM_ 000780),根据慢病毒RNA干涉载体pSicoR的要求,用Gene specific siRNA selector设计以CYP7A1编码区为靶点的特异性CYP7A1 siRNA三对序列,成功构建CYP7A1的慢病毒RNA干涉载体pSicoR-CYP7A1,并获得了稳定转染pSicoR-CYP7A1的人肝细胞(L-02 cells)。二、干涉效率的检测我们通过RT-PCR、实时荧光定量PCR、Western-blot和胆汁酸分泌功能检测等实验方法,以野生型L-02细胞和仅转染空载体pSicoR的L-02细胞作对照,检测了转染慢病毒干涉载体pSicoR-CYP7A1的L-02细胞目的基因CYP7A1在mRNA水平和蛋白质水平的干涉效率。半定量PCR结果显示:野生型和转染了空载体pSicoR的L-02细胞均有CYP7A1的表达,相对表达量基本一致,而转染了慢病毒干涉载体pSicoR-CYP7A1-1的L-02细胞CYP7A1的表达量明显减低;实时荧光定量PCR结果显示转染慢病毒干涉CYP7A1载体1的L-02细胞CYP7A1基因的表达受明显抑制,与对照组相比,转染慢病毒干涉pSicoR-CYP7A1-1的L-02细胞表达量仅为野生型L-02细胞表达量38.08%,即干涉效率为61.92%,为转染pSicoR空载体的L-02细胞表达量的44.81%,干涉效率为55.19%,均具有显著差异(p<0.05);Western-blot结果发现,转染pSicoR-CYP7A1-1载体的L-02细胞检测到CYP7A1蛋白与转染空载体pSicoR对照组相比明显降低,仅为其40.05%,比野生型L-02细胞也低,仅为其35.07%,可见通过RNA干扰有效抑制了肝细胞CYP7A1蛋白的表达,结果与实时荧光定量PCR一致。用CCK-8法绘制野生型、转染空载体pSicoR对照组及转染pSicoR-CYP7A1-1三种L-02细胞的生长曲线,并检测了各自的细胞培养上清的胆汁酸含量,结果表明转染pSicoR-CYP7A1-1载体的L-02细胞的细胞增值能力和前两者无显著差异,但胆汁酸分泌量明显低于前两者。综上所述,本研究中我们应用siRNA技术成功构建CYP7A1的慢病毒干涉载体pSicoR-CYP7A1-1,转染人肝细胞L-02后下调(knock down, KD)CYP7A1的表达水平,并有效降低肝细胞的胆汁酸分泌,为生物人工肝进一步的实验研究和临床应用奠定基础。更多还原

【Abstract】 At present, there are thousands of liver patients suffering from acute and chronic hepatitis, liver cirrhosis and liver cancer for heredity, hepatitis virus infection, drugs and alcohol abuse in the world. These liver damage at the different degree always associated with other organs damage, will develop inevitably into terminal stage liver failure, endangering their health and lives.It is difficult to cure liver failure patients completely, with a survival rate of orthodox expectant treatment only between 15%- 25%. A survival rate is increased by 60%-70% and 60% for one year and five years, respectively after emergency orthotropic total live transplantation (EOTLT) since1985. However, some limitation exists with emergency orthotropic total live transplantation (EOTLT): (1) Donator liver is of limit, devoid of longer preservation and needed much complicated operations. (2) Patients after operation need to take immunosuppressant lifetime. (3) Part of patients might lose the chance of cure by self-liver- regeneration. (4) Part of patients could not accept EOTLT in short term because of a bad general state of health. (5) Graft liver nonfunction often occurs when some patients whose liver functions promptly deteriorate are transplant liver which ABO is mismatch or others. These patients have to face with secondary liver transplantation. Taken together, the application of EOTLT is restricted. Recently, lots of researchers are searching for the new methods to treat liver failure. For example: (1) emergency auxiliary liver transplantation (EALT). (2) Emergency auxiliary hepatocytes transplantation. It is expected to replace EOTLT by some new approaches.Bioartificial liver system (BAL) provides a fair perspective for curing patients with liver failure by utilizing liver stem cell transplantation and liver tissue engineering. BAL is an in vitro biological response system in which hepatocytes can be maintained, that is connected with circulation of patients. It is composed of artificial part(bioreactor)and biological part(hepatocytes).BAL is based the exchange between extracorporeal circulation system (bioreactor) in which hepatocytes are cultured and blood plasma of patients directly or indirectly through semi permeable membrane. BAL was initially designed for eliminating cytotoxins temporarily in acute liver failure. A great improvement of BAL has taken place in recent years. It is coming true that maintaining liver function for a long-stage, such as the detoxication, synthesis and secretion of active enzymes and blood clotting. BAL is mainly suited for treatment of acute liver failure, and also plays an important role in pre-treatment and support of liver transplantation. Taken together, BAL can efficiently improve the consciousness and blood biochemical indicator of patients based on clinical studies.The conversion of cholesterol to bile acids in the liver is a major route for the elimination of cholesterol from the body. The secretion of bile acids is a specific physiologic process of hepatocytes. Bile acids are not only essential for digestion and absorption of lipids, but also are important for eliminating environmental toxins, carcinogens, drugs, and their metabolites such as cholesterol, bilirubin, and hormones. Bile acids are synthesized via the classic pathway and alternate pathway by which few bile acids are produced. The cholesterol 7αhydroxylase (CYP7A1) located in endoplasmic reticulum initially catalyzes the conversion of cholesterol to bile acids as a rate-limiting enzyme in the classic pathway. The expression level of CYP7A1 reflects the amount of bile acids synthesized.The bioartificial liver system (BAL) support liver failure maintenance therapy and strive for liver transplantation. But BAL has no biliary tract, bile acids from hepatocytes have no way to secret, which inevitably lead to cholestasis in BAL. The more hydrophobic bile salts may be cytotoxic acid and when present in abnormally high concentrations. To avoid the cytotoxic effect of bile acids to hepatocytes in BAL, we designed to reduce the expression of CYP7A1 in Human Hepatic cell lines-L-02. Three small interference RNA expression vectors of CYP7A1 were constructed and transfect into L-02 cells via lentiviral system. The efficiency of virus transfection was assayed by the level of expression of enhanced green fluorescence protein (eGFP) visualized by fluorescence microscope and the transfected cells were sorted for selecting a high and stable expression of eGFP-L-02 cells by fluorescence-activated cell sorting (FACS). The effects of RNA interference on CYP7A1 was identified by real time quantitive PCR and western blotting. The expression of CYP7A1 in L-02 cells transfected with pSicoR-CYP7A1-1 lentivirus is down regulated up to 31.2% and 34.1%, compared with that in untransfected L-02 cells or control lentivirus transfected L-02 cells at mRNA level. And the secretion of CYP7A1 was obviously suppressed after pSicoR-CYP7A1 transfection at protein level by western blot analysis. Taken together, we succeed to down regulate the expression and secretion of CYP7A1 by transfecting lentivirus plasmids of pSicoR-CYP7A1 into L-02 cells. Our system may provide a useful tool to reduce the secretion of bile acids in human hepatocytes and may play an important role in clinical usage of bioartificial liver.更多还原