【作者】 梁旭竞；

【导师】 杨冬华；

【作者基本信息】 暨南大学 ， 消化内科， 2009， 博士

【摘要】 生物型人工肝(bioartificial liver,BAL)治疗需要合适的种子细胞,正常人肝细胞是BAL较为理想的细胞源。然而肝细胞属于终末分化细胞,体外生存期短、来源受限,因此在应用方面受到限制。永生化肝细胞具有增殖优势、高分化功能及培养费用低等特点,有着广阔的应用前景。然而,即使在最佳的培养条件下,单一细胞系也不能完成肝细胞所有的功能。新近报道人脐带间充质干细胞(humanumbilical cord mesenchymal stem cells,hUCMSCs)具有多向分化的潜能,且容易获得、取材方便,无伦理方面争议,极可能诱导分化成肝细胞,因此,hUCMSCs可能是BAL一种新的细胞来源。然而MSCs与机体其它正常体细胞一样,也存在Hayflick界限,即在经过有限次的细胞传代后,就会停止增殖,且发生衰老和死亡。人端粒酶是一种由RNA和蛋白质两种组份构成的核糖核蛋白复合体,其中RNA组份是端粒酶复制时的模板,蛋白质组分主要是具有反转录酶活性的端粒酶催化亚基(human telomerase reverse transcriptase subunit,hTERT),其功能是利用自身RNA为模板,催化合成端粒DNA并加到染色体末端,使端粒延长。端粒的长度和细胞分裂时端粒损失的速度决定了细胞的衰老和寿命,通过诱导端粒酶的产生,端粒的长度可以得到修复和保持,使细胞的生命延长甚至永生。本研究拟通过人端粒酶逆转录酶催化亚基(hTERT)慢病毒表达载体构建hTERT-L02肝细胞和hTERT-hUCMSCs两种细胞,并对它们的生物学特性及功能特征进行研究,探讨利用新的肝细胞来源应用于BAL的价值,为共培养体系提供细胞学基础,同时探究体外分离小块肝组织培养正常成人肝细胞的方法,为研究成人原代肝细胞的永生化提供细胞学基础。本文主要分为两大部分:(1)利用慢病毒载体将人端粒酶逆转录酶催化亚基(hTERT)转染人肝细胞并对其生物学特性及功能特征进行研究,同时探讨体外分离小块肝组织培养正常成人肝细胞的方法;(2)构建转hTERT脐带间充质干细胞,并对其增殖及肝细胞方向分化能力进行检测,同时初步评估其应用安全性。第一部分利用基因重组、定点突变及Gateway等技术制备了表达hTERT基因的重组慢病毒,将其转染人肝细胞L02,通过杀稻瘟筛选获得阳性克隆细胞,分别采用实时荧光RT-PCR,TRAPeze(?) ELISA方法检测转染前后L02肝细胞的hTERT-mRNA水平和端粒酶活性的变化,在此基础上,通过MTT法、流式细胞法及RT-PCR等方法观察其形态和生物学功能的变化,结果显示该细胞在体外培养条件下基本维持肝细胞原有形态,存活期显著延长,增殖能力较强,在生物学功能方面,转染肝细胞同样具有表达和分泌ALB、LDH、ALT、AST及糖原合成的功能且未检测到AFP的表达。成功构建的hTERT-L02肝细胞有望解决人肝细胞来源稀少的难题。与此同时,建立体外分离小块肝组织,培养成人肝细胞的实验体系,采用胶原酶联合分散酶法分离的肝细胞具有操作简单、价格低廉的特点,同时获得的肝细胞量相对较多且存活率较高,为初步研究成人原代肝细胞的永生化提供了细胞学基础。第二部分使用胶原酶消化法分离脐带中的MSCs,进一步培养并鉴定,采用含hTERT的重组慢病毒感染hUCMSCs,建立过表达端粒酶的hUCMSCs,通过光镜、原子力显微镜、MTT等方法观察该细胞基本生物学特性,探讨其向肝系细胞分化的可能性。结果显示转染后的hUCMSCs基本维持MSCs原有形态,存活期显著延长,增殖能力较强,在体外具有原代MSCs相同的向脂肪细胞,成骨细胞分化潜能,且无裸鼠致瘤性,加入细胞因子可成功将hTERT-hUCMSCs向肝样细胞诱导分化,分化的细胞可表达肝细胞表面标志物ALB、AFP、CK18。构建的hTERT-hUCMSCs细胞具有向肝细胞分化的潜能,可能成为BAL和肝细胞移植的新的种子细胞,并且为共培养体系提供了细胞学基础。更多还原

【Abstract】 Appropriate seed cells play an important role in bioartificial liver(BAL) systems as the main biomaterial.Untransformed human cells with hepatic function should respond appropriately to proliferative stimuli,which is an ideal cell source for BAL systems.Although the primary human hepatocytes would be an ideal cell source for BAL,the shortage of hepatocytes and the difficulty to increase the doubling time of human hepatocytes in vitro have limited the clinical application.Immortalized human hepatocyte cell lines might provide an unlimited resource for BAL systems.However, even under the most appropriate environment,the single cell could not fulfill the whole functions of the hepatic cells.Recently,multipotency of human umbilical cord mesenchymal stem cells(hUCMSCs) has widely been shown in vitro.Furthermore, The hUCMSCs are available without ethical considerations and might have an ability to differentiate to hepatocytes.Therefore,hUCMSCs may be a new source for BAL systems.However,just like the other normal somatic cells,MSCs have limited capacity,after which they become senescent-a phenomenon now known as the "Hayflick limit".Telomerase is a ribonucleoprotein consisting of RNA and proteins.The RNA components are templates of telomere replication.The protein components consist of telomerase catalysis subunits and other associated proteins.The telomerase catalysis subunit is also named telomerase reverse transcriptase(TERT)that plays an important role in the replication of telomere repeat sequences and the extension of telomeres. The cellular senescence and the life-span depend on the loss rate of telomeres during each cell division and on the primary length of telomere.It has been demonstrated that telomerase reconstitution,via hTERT-expression can extend the telomere, prolong the life-span of cells and even cause the immortalization of cells.In the present study,we exposed L02 hepatocytes and hUCMSCs to lentiviral vectors coding for hTERT in order to prolong the life-span of cells and even cause the immortalization of cells,and tested the cellular properties and functionalities of the resulting cell lines.Meanwhile,we investigated the application value of the transgenic L02 and hUCMSCs.We also study the isolation and culture methods of primary hepatocytes which provide a basic work for establishing immortalized adult human hepatocytes.This dissertation includes two parts:(1) we exposed L02 hepatocytes to lentiviral vectors coding for hTERT and tested the biological properties and function of the resulting cell lines.Meanwhile,we studied the isolation and culture methods of primary hepatocytes;(2)we constructed hTERT-hUCMSCs,tested the proliferation and the ability of the differentiation into hepatocytes and initially evaluated the safety of hTERT-hUCMSCs.In the first part,we obtained recombinant lentiviruses expressing hTERT gene through molecular biological technology.The L02 hepatocytes were infected with recombinant lentivirus and the stable cells were selected by blasticidin.The hTERT mRNA level was determined by Real-time RT-PCR and the expression of telomerase activity was detected by TRAP- ELISA in the transfeeted cells.The new hepatocytes were observed and evaluated by molecular biological and morphologic methods.The main research results are shown as following:the hTERT-L02 hepatocytes maintained original shape and exhibited prolonged life span,which had similar synthesis function, such as ALB,ALT,AST,LDH and glycogen synthesis.AFP had not been found in the hTERT-L02 hepatocytes.Meanwhile,we also established the isolation and culture methods of primary human hepatoeytes which provide a basic work for establishing immortalized adult human hepatoeytes.Combining eollagenase with dispase digesting method had the advantages of simplified procedure,large quantity of cells.In the second part,we isolated human UCMSCs by collagenase digesting method and characterized them in vitro by measuring the expression of mesenchymal stem cell(MSC) markers,and evaluating their abilities to differentiate into adipocytes and osteocytes. A line of hUCMSCs over-expressed hTERT is constructed by transducting hTERT genes into hUCMSCs with a recombined lentivims.We observed the basic biological characteristics of hTERT-hUCMSCs and invesgated the differentiation potential of hTERT-hUCMSCs into hepatic lineage.We found that the transduced cells had strong ability to proliferation,maintained the original shape and the capacity to differentiate into adipocytes and osteocytes.Moreover,the hTERT-hUCMSCs exhibited no oncogenicity.After exposure to hepatic differentiation media,the hTERT-UCMSCs could differenciate into hepatocyte-like cells,and express markers of hepatic lineage,including albumin,Alpha-fetoprotein, cytokeratin-18.The hTERT-hUCMSCs might be an alternative source for liver-directed cell therapy.更多还原