Study Of Human Immortalized Hepatocytes And Differentiation Of Umbilical Cord Mesenchymal Stem Cells Transfected By HTERT Gene

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.