| Hepatic failure,a clinical manifestation for various hepatic diseases at the end-stage,Seriously threatens the health of human.Liver transplantion is the only effective treatment for hepatic failure.Unfortunately,its application is limited by donor organ availability and the costly expense of the operation.So there is a need to develop artificial liver support system as bridging procedures to sustain patients with subacute or acute hepatic failure. On the one hand,it can provide time to find a suitable donor liver,on the other hand,to gain time for hepatocyte regeneration.Although physical-based artificial liver support system has certain effectiveness for patients with hepatic disease,it still cannot replace the complex function of liver.With the developing of cell biology and tissue engineering,the extracorporeal bioartificial liver support system(EBLSS) in the treatment of hepatic failure in the application, is increasingly subject to public attention.The primary hepatocytes of human are the ideal material for bioartificial liver support system, but proliferation of the primary hepatocytes is extremely difficult. Although animal-derived liver cells are able to obtain substantial, they have a great deal of risks,including zoonotic diseases(such as pig endogenous retrovirus infection),immune reactions induced by heterologous proteins.So it is a good way to establish a immortalized hepatocyte line to solve the problem of the source of hepatocytes.In this study,we transfected the hTERT(human telomerase reverse transcriptase)into adult hepatocytes(HL-7702) to establish a new immortalized adult hepatocyte line(pLN/HL-7702)and we did some research and contrast on the morphous,proliferation,fuction between two types of the hepatocyte lines,and we also did primary research on tumorigenicity of the new cell line.We hope to lay an experimental basis to resolve the sources of bioartificial hepatocytes.We transfected the hTERT into adult hepatocytes(HL-7702) by using the retroviral vector pLNCX2-hTERT.And then we detected gene by RT-PCR,and detected relevant the protein by western-bolt and indirect immunofluorescence assay.Morphous and ultrastructure of cells was observed by light microscope and electron microscope. Proliferation of the cells was examined by MTT staining method;the expression of some functional genes was detected by RT-PCR. Cytochrome P450 was detected by using western-bolt analysis. Tumorigenicity was test by the soft agar test and tumorigenicity test of nude rats.The results:1.Gene expression in liver cells and associated protein can be detected by RT-PCR,western-bolt,and indirect immunofluo-rescence assay;2.Morphous and ultrastructure of cells was observed by light microscope and electron microscope and there was no change after we transfected hTERT into HL-7702;3. Proliferation experiments suggested that the capacity of proliferation was significantly enhanced after transfecting the hTERT into HL-7702;4.Expression of functional gene could be detected by RT-PCR;5.The examination of hepatocytes supernatants showed that the general biochemical functions of cells were not infulunced by transfection;6.Cytochrome P450 was detected by using western-bolt analysis;7.The soft agar test and tumorigenicity test of nude rats showed that the new immortalized adult hepatocyte line (pLN/HL-7702) did not show the tumorigenicity.Discussion:In this research we successfully established a new immortalized hepatocytes line pLN/HL-7702.Its proliferative capacity in vitro was improved than hepatocytes line HL-7702.There is no difference in the morphology and biological function between pLN/HL-7702 and HL-7702.Cytochrome P450 was detected by western-bolt.In the primary research we did not find tumorigenicity of pLN/HL-7702.That Suggests it is much easier to culture mass hepatocytes in vitro for the new immortalized hepatocytes line pLN/HL-7702.It can be probably used as a safe and effective source of the bioartificial hepatocytes.
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