| Orthotopic liver transplantation (OLT) is the most successful treatment for end-stage liver disease. However, liver transplantation is limited by the shortage of available donor organs. For this problem, liver tissue engineering is a good solution. It is no doubt that the parenchymal cells in the liver are dispensable if you want to build a complete lobular functionally. It is well known that the main cells in the liver are hepatocytes and biliary epithelial cells (BECs). Although BECs constitute only 3% to 5%, they play the key function in the liver, for example bile secretion, adjust the composition of bile, control the flow of bile and transport the bile. Therefore, just like hepatocytes, BECs are essential in liver tissue engineering. But, liver tissue engineering is mainly concentrated on the differentiation of hepatocytes not on BECs at present. With their potential to develop into virtually any cell type, embryonic stem cells (ES cells) might be an ideal source of donor liver cells for cell therapies aimed at restoring or substituting lost hepatic mass in diseased livers.The academic dissertation can be divided into two parts. In part I, we construced a pCK19-Rluc-mRFP double report vector firstly, and then established a hepatic progenitor cells (HPCs) strain transfected by the report vector. In part II, we established a set of standand culture method of mouse ES cells and a mouse ES cells strain stably transfected by the double report vector. In this study, stable transfected liver progenitor cell line and mouse embryonic stem cell line are gotten by genetic modification and some methods are provided to basic research of liver regenerative medicine.This study is mainly include this two parts below.Part I. A pCK19-Rluc-mRFP report vector was construcedIn recent years, the gradually mature of genetic modification technology provided a convenient tool to the study of differentiation. The genetic modification technology can track the differentiation conveniently, so the differentiation changed to study easily. In this study, a expression vector is construced by renilla luciferase and monomeric red fluorescence protein which is controled by CK19 promoter. Second, the GFP positive liver progenitor cells were co-cultured with EPM-PT67 cells which could express the molecule-epimorphin for 5 days. Then, we found that the stable transducted liver progenitor cells'shape were not only transformed and arranged as cord like structure, but also renilla luciferase, red fluorescence protein, RT-PCR and Western-blot were detected. So, these results proved that the liver progenitor cells had been induced to bile duct epithelial cells. The vector enhanced by CK19 promoter can monitor the differentiation of liver progenitor cells in different environment.1.CK19 is a special marker in BECs in liver Hepatic lineage cells lack specific markers in the process of development and differentiation. They only have highly expressed markers which can help us identify cells which one had induced. specific markers play an importment role in affirmance of differentiated cells.Cytokeratins (CKs) are the important marker in epithelial cells, which is related to cell differentiation and cytoskeleton and is belonged to intermediate filament family. CK19, which is high expressed in BECs, is a special marker in BECs. CKs expressed in epithelial cells of many organizations, such as liver, pancreas, bladder. But CK19 is a special marker in BECs in liver. In order to diagnose cholangiocarcinoma earlier and more accurate, Michael Lie-A-Ling selected CK19 among many markers in BECs and constructed pShuttle-CK19-Luc. By measuring the expression efficiency of all kinds of marker, Michael Lie-A-Ling believed that CK19 is a special marker in BECs.2.A pCK19-Rluc-mRFP report vector was construcedCK19 is a special marker in BECs. In the study, the cytokeratin 19 promoter segment was cloned from hepatocellular carcinoma cell line HepG2, the renilla luciferase and a red fluorescence protein's fusion gene was cloned from pcDNA3.1-hrl-mrfp-ttk vector and poly A was cloned from pcDNA3.1 vector. Then the segments were inserted to finish the double report lentiviral vector. Four restriction enzyme digestion shown that there were 3 inserts, 744bp, 1650bp and 225bp, in the vector and sequencing results proved that the 3 inserts were correct.We could get stable transfected cell line from green fluorescent protein (GFP).3.Functional verification of expression vector through WB cells differentiated to BECs after co-cultureWB cells which are gotten from adult male Fisher rats'liver are a kind of diploid epithelial progenitor cells and express the marks of hepatocytes and BECs. WB cells can differentiated to hepatocytes and BECs in vivo and it is a very well liver progenitor cell model in the differentiation of hepatocytes and BECs. Through lentivirus infection we got stable transfected WB cell line which express a expression vector vector. Then, WB cells were induced to BECs through co-cultured with EPM-PT67 cells. The EPM-PT67 cells and the method of co-culture were created by Hailei Yao and Yali Jia in our lab. After co-culture, we found that not only WB cells formed into a ring, but also the result of RT-PCR and Western-blot proved WB cells differentiated to BECs. Compared with control group, we knew that red fluorescence and renilla fluorescence were started by CK19 promoter and the expression of experimental group was 13.6 times than control group through quantitative detection. So, the function of expression vector in cells can be studied easily and the situation of cell differentiation can be reflected conveniently.Part II. Establishment of mouse ES cell strain stably transfected with pCK19-Rluc-mRFPEmbryonic stem cells which are selected from embryonic cells and primordial germ cells have developmental totipotency. ES cells have differentiational potential like early embryo, and it has same feature with embryonic cells and somatic cells. ES cells are used as new materials widely in tissue engineering, animal cloning, mammalian gene expression and regulation and developmental biology. Evans established the mouse ES cells firstly in 1981. We established a set of standand culture method of mouse stem cells on a feeder layer of mouse embryonic fibroblast (MEF) cells.In this study, pCK19-Rluc-mRFP report vector was transfected into mouse embryonic stem cells through lentivirus, and then we enriched the mouse embryonic stem cells which express GFP using flow cytometry technique. The application of the results provides a new method for purificate embryonic stem cells and epithelioid cells which came from embryonic stem cells.So, these results proved that the liver progenitor cells had been induced to bile duct epithelial cells. The vector enhanced by CK19 promoter can monitor the differentiation of liver progenitor cells in different environment. In a word, this lentivirus vector can help us study the differentiation mechanism of liver progenitor cells, and scan the molecules which can do a help in differentiation.Further, this result will provide the seed cells in bioartificial liver, the transplantiation of hepatic oval cells and gene therapy.
|
PDF Full Text Request