| Liver transplantation is currently the only effective treatment for end-stage liver disease. Clinical application of liver transplantation is limited by lack of donor livers. In the past two decades, as an alternative to liver transplantation, clinical hepatocyte transplantation has draw much attention and has achieved a certain effect. However, Resources for the isolation of cells for transplantation are limited and restrict the widespread application of liver cell therapies. The biological studies on stem cell have shown that stem cells divide exponentially, form a new nest, and differentiate into cells owning specific function and rebuild the organization. It is particularly encouraging that embryonic stem cells and hepatic stem cells derived from fetal liver may differentiate into liver precursor cells, mature hepatocytes and bile duct cells in vitro, and in vivo transplantation experiments also observed injury liver repopulation by exogenous stem cells, so that the liver damage on the structure and function is effectively recovered. The success achieved, which fully demonstrates the use of stem cells to human liver disease and the possibility of clinical treatment.The laboratory used human fetal liver of 4.5-6 weeks as the material to isolate stem cells by the method of cloning, then established a cell line named human fetal liver-derived stem cell lines (hFLSC). hFLSC has the following characteristics:①powerful proliferation;②expression of multiple types of molecular markers:not only the liver stem cell-related markers (such as AFP, ALB, CK8, CK18, CK19, GGT, DPPIV, c-Met, Thy-1 and c-kit, etc.), but also the interstitial cells of some molecular markers (such as CD44, CD29, SDF-1, etc.), but it did not express stem cell markers (such as CD44, CD29, SDF-1, etc.);③with multiple potential resistance:it can differentiate into hepatocyte, bile duct cells, fat cells, osteoblasts in vitro;④it can participate in the regeneration of liver damage in mice:using the mice model of acute liver damage induced by carbon tetrachloride(CCL4) with severe combined immunodeficiency, we detected hFLSC cells with green fluorescent protein marker gene. However, due to the defects of acute liver injury model induced by carbon tetrachloride, we are not able to fully evaluate the ability of implantation, homing, repopulation of hFLSC as well as its function. The model of Fumaric acid acetoacetate hydrolase (fuarylacetoacetate hydrolase, FAH) knockout mouse is an ideal animal model of liver injury to evaluate the repopulation of exogenous stem cells, FAH knockout mice has been widely used in the cell transplantation researches, the mice existent extensive and continuous mouse liver injury that caused liver micro-environment which is particularly suitable for the proliferation of transplanted cells. Our researches establishes on the basis of hFLSC, and choose FAH knockout mouse model as the basic animal models to evaluate the repopulation of hFLSC in liver injury, and further explore the biological characteristics of in mice and the possibilities of hFLSC as a cell source for liver cell transplantation, and establish a basis for the basic research and clinical application of hepatic progenitor cells derived from fetal liver.The research is divided into two parts:in the first part of the experiment, by transplantation of hepatocyte isolated from wild-type strains mice into Fah-/-mice, we observe the role of liver regeneration environment of Fah-/- mice in stimulating the proliferation of transplanted hepatocytes, and preparing for the next study that is transplantation of hFLSC. Furthermore, though we knew that the injury of liver cell of Fah-/- mouse is the major factor which cause the proliferation and repopulation of exogenous cells, however, its pathological changes, particularly analyzing the characteristics of liver cell injury by electron microscopy has not been reported. In our research, we obtained the liver of Fah-/- mouse at different time after the withdrawal of drug, then HE staining and electron microscopy were performed on liver section, in order to observe preliminarily the pathological changes of Fah-/- mouse liver after the liver injury. In the second part of the experiment, in order to obtain immunodeficiency FAH knockout mouse strains which is prepared for the latter hFLSC transplantation, the Fah-/- mice was hybridized with Rag2-/- immunodeficient mice, Fah-/- Rag2-/- mouse strain was established, the result of flow cytometry showed that Fah-/- Rag2-/- mouse lose the function of T cells and B cell, it can be used in hFLSC transplantation. HFLSC cells were cultured in vitro and transplanted into Fah-/-Rag2-/-mice through spleen, mouse livers were obtained at different time points of, the implantation, homing and the efficiency of repopulation of hFLSC were detected by the method of immunohistochemistry, the capability of hepatic differentiation is evaluated.The first part of the study show that after the withdrawl of NTBC, Fah-/- mice showed spikes, bowing, body weight decreased significantly mice died after 6-7 weeks after drug withdrawal; and Fah-/- mice with normal feeding NTBC all survive healthily, their weight growth stably, and have normal reproductive capacity. We successfully achieved the liver repopulation of wild-type mouse's hepatocyte in Fah-/-mouse after the withdrawal of the drug. The result of immnuohistochemistry of Fah protein showed that wild-type hepatocyte with the expression of Fah protein was detected in the Fah-/- mice liver after 1 week of liver cell transplantation. As time passed by, the degree of liver repopulation of transplanted hepatocyte gradually increased, the yield level is 22±6%in 3rd week, In the 4th weeks, the liver repopulate about 38±11%, in the 6th week, the liver is repopulated 67±8%, in the 8th week, the liver repopulate about 78±17%. In the 8th week, the Fah-/- mice liver is almost rebuilt completely by transplanted hepatocytes. The transplanted hepatocytes engraft into the Fah-/- mouse liver structure, but does not damage the liver lobule structure, chimeric liver's board structure and the hepatic lobules was normal, without the formation of pathological nodular hyperplasia. HE staining and electron microscopy further demonstrated that the progressive, irreversible damage of Fah-/- mouse hepatocyte is the internal mechanism of promoting the proliferation of transplanted hepatocytes.In the second part of the study, first of all, we have successfully established a Fah-/- Rag2-/- mouse strains:using CD3, CD 19 as markers, flow cytometry detects the changes in number of T cells (CD3+), B cells (CD 19+) in the bone marrow cells of Fah-/-Rag2-/-, compared with Fah-/- mice. The results show that the function of T cells and B cells are lost in Fah-/-Rag2-/-mouse. Secondly, hFLSC was transplanted into Fah-/-Rag2-/-mice, in 1,3,5,6,8 weeks, through the immnuohistochemistry of Fah enzyme, hFLSC can be detected, because the expression of Fah enzyme is a good sign for the detection of transplanted cells (Fah-/-). We further apply the immnuohistochemistry of liver cell functional protein-albumin, we preliminarily evaluate the ability of hepatocyte differentiation of hFLSC in the liver of Fah-/-Rag2-/- mice. The preliminary resulst shows that hFLSC could engrafte into the liver of Fa h-/-Rag2-/- mice, but its repopulation of the liver is less efficient, mice cannot restore the normal liver function.The results of this study show that:Fah-/- mice is an ideal model for the study of liver cell transplantation. The progressive, irreversible damage of Fah-/- mouse hepatocyte is the internal mechanism of promoting the proliferation of transplanted hepatocytes. Hepatocyte progenitor cells from human fetal liver (4.5-6 weeks) may be one of the candidate cells, but its efficiency may be related to the cell's mature degree. Hepatocyte differentiation induced in vitro may be necessary before its clinical application.
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