| The cancer stem cell is the cancer cell that has the potential to transfer disease or to form tumours following transplantation. It has the potential to self-renew, forming additional tumorigenic cancer cell of similar phenotype and to give rise to phenotypically diverse cancer cells with more limited proliferative potential. The concept was frist advised in the later of 20th century, But many factors such as the lack of knowledgement of stem cell, the technique and the condition of experimental limits, hinder the research of it. Until recent years, as the discoveres of acute leukemia-initiating cells, breast cancer stem cells, brain tumor stem cells and chronic myeloid leukemia stem cell, The cancer stem cell has attracted the interestes of researchers again.The primary of Hepatocellular Carcinomas is one of the common malignancy tumours in our country, especially the large liver cancers. The patients often have short life span and the cure has little effect. The mortality of Hepatocellular Carcinoma in our country is third. As the theory of cancer stem cell says: The occurrence, The depravation, The metabasis and The curative effect are all related to the cancer stem cells. The aim of this research is to knowe the existence of liver cancer stem cells or not and its basic biological features.At present, researchers have found the existence of liver progenitor cells in the liver preneoplastic foci which was induced by chemical carcinogens in various rodent models. The liver oval cell was first found in the liver of rat which was induced to form tumor by chemical carcinogen. What is more, researchers also found the existence of liver oval cells or progenitor cells in the human livers which have the disease of chronic hepatitis C, genetic hemochromatosis, alcoholic liver, hepatocellular adenomas and hepatocellucar carcinoma. These progenitor cells often emerge in the middle of cancer cells and the more serious of the diseases, the more numbers of these cells. Because the diseases above are all related tothe happenence of hepatocellucar carcinoma or it is the hepatocellucar carcinoma. Researcheres speculate that these progenitor cells which are observed in these diseases are probably to be the "liver cancer stem cells". In order to find many more trustful evidences which can prove it. This research try to isolate these progenitor cells from fresh specimens of human hepatocellucar carcinoma and establish a cell line which can be cultured long time in vitro. The specific way is to divide the fresh tissue into two partes. One is to do the primary cell culture, The other is to be transplanted into the skin under of NOD/SCID mice, When it formes secondary tumors, Do the primary cell culture again. By the way, The four cell lines derived from human liver cancer tissues are established. One of them, named P2-HCC (passaged 2- hepatocellucar carcinoma ) has some features of cancer stem cells. It derived from the tissues of passaged twice in NOD/SCID mice. This cell line maybe is a progenitor cell line or a tumor cell line which has many more liver cancer stem cells.(1) Establishement of P2-HCC and its basic phenotype. The fresh samples of human Hepatocellular Carcinomas from surgery was passaged twice in NOD/SCID mice, then we conducted primary cell culture on the passaged tumor samples. By the way of single cell cloning experiment, We established the cell line of P2-HCC. Cells of P2-HCC show a typical epithelial morphology, with 3-4 nucleoli. The size and the shape of it is homogenous. The juncture of cells is tight. What is more, This cell line has the characterization of overlapping, which is the symbol of tumour cells. The proliferation capability of it is powerful.(2) P2-HCC is a tumor cell line and has the ablity to generate the same tumor as the initial tumor. The P2-HCC has a abnormal karyotype and the number of chromosomes varies greatly, from 35 to 120. But many of them have chromosomes between 70-85. The tumorigenic ablity of P2-HCC is powerful. After implantation of only 100 cell to the NOD/SCID mice, It can form tumors within 6 weeks. The type of this secondary tumors is same as the primary tumor. The breast cancer stem cell has only 17% of forming tumor after implantation of 100 cells.(3) The P2-HCC can differentiate into hepatocytes in vitro after induction. Some small molecule substances such as Sodium butyrate, dimethyl sulfoxide, hydrocortisone and so on can induce liver stem cell differentiate into hepatocytes. After 5 days of induction with hydrocortisone, The morphology of P2-HCC changes greatly, The border and the nucleolus become distinct, Binuclear cells could be observed at a percentage of 10%, By 7days, The percentage could reach 30%. The characterization of hepatocyte is more obvious. The most important change is that the cell could not continue to proliferate while confluency was reached. This indicats that the cells after induction have lost the features of cancer cells and obtain the characterization of normal hepatocyte. There are also emergence of Binuclear cells after induction with dimethyl sulfoxide, but the ratio is lower than induction with hydrocortisone, By 10 days after induction, there was only 5%. This indicates that P2-HGC has the features of liver stem cell and can differentiate into hepatocyte.(4) The immunohistochemical results indicates that the P2-HCC has the potential of auto-differentiation in vitro. The protein of albumin is one of the functional proteins of hepatocytes. It is expressed not only in the mature hepatocytes but also in the type II oval cells which have the potential to differentiate into hepatocyte. Although the expression is less than the mature hepatocytes, The results of immunohistochemistry showes that the strong positive of ALB cells always emerge at the edge of the cell cluster, while other cells lie in the middle. This is maybe the results of auto-differentiation as the cells proliferate. Additionally, by the way of RT-PCR, We also compare the expression changes of some proteins before and after induction of P2-HCC. The result suggests that the expression of albumin is higher in induced cells than in P2-HCC. The Glucose-6-phosphatase is not detected in P2-HCC. But it can be detected in induced cells. So we suppose that the P2-HCC lose some characterizationes of liver cancer cells after induction and obtain the features of hepatocytes.(5) The P2-HCC has side population (SP) cells. Some cells have the potential to efflux lipophilic dyes, like Hoechst 33342 dye, from nuclear to outer.This feature is called "side population phenotype". It is related to the expression of the ATP-binding cassette transporter protein ABCG2. The cells which have this phenotype are called "SP cells". The resultes of many experimentes indicated that this phenotype is maybe another stem cell marker. As the way of reportes, we analysis the SP phenotype of P2-HCC, The result showes that there are SP cells in this cell line, but the percentage is very small. The SP cells often disturb at the middle of a cluster. What is more, There is another cell type which can be dyed by Hoechst 33342, but the colour is lighter than other dyed cells. By the way of single-cell culture, we isolated this cell from P2-HCC and established another cell line, named P2-HCCS1. This cell line is easier to differentiate into mature hepatocyte than P2-HCC. It also express some progenitor cell markers such as c-kit. We also account the number of SP cells and the light dyed cells in the single cell suspension. The percentage is 6.4% and 14% respectively.(6) At the appropriate condition, The P2-HCC can take part in the restoration of injured liver, not forming tumors. As the P2-HCC has the potenth' tc differentiate into normal hepatocyte in vitro, We suppose that it maybe also can differente into normal hepatocyte in vivo at the particular condition. With these considerationes, we implant P2-HCCf which is marked genetically with enhanced green fluorescent protein (EGFP) into SCID mice. This recipient mouse had made operation of liver partial hepatectomy or treated by carbon tetrachloride. After 20 days of implantation, we killed the recipient mouse and made the cryostat sections of its liver. The results show that the P2-HCCf has located in the recipient mouse liver and took part in the regeneration. The located foci often has 2-3 cells and there is no morphology differences between implanted P2-HCCf and mice's hepatocytes. The interesting point is that although the P2-HCC is the tumor cell, There is no sign of forming tumors. This maybe indicated that the tumor cells or the cancer stem cells can transform into normal cells at the special environment.From this research, We can make conclusiones as follows: There is really liver cancer stem cells in liver tumors; This cell has powerful potential of forming...
|
PDF Full Text Request