| IntroductionIt is estimated conservatively that there are 350 million persistent carriers of HBV worldwide, 25% of whom have chronic liver disease and cirrhosis, which could progess to hepatocellular carcinoma. The annual mortality from hepatitis B infection and its sequelae is 1-2 million people worldwild. With high rate of prevalence of HBV in China, there are 150million carriers and 30million chronic hepartitis B, which represents a major public-health concern because of its propensity to progress to liver cirrhosis and hepatocellular carcinoma. The precise pathogenetic mechanisms responsible for the acute and chronic hepatitis B are only partly defined.Although the improvement of the surgical skills and the adjuvant therapies, hepatic function failure and hepatocellular carcinoma is still major cause of HBV-associated mortality which is difficult to cure because of recurrence after resection. Therefore, liver fibrosis, liver cirrhosis and hepatocellular carcinoma are the hot points of basic and clinical research. In spite of the reversal of early stage liver fibrosis under some circumstances, these results just limited to the animal models. After the formation of liver cirrhosis, it almost is impossible to reverse pathological changes.It is well-known that stem cells exist in many organs, which are responsible for self-repair and regeneration in various organs of the human body. It has been demonstrated that they have great relationship with tumor cells and have some characteristics in common. Recently, a number of investigations demonstrate that adult stem cells may be targets for malignant transformation. Chronic infection and regeneration in the damaged organs can lead to the transformation of normal stem cells into cancer cells, which may include 'cancer stem cells (CSCs)'.Hepatitis B virus (HBV) often causes chronic infections of liver which can lead to the liver cirrhosis and HCC. The stem-like cells in chronic liver diseases can rise with disease severity and these cells directly participate in the formation of hepatocellular carcinoma in animal models. Therefore, hepatic stem cells might play a role in hepatic carcinogenesis. Identification of these cells will add to our understanding of hepatocarcinogenesis.Multipotential mesenchymal stem cells (MSCs) can be induced to differentiate into hepatocytes which meant the mesenchymal-epithelial transition (MET). Mesenchymal stem cells have intimate relationship with fibroblasts and the comparison of their characteristics between MSC and fibroblasts demonstrate that they represent probably different functional states of the same cells. These fibroblasts express stem cell markers and possess the capability for further differentiation. Under some circumstances, the fibrobalsts exhibit multilineage differentiating potentiality. Therefore, the name of fibroblast is often used interchangeably with mesenchymal stem cell. These studies on fibroblasts remind us if the fibroblasts in the cirrhotic liver possess the characteristics of stem cells.In the present investigation, we firstly investigated the expression of varied stem cell markers in the tissues of cirrhotic liver and hepatocellular carcinoma as well as the adjacent tissues to them. Next, we examined the capacity of tumorigenicity of the cells in cirrhotic liver and tissue adjacent to HCC in vivo by directly transplanting them into BNX mice. Then, we further identified the characteristics of proliferation, self-renewal and cloning formation of the stem-like cells in these tissues. Finally, we investigated the hepatic differentiation potential of the fibroblasts in cirrhotic liver and HCC after the successful isolation of the fibroblasts. We hypothesize that, fibroblasts in cirrhotic liver and hepatocellular carcinoma are capable of proliferating and differentiating into functional hepatocytes cells in vitro. Material and MethodsTwenty Surgically resected HCC and corresponding adjacent tissues as well as 10 cirrhotic liver tissues were collected from our hospital. We select 5 cirrhotic tissues and 5 HCC with their paired samples for tissue transplantation and cultured fibroblasts.The tissues had been processed routinely and were cut into 5-mm thick sections. Then, conventional IHC was performed using the following antibodies: c-kit, CD133, CD90, CD34, CK18 and CK-19. Negative control experiments were performed by replacing primary antibodies with PBS only. After the isolation of fibroblasts in different tissues, we analyzed the expression of CD73, CD44, CD34, CD45, CD90, c-kit, CD133, vimentin, desmin,fibronectin,α-SAM, collagen prolyl 4-hydroxylase, CK-18, CK-19 and albumin by cell immunohistochemistry, FCM, FI and WB.Under varied conditions, we investigated the proliferation, self-renewal and cloning formation of the cells from the tissues of cirrhotic liver and hepatocellular carcinoma as well as their adjacent tissues. Moreover, we also examined the morphological changes and the specific genetic expression as well as proteins in the fibroblasts from the tissues of cirrhotic liver and hepatocellular carcinoma when the cells were cultured under different media and time points.To assess the in vivo capacity of tumorigenesity of the tissues and the cells from cirrhotic liver and hepatocellular carcinoma, we selected five tissue samples of each tissue type respectively and then cut them into tissue pieces of 1-2 mm~3 in the media of MEM and then washed them twice in PBS. The tissue samples were transplated into BNX mice by 18 transfixion pin. After long-term culture, 1X10E6 fibroblasts from hepatocellular carcinoma were also injected into BNX mice subcutaneously. Record the time of tumor formation, size, morphology, texture and activity. Five months later, the mice were executed and got the tissue samples of tumors and were submitted to the examination of HE and IHC.Under inducible conditions, we investigated the hepatic differentiation of the fibroblasts from cirrhotic liver and hepatocellular carcinoma. ResultsAnalysis of stem cell markers in the tissues of cirrhotic liver and HCC as well as their adjacent tissues by IHC showed the different expression patterns. In general, stem cell markers in noncancerous tissues appeared more common as compared to HCC. Most CD34 and CD90-positive cells were located in proliferating bile ductules and they represented hepatic progenitor cells morphologically. Expression of CD133 and c-kit distributed in the parenchyma of cirrhotic liver and HCC and they shaped as intermediate hepatocytes morphologically and spatially. Almost all the tissue samples expressed CK-18 and CK-19 and some of the cells they represented were located in the parenchyma which looked like intermediate hepatocytes.The in vitro experiments also demonstrated that the cells in cirrhotic liver and adjacent tissues to HCC possess the great capacities of proliferation, self-renewal and cloning formation.Our results revealed that very few fibroblasts expressed CD73 and CD44, which are markers specifically exposed on the surface of mesenchymal stem cells. Expression of CD34 and CD45 representing haemopoietic stem cells was not observed. A small subpopulation of Thy-1-positive cells was observed while the fibroblasts in lung bronchial homogeneously expressed Thy-1. In addition, some of c-kit and CD133-positive cells were also identified. Further evaluation of cystoskeletal and extracellular matrix elements by immunofluorescent staining showed that a few cells stained positively for the myofibroblast marker a-smooth muscle actin in microfilaments. Moreover, all fibroblasts expressed vimentin, desmin, fibronectin, and some of them were positive for epithelial markers CK-18 and CK-19, but albumin expression was not observed. Additionally, a homogeneous staining for collagen prolyl 4-hydroxylase and fibronectin was observed. Under specific conditions, the fibroblasts gradually progressed toward the polygonal morphology of hepatocyte in a time-dependent manner accompanied the expression of different hepatic lineage markers. The fibroblasts in the beginning of culture showed typical spindle-shaped morphology. After 2-week induction, the morphology was lost and cells developed a broadened, flattened morphology. 4 weeks later, a retraction of elongated ends was observed and then cuboidal morphology of hepatocyte developed with increasing time of differentiation.Three mice from two groups transplanted with tissues adjacent to HCC gave rise to tumors. The solid tumors were about 1-cm in diameter and histological examination showed a highly cellular mass with necrotic center. The high nuclear/cytoplasmic ratio of the cells and the loss of normal tissue structure indicated they were malignant tumor. CD133 and c-kit expression was also identified by IHC, these cells were morphologically oval cells. Long-term cultured fibroblasts did not initiated tumors in mice. ConclusionsAnalysis of stem cell markers in the tissues of cirrhotic liver and HCC as well as their adjacent tissues by IHC showed the different expression patterns. The positive cells in these tissues may represent different cell types. The stem-like cells in cirrhotic liver and adjacent tissues to HCC possess the great capacities of proliferation, self-renewal and cloning formation. They also possess the capacity of tumorigenicity and may contain the candidates for cancer stem cells of HCC. These findings provide a novel model of hepatocarcinogenesis which will facilitate the understanding of the mechanism of hepatocarcinogenesis and multicentric origination as well as multicentric recurrence of HCC. It is of great significance to the prophylaxis and therapy for HCC.Human fibroblasts in cirrhotic liver and HCC exhibit stem cell phenotypes and differentiating potentiality to mature hepatocytes in vitro, which imply the mesenchymal-epithelial transition. Our results shed new light on effect of fibroblasts in process of liver fibrosis. Most important of all, fibroblasts may serve as a new target for reversion of liver fibrosis or cell source for tissue engineering even cell therapy of severe liver disease.
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