| BackgroundHepatcellular carcinoma (HCC) is a common malignant tumor in China, the mortality caused by which ranks the third among all malignant tumors. Researchers have long been searching for the cellular origin and causes of HCC to try to find effective preventive and treatment measures under the hints of the pathogenesis of HCC. As for the cellular origin of HCC, there are two major hypotheses: one is that HCC is caused by abnormal differentiation of hepatic stem cells, i.e., oval cells, and the other is that HCC results from dedifferentiation of mature hepatic cells. Each hypothesis has its own supporting evidence. In their experiment on carcinogenesis induced by fumigacin B1 in mice, Sell S. et al found that vast necrosis of hepatic cells occurs first accompanied by proliferation of oval cells, which is followed by pseudolobule formation in the necrosis region as well as rapid increase in the number of oval cells. Meanwhile, positive reaction of mRNAs of oncogenes c-myc and AFP, TGF-a, and TGF-P is present in oval cells and the reaction is tended to enhance. After that, the number of oval cells curtails with accordingly reduced expression of oncogenes. Then basophilic hepatocellular nodules form and finally carcinomatous nodules take shape. The authors thus holdthat oval cells, induced by carcinogenic factors, can differentiate directly into cancer cells. Accordingly, many authors think that hepatic oval cells are the cellular origin of HCC. In studying the detrimental effects of a carciongen, methyleugenol, on organs of rat, Johnson JD et al found that the rat develops various diseases, such as HCC, cholangiocarcinoma, neuroendocrine malignancies, hemosiderosis, normoblastocytosis, and bone marrow hyperplasia. Additionally, it has reported that patients with hemosiderosis are highly risky of HCC. Why do a carcinogen induce carcinogenesis in multiple organs? The reason may lie in bone marrow stem cells (BMSCs). Genetically mutated stem cells can abnormally differentiate and migrate to multiple organs with blood flow, leading to carcinogenesis in multiple organs. If it is the case, BMSCs should be the mother cells of hepatic oval cells. Petersen BE et al. first damaged BMSCs in female rats using large doses of irradiation, and then performed bone marrow transplant on these irradiated rats with bone marrow from allogenic male rats. Following transplant, the irradiated rats were orally administered with 2-acetaminofluorene (2-AAF) to suppress hepatocellular proliferation. At day 13 of transplant, positive expression of Y-chromosome was found in a small number of hepatic cells and oval cells. Having performed biopsies on liver tissues from female patients who received transplant of bone marrow from male donators, Malcolim RA et al found that 0.5% to 2% of the recipient hepatic cells show positive expression of Y-chromosome. The researches above confirm that part of oval cells derive from BMSCs. Accordingly, many authors think that liver oval cells are the cellular origin of HCC. However, some researches reveal that oval cells express some antigens associated with BMSCs, a finding further supporting the derivation of oval cells from BMSCs. These experimental data pose the question that whether BMSCs are the cellular origin of HCC. Up to date, a systematic research report in this field is stillunavailable. Hence, the Foundation of Natural Sciences of Guangdong Province funded this project.ObjectiveTo investigate the cellular origin of HCC, a group of BMSCs were isolated and purified, the differentiation of BMSCs transfected with oncogenes in vivo and in vitro and their colonization in rat liver were observed, so as to provide a rationale on the stem cell origin in the pathogenesis and progression of HCC.MethodsSince the composition of BMSCs is complex and there are many subpopulations with different surface markers, the research on BMSCs is somewhat difficult. Under specific conditions, marrow mesenchymal stem cells (MSCs) can differentiate into various tissues and cells. Exogenous genes can be easily introduced into and expressed in these cells, making them potential target cells for gene therapy and stem cell research. Therefore, they were studied in this research as the representative of BMSCs.1. Rat MSCs were obtained through gradient centrifugation. Rat hepatic cells were obtained through modified two-step method. Cell growth curves were plotted with the use of MTT colorimetry and cell type was identified using alkaline phosphatase staining and flow cytometry.2. The ability of MSCs to differentiate into mature hepatic cells was tested by in vitro induction of MSCs under different conditions.(1) Following cell identification, MSCs and hepatic cells, separated by a semipermeable membrane, were co-cultured. Separately cultured MSCs were used as the control. At day 1, 3, 7, 14, 21, and 28, the gene and protein expression of AFP, albumin, and CK-18 was detected using RT-PCR and immuocytochemistry.(2) Differentiation of MSCs to hepatic cells induced by HGF: MSCs werecultured in medium with various concentrations of HGF: Ong/ml, 10 ng/ml, 20 ng/ml, and 40 ng/ml. At day 1,3,7, 14, 21, and 28, the gene and protein expression of AFP, albumin, and CK-18 was detected using RT-PCR and immuocytochemistry.3. MSCs were labeled with green fluorescent protein (GFP) and grafted allogeneously in different ways.Differentiation and colonization of MSCs in rat liver was observed under different conditions.MSCs, which were obtained from rat bone marrow, were cultured and labeled with GFP, and were amplified in vitro. Then they were infused into rats via vena caudalis and vena portae. The recipient rats were divided into the hepatic impairment group, which were fed with tetrachloromethane and the healthy control group. At day 7 of grafting, the complete liver was obtained and examined using fluorescence detection, conventional pathology, and immunohistochemistry. At day 3 and day 1, the expression of grafted MSCs in rat liver was examined using quantitative PCR assay. The colonization and distribution of stem cells in rat liver were also studied.4. Differentiation of MSCs that were transfected with oncogenes using transient transfection was observed in vitro.(1) MSCs were transfected with c-myc, K-ras, c-myc, and K-ras, respectively, while MSCs that were not transfected were cultured as the control.(2) Transfected MSCs were cultured routinely. At day 1, 3, 7, 14, 21, and 28, the gene and protein expression of AFP, albumin, and CK-18 was detected using RT-PCR and immuocytochemistry.(3) Transfected MSCs were co-cultured with hepatic cells. At day 1, 3, 7, 14, 21, and 28, the gene and protein expression of AFP, albumin, and CK-18 was detected using quantitative RT-PCR and immuocytochemistry.5. MSCs, transfected with oncogenes, were grafted allogeneously in different ways to observe the differentiation and colonization of MSCs in rat liver.MSCs, transfected with different oncogenes and amplified in vitro, were infused into rats via vena portae. The recipient rats were divided into the hepatic impairment group, which were fed with tetrachloromethane and the healthy control group. At day 7, 14, 21, 28, and 42 following grafting, the complete livers were obtained and examined using fluorescence detection, conventional pathology, and immunohistochemical detection of GFP, c-kit, and AFP to study the colonization and distribution of stem cells in rat liver. Results1. MSCs, isolated by gradient centrifugation, were of high purity and viability. AKP stain was strongly positive. Flow cytometry showed that 98% of the cells expressed the surface markers of MSCs : CD29+, CD44+, CD34—, CD45—, CD49a+, and CD49b+.2. The process of cell proliferation and the time of morphologic changes varied according to the frequency of medium change. The cells grew better and proliferated faster in medium changed daily than in medium changed every two to three days. The time to reach a certain cell number for cells cultured in daily changed medium was 2 to 3 days ahead of cell cultured in medium changed every 2 to 3 days.3. Rat MSCs differentiated successfully into hepatic cells in vitro under differentconditions.(1) During co-culture with hepatic cells, MSCs changed in morphology, size and number. Cells with two or more nuclei were found and the cell contour was clear. RT-PCR revealed that at day 7 of co-culture, the expression of AFP gene was present and enhanced at day 14 and then decreased at day 21, and that from day 14, the expression of albumin and CK-18 genes was present and persisted from then on. No such expression was found in separately cultured MSCs. Co-cultured MSCs showedpositive AFP stain on immuohistochemical examination at day 7, positive albumin and CK-18 proteins at day 14. But separately cultured MSCs showed negative results.(2) In HGF-induced MSCs (HGF: 20 ng/mland 40 ng/ml), the expression of AFP gene was present at day 7 and enhanced at day 14 and then decreased at day 28 and that from day 14, the expression of albumin and CK-18 genes was present and persisted from then on When HGF concentration was 0 ng/ml or 10 ng/ml, no AFP, CK-18 or albumin was expressed during the course of culture.4. MSCs, labeled with GFP, were grafted allogeneously in different ways.(1) All the rats that received allogenic MSC graft survived to the end of the designed time. No accidental death occurred. Nor did immunological rejection(2) MSCs, grafted in different ways, colonized in the recipient rat liver. In impaired rat liver, MSCs successfully differentiated into mature hepatic cells and oval cells and these cells survived permanently; while in normal rat liver, only a few oval cells were differentiated from MSCs. Therefore, MSCs' colonization and differentiation into mature hepatic cells and oval cells in rat liver are associated with hepatic impairment, but not with the ways to graft.(3) Detection of GFP using quantitative RT-PCR: Following infusion of MSCs through vena portae and vena caudalis, more of them colonized in impaired rat liver than in normal rat liver (p<0.05). In impaired rat liver, there was no significant difference in the amount of colonization of MSCs between infusion via vena portae and via vena caudalis (p>0.05). In normal rat liver, there was significant difference in the amount of colonization of MSCs between infusion via vena portae and via vena caudalis (p<0.05) and the difference was related to the time to grafting.5. Differentiation of MSCs that were transfected with oncogenes using transient transfection was observed in vitro.(1) Lentivirus vector- and lentivirus vector plus double gene- transfected MSCswere both successful with high efficiency of transfection. Both methods were simple.(2) Conventionally cultured MSCs transfected with or without oncogenes did not express AFP, ALB, and CK-18 during the course of culture.(3) MSCs, which were transfected with c-myc and co-cultured with hepatic cells, expressed AFP gene at day 7 and the expression enhanced at day 14 and reduced at day 28. From day 14 on, the albumin gene began to express, and the expression persisted from then on. From day 14 on, the CK-18 gene began to express, and the expression persisted till day 28.(4) MSCs, which were transfected with K-ras and co-cultured with hepatic cells, expressed the AFP gene at day 7 and the expression enhanced by degrees, at day 14 ALB and CK-18 reduced gradually and at day 21, their expression disappeared..(5) MSCs, which were transfected with c-myc and K-ras and co-cultured with hepatic cells, expressed the AFP gene at day 7 and the expression enhanced rapidly, and at day 7 ALB and CK-18 reduced rapidly and at day 14, their expression disappeared.6. MSCs, transfected with oncogenes, were grafted allogeneously in different ways to observe the differentiation and colonization of MSCs in rat liver.(1) Following grafting of allogenic MSCs, a total of 6 rats died from other causes in the designed duration. No immunological rejection occurred.(2) MSCs,which were transfected with oncogenes and infused through vena portae, colonized in the recipient rat liver.(3) In the group of rats with hepatic impairment, 2 rats were grafted with MSCs that were transfected with double oncogenes and 1 rat with MSCs that were transfected with K-ras. In the group of rats with normal liver, 1 rat was grafted with MSCs that were transfected with double oncogenes and 1 rat with MSCs that were transfected with K-ras. During day 21 to day 28, grain-like, gray granules werepresent at the liver surface and the liver stiffened. It can be found microscopically that liver cancer, primarily hepatocellular carcinoma formed. Positive results of GFP, c-kit and AFP were present on immuohistochemical staining, with consistent distribution regions and sites of positive cells.(4) Regardless of hepatic impairment prior to grafting, rats grafted with MSCs that were transfected with c-myc gene had swollen livers with no obvious tuberosity or tumor. Small oval cells were found microscopically in the periphery of central veins and vena portae. Immunohistochemical staining of c-kit was positive, while that of AFP negative. Conclusions1. Gradient centrifugation can be used to isolate MSCs. This method is simple and convenient, which can produce highly pure MSCs. Compared with change of medium every 3 to 4 days, daily change of medium helps MSCs proliferation and the morphologically unified colonies form.2. Rat MSCs, co-cultured with hepatic cells, can be induced to differentiate into hepatocyte.3. Induced by relatively high concentrations of HGF, marrow mesenchymal stem cells can differentiate into hepatocyte.4. GFP-labeled rat MSCs can colonize in allogenic rat liver following grafting in various ways. The grafted MSCs are the cellular source of oval cells in liver. They can also differentiate into mature hepatocyte. The aggregation time and number of stem cells in liver are closely associated with hepatic impairment but not with the ways to graft.5. Under the conditions favoring cellular induction to hepatic cells, MSCs, transfected with oncogenes, can differentiate into liver cancer cells under the influence of some oncogenes. MSCs, which are transfected with multiple genes, aremore likely to differentiate into cancer cells.6. Rat MSCs, transfected with oncogenes and grafted allogeneously, will be induced to form liver cancer cells under the influence of some oncogenes. Synergism of multiple genes will increase the possibility of carciongenesis. Hepatocellular carcinoma may derive from genetically mutated MSCs. |
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