Primary Study On The Identification Of Hepatoma Stem Cells And The Effects Of Routine Chemotherapy On Hepatoma Stem Cells

It is generally thought that tumor consists of homogeneous tumor cells, which all possess the same unlimited reproductive activity. But recently an original theory about tumor stem cell thinks that tumor cells have heterogenicity, and only a minority of subgroup, having oncogenicity and acting as the role of tumor stem cell, possess the potentiality of self replication, multidirectional differentiation and unlimited proliferation, the ability of tumor-initiating and the characteristics of metastasis, but the majority go to death after temporal differentiation. The former is named tumor stem cell(TSC) derived from the similar function with stem cell. Presently, TSCs have been successfully isolated from many tumor tissues such as leukemia, breast cancer, neuroglioma, prostatic carcinoma and so on, so the research of tumor has entered an era of TSC.Increasing evidences show that hepatoma stem cells certainly exist and possibly become a key target in liver cancer therapy. It is difficult and critical for the research of liver cancer to find out specific surface markers, but the following findings published in recent years are exciting: the marker molecule ABCG2 of liver SP cells, which have the biological characteristics of hepatoma stem cells, acts as a marker of hepatoma stem cells; CD133, as the marker of breast cancer, colon carcinoma and neurospongioma stem cells, is also considered as a marker of hepatoma stem cells; and the expression of CD90, as another specific marker of hepatoma stem cells, is found in the hepatoma tissue and peripheral blood. To sum up, ABCG2, CD133 and CD90 have been focused on the research of hepatoma stem cell surface marker. Yet, the study on hepatoma stem cell just is on the beginning and still remains a good many questions to be resolved. What are the characteristics of existence and distribution of hepatoma stem cells in the hepatoma tissue and peripheral blood? Is hepatoma stem cell related to the aggravation of liver cancer? What is the relationship between chemotherapy drug resistance cell and hepatoma stem cell? How are hepatoma stem cells, which are rare in the tissue of liver cancer, isolated and enriched? It is a pity that we still can not find any answers to the above questions from current researches.Hepatoma stem cell is generally resistant to chemotherapeutic drugs, and current therapeutic measures for liver cancer have no effects on hepatoma stem cells. It is possible that the treatment failure or the relapse of liver cancer is due to the chemotherapy escape. So, investigating the biological characteristics and the chemotherapy escape mechanisms of hepatoma stem cell are important in seeking hepatoma stem cell-targeted therapy. In this study, based on the latest advancement of the current researches on surface markers of hepatoma stem cell we will focus on hepatoma stem cell, analyze the relationship between the aggravation of liver cancer and the existence and distribution of hepatoma stem cell in the human liver cancer tissues and peripheral blood. We also try to investigate the therapeutic reaction of routine chemotherapy on human hepatoma stem cells in vitro in order to help the isolation, enrichment and targeted therapy of hepatoma stem cells.Objective:1. To detect the expressions of hepatoma stem cell surface marker CD133 and CD90 in the tissues of hepatocellular carcinoma (HCC) and evaluate their significance in clinical pathology.2. To investigate the relationship between the positive expressions of CD133 and CD90 and the aggravation of hepatocellular carcinoma(HCC) by comparing the percentages of CD45-CD133+ and CD45-CD90+ peripheral blood cells from HCC patients with different features of clinical pathology.3. To investigate the effects of routine chemotherapy on hepatoma stem cells of human liver cancer and seek new methods and approaches for the isolation and enrichment of hepatoma stem cells. Methods:1. The expressions of CD133 and CD90 in the liver cancer cells taken from 93 HCC patients and the normal liver cells taken from 10 healthy people were detected by immunohistochemical method.2. The percentages of CD45-CD133+ peripheral blood cells in 65 HCC patients compared with that of 20 healthy people were detected by the flow cytometry; the percentages of CD45-CD90+ peripheral blood cells in 49 HCC patients compared with that of 10 healthy people were detected by the flow cytometry.3. Human hepatoma cells were treated in vitro by 6 cycles of the chemotherapy combined with adriamycin, fluorouracil and cisplatin at their plasma peak concentration; the expressions of ABCG2 and CD133 of drug surviving cells (DSCs) were detected by immunohistochemistry; the percentages of ABCG2+ and CD133+ cells were evaluated by the flow cytometry; the expression of ABCG2 protein was detected by Western blot.Results:1. Among 93 HCC cases, the positive expressions of CD133 in 71 cases and CD90 in 64 cases were found with the percentages of average positive cells, (6.4±3.3)% and (4.3±3.9)% respectively, and no positive expressions of CD133 and CD90 were found in the normal liver tissues(P<0.01); the percentages of CD133 and CD90 positive cells in the HCC tissues of TNM stageⅢandⅣ[(8.1±3.7)%, (5.7±4.2)%] were significantly higher than those of stageⅠandⅡ[(4.1±2.3)%, (2.3±1.9)%]( P< 0.01) ; Spearman correlation analysis indicated that the expressions of CD133 [ (0.1±0.1)% in gradeⅠ, (1.9±0.7)% in gradeⅡ, (5.9±2.3)% in gradeⅢ, (16.1±4.7)% in gradeⅣ] and CD90 [(0.3±0.3)% in gradeⅠ, (1.8±1.7)% in gradeⅡ, (3.3±2.9)% in gradeⅢ, (10.8±4.2)% in gradeⅣ] were up-regulated with the upgrading of the pathohistology grade (P<0.05); the positive correlation was observed between the expressions of CD133 and CD90 (r=0.402,P<0.01).2. The percentage of the peripheral blood mononuclear cells(PBMC) with the CD133 expression in 65 HCC patients was (2.0±1.3)%, no CD133 expression was found in the controll group, and there was the difference between them(χ2=20.20,P<0.01);the percentage of CD45-CD133+cells was significantly related to the tumor amicula integrality(P<0.01), the portal vein tumor embolus(P<0.01)and the TNM stage(P<0.01), but not to the tumor diameter as well as the AFP value(P>0.05); the Spearman correlation analysis indicated that the expression of CD133 was up-regulated with the upgrading of the pathohistology grade ( P<0.01).3. The percentage of CD45-CD90+ peripheral blood mononuclear cells(PBMC) in 49 HCC patients was ( 3.3±1.2) %, no CD45-CD90+ expression was found in the controll group(10 cases), and there was the difference between them (χ2=33.55 , P < 0.01 ); the percentage of CD45-CD90+ cells was significantly related to the tumor amicula integrality( P < 0.01 ) , the portal vein tumor embolus ( P < 0.01 ) and the pathohistology grade(P<0.01), but not to the tumor diameter as well as the AFP value(P>0.05); the Spearman correlation analysis indicated that the expression of CD90 was up-regulated with the TMN stage ( P<0.01).4. The combined chemotherapy inhibited hepatoma cells. The percentage of cells with ABCG2 positive expression in DSCs group was (71.2±14.7)%, the percentage of cells with ABCG2 positive expression in control group was (2.4±1.4)%, and there was the difference between them(χ2=1906.25,P< 0.01); the percentage of cells with CD133 positive expression in DSCs group was (94.5±13.8)%, the percentage of cells with CD133 positive expression in control group was (3.3±1.7)%, and there was the difference between them(χ2=1664.29,P<0.01);the percentage of CD45-ABCG2+ cells in DSCs group was (57.32±4.63)%, the percentage of CD45-ABCG2+ cells in control group was (1.31±0.64)%, and there was the difference between them(χ2=756.94,P<0.01);the percentage of CD45-CD133+cells in DSCs group was (87.63±7.29)%, the percentage of CD45-CD133+ cells in control group was (2.03±0.96)%, and there was the difference between them(χ2=1481.50,P<0.01). The strip of ABCG2 protein expression, which was found in relative molecular mass 70 000 Da or so, significantly increased in DSCs group as comparison with control group, and there was the difference between them(P < 0.05).Conclusion:1. The hepatoma stem cells with CD133 and CD90 positive expression exist in the HCC tissues, and the expressions of CD133 and CD90 are positively related to the TNM stage and the pathohistology grade of HCC patient.2. The hepatoma stem cells with CD45-CD133+ and CD45-CD90+ positive expression exist in the peripheral blood of patients with liver cancer, and the percentage of these stem cells is closely correlated to the aggravation of hepatocellular carcinoma. Detecting the percentage of cells with CD45-CD133+ and CD45-CD90+ expression in the peripheral blood of patients with liver cancer has clinical significance in predicting the hematogenous dissemination and the metastasis of cancer cells.3. The significant increase of the percentages of cells with ABCG2 andCD133 positive expression and the protein expression of ABCG2 in DSCs of human hepatoma cells indicates that routine combined-chemotherapy has no effects on hepatoma stem cells.