| Background and objective:Researchers have reported that bone marrow stem cells (BMSCs) possess the ability of lateral differentiation in vitro, which can be directly differentiated into cardiac cells, pancreatic cells, muscle cells and liver-like cells, but also some scholars have found BMSCs can transdifferentiate into liver cells in vivo, however, this transverse differentiation doesn’t directly differentiate from BMSCs into liver cells, which achieve this goals through the BMSCs infusion with liver cells in the liver tissue. CD34 and CD 133 are molecular markers of BMSCs, Chinese scholars have induced CD34+cord blood cells into liver-like cells. The conception of irreversible cirrhosis has been changed by anti-hepatitis B virus (HBV) nucleoside drugs therapy. A large number of studies have confirmed that nucleoside antiviral therapy could reduce the risk of HBV-related hepatocellular carcinoma (HCC), but the mechanism that induced reversal of liver fibrosis and reduced the occurrence of liver cancer is still unclear. Hepatic stellate cells (HSCs) and fibroblasts are effector cells of liver fibrosis. The essence of regenerating liver depends on the liver cells and liver stem cells. Researchers have found that HCC was closely associated with liver cancer stem cells. Promoting the differentiation of stem cells into the tissue cells that patients need is becoming a new way for medical workers treating liver cirrhosis and even HCC. Studies have showed that the anti-tumor nucleoside analogue 5 aza-2’-deoxycytidine (5-AZA) can induce WB rat liver stem cells into insulin-producing cells. Based on this research, do anti-HBV nucleoside drugs whose chemical structure is similar to 5-AZA have the same effect? This study aimed to explore whether the anti-HBV nucleoside drugs can influence the BMSCs of the patients in the treatment of chronic hepatitis B (CHB), hepatitis B-related liver cirrhosis and HCC, which will find new principle for improvement liver function with nucleoside antiviral treatment of CHB and cirrhosis, explore the possible mechanisms of HBV-related HCC generation.Methods:1. Enrollment and exclusion criteria for the patients were according to the standard required to establish experimental study:394 cases of patients with CHB, liver cirrhosis and HCC were accepted in this study and 12 healthy volunteers were recruited. All the patients and volunteers were divided into seven groups:Group 1:83 cases of CHB patients without anti-HBV treatment; Group 2:64 CHB patients with anti-HBV treatment; Group 3:67 cases of cirrhosis patients without anti-HBV treatment; Group 4:78 liver cirrhosis patients with anti-HBV treatment; Group 5:49 cases of primary HBV-related HCC patients without anti-HBV treatment; Group 6:53 cases of primary HBV-related HCC patients with anti-HBV treatment; Group 7:12 healthy volunteers. Groups with anti-HBV therapy were sustained with antiviral nucleoside drugs for more than 2 years.2. Peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy volunteers and CD34 and CD133 positive cells in them were detected by flow cytometry.3. Total RNA was extracted from the liver tissue in some of the enrolled CHB and liver cirrhosis patients and, reversely transcribed into cDNA. Real-time quantitative PCR (Real-Time PCR) was amplified to detect the CD34 and CD133 mRNA level in CHB and cirrhosis patients. 4. Immunohistochemical staining was used to detect the expression of CD34 and CD 133 in parts of CHB, cirrhosis and HCC pathology paraffin slice samples. 5.Serological markers were measured and analyzed from the 7 groups, including ALT, AST, ALB, HbsAg, HbeAg, HBeAb, HBVDNA and immunology indicators CD4, CD8 absolute value and percentage. AFP indicators were only detected in the HCC patients.Results:1. Compared with non-anti-HBV treated groups, patients with CHB, cirrhosis or HCC taking nucleoside(s) for antiviral treatment whose clinical liver function had been improved in varying degrees, but there was no difference of CD4 cells and CD8 cells in the CHB, cirrhosis or HCC groups comparing with their counterparts (P> 0.05).2. The percentage of CD34 in patients’PBMCs of CHB and liver cirrhosis groups taking nucleoside(s) for antiviral treatment were significantly higher than the non-anti-HBV treated ones (P<0.05, P<0.01), in contrast, the expression of CD34 in antiviral HCC group is lower than that without antiviral therapy (P<0.01); there was no statistical difference between CHB nucleoside group and the non-anti-HBV treated CHB group of CD133 in PBMCs (P> 0.05), the expression of CD 133 in liver cirrhosis and HCC groups with antiviral treatment was statistically lower than the untreated group (P<0.01). Using ANONA analysis, we found that there was significant differences between the healthy control group (P <0.001) in patients with CD34 and CD133 expression of HBV infection.3. The levels of CD34 mRNA in the antiviral groups of CHB and liver cirrhosis had upregulated obviously than non-anti-HBV treated ones (P<0.01).4. Immunohistochemical staining results showed that CD34 index of CHB and liver cirrhosis groups taking nucleoside(s) for antiviral therapy were higher than their counterpart groups (P<0.05, P<0.01) and in HCC groups, patients taking nucleoside(s) for antiviral therapy had a lower CD34 index than patients not. Cirrhosis patients taking nucleoside(s) for anti-HBV therapy whose CD133-positive rate showed a declining trend (P<0.05), we didn’t find any statistical significance in CD 133 expression in the liver tissue of CHB groups (P> 0.05).The expression of CD 133 in HBV-related HCC group with antiviral therapy even showed a sharply downward trend than the non-anti-HBV group(P <0.01).Conclusion:1.Compared with healthy volunteers, expression of CD34 and CD 133-positive cells of PBMCs in patients with HBV-associated diseases were significantly increased, indicating that liver injury promote the mobilization and release of BMSCs. 2. Anti-HBV nucleoside drugs promote the BMSCs release to the peripheral blood.3. Anti-HBV nucleoside drugs can promote the recovery of liver function in patients with similar immunization by inhibiting HBV replication and influencing stem cells.4. CD34+cells associated with liver function restoration, while the expression of CD133 is associated with the occurrence of liver cancer.5. CD34 positive cells may promote angiogenesis in the liver involved in the regeneration of liver cells.6. Nucleoside antiviral therapy reducing the incidence of HBV related HCC may be associated with the down-regulation of liver cancer stem cell, CD133+cells.7. Nucleoside antiviral drugs may inhibit the occurrence of liver cancer stem cells and thus indirectly lowered the HCC tumor angiogenesis. |
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