| Backgrounds:Poison is the central of tumor incidences; detoxification cancer method is an effective method of traditional Chinese medicine for treating cancer, and detoxification cancer prescription is remains unclear of the anticancer mechanism. Follicular helper T cells(Tfh) play a major role in B cell activation; we found HBs Ag and CD57 are closely related in the early experiments, and believe that the presence of CD4+CXCR5+CD57+T cells subsets in the human body, and detoxification cancer prescription can adjust the T cells subsets. CD4+CXCR5+CD57+T cells play an important role in the treatment and prognosis of Hepatic carcinoma.Objectives:Detoxification cancer method is an effective method for therapy cancer,mechanism of detoxification cancer prescription treatment cancer is remains unclear. This study was to analyze the role and prognosis of CD4+CXCR5+CD57+T cells in hepato-carcinogenesis. To discuss anti-tumor effect of detoxification cancer prescription and regulate CD4+CXCR5+CD57+T cells of mechanism for treatment tutor.Methods:Clinical trials:This study screened subjects 65 cases liver cancer patients under the study program and 32 cases of healthy volunteers as a control group. Express ofCD57, HBs Ag, CXCR5 and other proteins by HE staining and immunohistochemical detection of liver cancer specimens by surgical specimens; adding a fluorescent marker CD57, CXCR5 and CD4 antibody after isolated peripheral blood lymphocytes, performed flow cytometry to analysis of CD4+CXCR5+CD57+T cell expression; ELISA method to detect IL-21, IL-6, IFN-γ, CXCL13 and CXCR5 of concentration in serum. ALT,AST, bilirubin, Ig G of concentration in serum with biochemical tests analyzed serum of patients and concentration in serum.HBV-DNA expression of HCC with PCR-fluorescent probe assay; a nalysis IL-10, TGFB1, RORC, IL-17 A, IL-21, the expression of IL-6,CXCR5 and CD57 and other gene of tissue samples by using fluorescence quantitative QPCR in liver cancer specimens.Cell research:Lymphocyte separation from healthy umbilical cord blood or peripheral blood, after IL-2(300U / ml), IL-1α(100U / ml) and IL-21(40ng / ml)induced, isolated by screening the beads, the addition of CD57, CXCR5 and CD4 fluorescently labeled antibodies to detect CD4+CXCR5+CD57+T cell expression with flow cytometry.Lymphocytes Cell culture fluid 0.5ml mixed in Hp G2 Cell culture fluid,the ratio was 20%, after the 24-48 hours to Hp G2 cells apoptosis detection.Analysis of shuganjianpi prescription, detoxification cancer prescription, and DDP inhibition of Hp G2 cells growth by MTT. ELISAdetects L-21, IL-6, IFN-γ and CXCR5 of concentration cell culture fluid.Animal experiment:Kunming mice, C57BL/6(purchased from Guangdong Medical Experimental Animal Center), C57BL/6-HBv(purchased from Shanghai South Model Organism Research Center), and C57BL/6-Foxp3GFP(purchased from the Jackson Laboratory). H22 hepatoma cells(Guangdong Medical Experimental Animal Center) injection 0.1ml in left forearm armpit, 48 hours after giving drug intervention and intervention time is 10 d.After the mice were sacrificed, the liver, lung and tumor tissue HE staining and immunohistochemical analysis expression of CD57, CXCL13protein; after grinding aseptic isolation spleen, separation of lymphocytes,detect CD4+CXCR5+CD57+T cells with flow cytometry. ELISA detect IL-21, IL-6, IFN-γ, CXCR5 and CXCL13 of peripheral blood; PCR fluorescence probe to detect HBV-DNA expression; analysis of IL-10,expression TGFB1, RORC, IL-17 A, IL-21, IL-6, CXCR5, CD57 and other genes of tumor tissue by fluorescence quantitative RT-PCR.Statistical methods:Using SPSS(statistics package for social science) 19.0 software for data analysis, quantitative data if they meet the normal distribution, descriptive statistics available indicators mean and standard deviation, if not normal distribution, the median index selection and differential(ie: difference between maximum and minimum). For normally distributed of quantitativedata, such as the two groups using t test.P<0.05 was considered statistically significant.Result:Clinical trials:Pathological examination results show that HBs Ag positive and CD57-positive results for 83%, indicating that there is a close correlation between the two groups. CD4+CXCR5+CD57+T cells in the normal population is 5.95%, in stage I-IIIA HCC patients is 5.39%, in stage IIIB-IV HCC patients is 3.21%, and it is statistically significant differencebetween the normal population and IIIB-IV liver cancer(P <0.05).CD57+CXCR5+T cells in the normal population is 20.9%, it is 13.8% in stage I-IIIA HCC patients, it was 8.44% IIIB-IV patients with hepatocellular carcinoma, and it is statistical significance among the three groups;CD4+CD57+T cells in the normal population is 3.5% and 3.18% in stage I-IIIA HCC patients and 2.02% in stage IIIB-IV HCC patients, it is a significant difference between the three groups.CD4+CXCR5+CD57+T cells expression of in the normal population is22.9%, and I-IIIA patients with liver cancer are 8.18%, and IIIB-IV is 2.79%of liver cancer patients, with statistical significance. ELISA analysis found that the number of IL-21 is significantly different, and INF-γ was no significant difference; RT-PCR results showed that Hbs Ag positive samples of IL-6, LI-21, IL-17 A, CD57 and CXCR5 of gene expression wassignificantly decreased, with statistical significance.Regression analysis showed that CD4+CXCR5+CD57+T cells is closely related to the level of ALT, and CD4+CXCR5+CD57+T cells will be increased when elevated levels of ALT, with statistical significance.CD4+CXCR5+CD57+T cells levels are closely correlated with the level of Ig G; there is no significant relationship with HBV-DNA.CD4+CXCR5+CD57+T cells increase survival time and the longer the survival time of patients.Cell Research:Peripheral blood stem cells differentiate into CD4+CXCR5+CD57+T cells by IL-2, IL-21 and INF-γ induced, and increasing the number of cells increases with culture time, accounting for CD4+cells count of 22%; induced the CD4+CXCR5+CD57+T cells secrete IL-6, IL-21, INF-γ, with time correlation, and no secreted CXCL13.Flow analysis showed CD4+CXCR5+CD57+T cells induced 71.3% of Hp G2 apoptosis in 24 h hours, 89.2% in the 48 hours with statistical significance, the analysis found that the expression of the mechanisms associated with FAS. CD4+T-cell count 21.4 percent of the total,CD4+CXCR5+T cells was 5.9%, while the CD4+CXCR5+CD57+T cells was9.7%, CD57+T cells was 28.2 % when induce 12 d. With the incubation time,CD4+, CD57+, CD4+CXCR5+and CD4+CXCR5+CD57+T T cells was significantly increased, with statistical significance.Shuganjianpi prescription inhibition rate of 11.1%, detoxification cancer prescription is 72.8%, DDP 74.7%, with significant differences between the detoxification cancer prescription and Shuganjianpi prescription groups.Detoxification cancer prescription can be suppressed secretion of IL-6; and inhibiting the secretion to IGF-IR, the effect is quite DDP.Animal Research:Detoxification cancer prescription can inhibit the growth of tumor tissue, but its role is lower than DDP; liver and lung of C57BL/6-HBV appear lymphocytes and plasma cells infiltratied. H22 of C57BL/6-HBV mice that Ki67 immunohistochemistry the positive rate was 80%, TCM intervention later positive rate was 30%, DDP was 10%, IL-21-positive rate of 10% or less, with statistically significance(P<0.05). ELISA results show that TCM can improve the IL-6, IL-21, INF-γ, CXCL13 levels, with statistical significance. HBV can significantly inhibit the expression CD4CXCR5, prompting IL-6 was significantly increased, IL-21 decreased,TCM intervention after, was significant increased in CD4CXCR5 of cell number. It reduced the number of cells CD4CXCR5 after DDP intervention.It can significantly increase the number of CD4CXCR5, with IL-21 intraperitoneal injection.CD4+CXCR5+CD57+T cells of C57BL/6 mice were was 6.2%, while C57BL/6-HBV was 2.16%, there are significant differences between the two groups. Infect H22 cells to C57BL/6-HBV, d5, d10 and d14 to detect flowcytometry, and the results show, CD4+CXCR5+CD57+regulatory T cells did not change significantly, give detoxification cancer prescription interventied,CD4+CXCR5+CD57+T significant increase in the number of T cells,CD4+CXCR5+CD57+T cells showed no change after DDP interventied,CD4+CXCR5+CD57+T cells increased significantly after IL-21 interventied.Real-time quantitative PCR analysis of the expression of H22 tumor in Tfh, Th17 and Treg-related genes found that IL-6, LI-21, IL-17 A, CD57 and gene expression of CXCR5 decreased significantly in the C57BL/6-HBV;IL-6 and IL-21 significantly improve, CXCR5 and gene expression of CD57(P <0.01) after detoxification cancer prescription interventions; after DDP intervention, IL-6, IL-21, CXCR5, CD57 gene decreased expression. Gene expression analysis that HBV significantly inhibited Tfh, Treg, Th17-related genes, but in DDP and detoxification cancer prescription interventions after,there are very significant differences, the significant increase CD4+CXCR5+CD57+T cells in detoxification cancer rescription.Conclusion:1. CD4+CXCR5+CD57+T cells are a subpopulation of CD4+CXCR5+T cells;inhibit liver cancer effects and induced Hp G2 cells apoptosis through CD95.HBV can inhibit CD4+CXCR5+T cells and CD4+CXCR5+CD57+T cells function, which may be an important factor in the formation of liver cancer.2. The toxin is central place of liver cancer pathogenesis, and based on the toxin can inhibit the body’s immune function, CD4+CXCR5+CD57+T cellsand CD4+CXCR5+T cell function decreased so that the body showed righteousness deficiency.3. Detoxification cancer prescription can kill Hp G2 cells; reduce the secretion of IL-6 and IGF-IR. Detoxification cancer prescription significant increased number and function of CD4+CXCR5+T cells and CD4+CXCR5+CD57+T cells. It may be the mechanism of action against cancer. |
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