| Background and aims:Liver cancer is one of the most common malignant tumor in the world, and it s incidence in third place, the cases of deaths each yearMore than 500000.Because of its high malignant degree, prognosis is poor, known as the king of carcinoma.Wi th the development of liver cancer research and treatment of constantly updated, ha s obtained certain improve clinical curative effect of hepatocellular carcinoma.But is period, when most of the patients with liver cancer diagnosis is often missed oper ation time, surgical resection only 20-30%, and the conventional radiation and chem otherapy sensitivity is poorer, and the high rate of recurrence of liver cancer metast asis, these factors make the prognosis of patients with liver cancer is still unsatisfa ctory. Statistics show that liver cancer recurrence after 5 years was 70%, and the rec urrence of transfer has become the biggest obstacle to increase long curative effect in liver cancer, further is to conquer one of the key problems of liver cancer.Early liver cancer research mainly focused on gene mutations, such as the acti vation of proto-oncogenes or the inactivation of tumor suppressor genes, etc., hopin g to find play a decisive role in the development of liver cancer node molecules, a lso hope to like other tumors clinically a detailed classification of liver cancer.Due to the heterogeneity of cancer of the liver, the current research was disappointing.R ecent studies have found that the immune microenvironment in HCC development a lso plays an important role in the process, related to the tumor infiltrating immune cells such as Treg cells, macrophages, neutrophils and Thl7 cells in liver cancer de velopment has an important reuse, by secrete inflammatory factors associated applie s to promote or inhibit tumor cells.Treg cells plays an important role in maintaining the stability of the immune microenvironment, and participate in peripheral immune tolerance to maintain and p revent autoimmune reaction, and its main marker is Foxp3.Related studies have sho wn that infiltration of Treg cells in tumor tissue of the host immune response plays an inhibitory effect, so it plays a negative effect in the treatment of tumor.TGF -beta mainly activated macrophages to secrete, some damaged liver cells can secrete a small amount of TGF-beta, but by acting on TGF-beta receptors activate downst ream smad2/3, promote smad2/3 into the nucleus, in the development of the occurr ence of cancer of the liver plays an important role.Related studies have shown that TGF-beta became involved in the nature of CD4 + T cell differentiation process of Treg cells, its regulation in the host immune response and immune tolerance also play an important role in the process.Early research has shown that TGF-beta can i nduce the TCR defects of CD4 + CD25- nature of Foxp3 expression in T cells, cau sing peripheral CD4 + CD25- T cells respond to CD4 + CD25－Treg cells differentia tion, with a strong potential of immunosuppression.In addition, the study also found that TGF-beta in human peripheral blood Treg cells extend and amplification also plays an important role in the process.SM-16 is the specificity of TGF-beta type 1 receptor inhibitor, related studies have found that SM-16 by blocking TGF-beta pathway restrain the progress of liver fibrosis, however, SM-16 in the role of liver cancer are not reported.Therefore, thi s topic will be through the TGF-beta type 1 receptor inhibitor SM-16.TGF-betaresea rch on the function of Treg cells in the progression of liver cancer, and combined with the clinical sample analysis TGF-beta and IL 10 correlation.Methods:We divided the mice into three groups, namely healthy group, DEN group and DE N + SM-16 block group. Development of HCC, expression levels oF TGF-β and Fo xp3, serum levels of TGF-β, IL-10 and GP73, and percentage of Treg cells of the mice in the healthy group, DEN group and DEN + SM-16 group were analyzed. R ole of TGF-P in the polarization process of Treg cells was analyzed in vitro by flo w cytometry. TGF-P and IL-10 expression levels in tumor samples from HCC patie nts were identified by IHC, and their correlation was analyzed.Results:In the DEN-induced HCC mouse model, we proved whether SM-16 can successfull y block TGF-β. We sacrificed the mice in three groups at different time periods, a nd harvested their liver tissues. PCR results showed that the DEN group had a sig nificantly higher TGF-P expression level than the other two groups. ELISA results r evealed that the TGF-P protein concentration in peripheral blood of mice in DEN g roup was significantly higher than the other two groups. As for protein level, we al so found markedly higher TGF-β protein level in liver tissues of mice in DEN gro up compared with the other two groups by WB experiments. Observation of histolo gical changes in livers of mice in three groups by HE staining found evidently sev erer liver damage in DEN group than the other two groups, which was manifested as compact liver tissues and occurrence of massive neutrophil infiltration. Degree of liver damage in DEN + SM-16 group was almost the same with the healthy grou p. It was thus clear that the TGF-P blockade might play a certain hepatoprotective role. From the perspective of biological indicators, GP73 is a common serum marke r for early liver cancer. We detected its serum level by Elisa, and found markedly lower serum GP73 level in DEN+SM-16 group than the HCC groups, thereby dem onstrating that the malignant degree of HCC was decreased after SM-16 treatment. We used flow cytometry to detect the expressions of CD4T subtype-related genes T h1, Th2, Th17 as well as Treg cells in spleens of mice in three groups. The result s showed that the Treg cells changed substantially before and after TGF-β blockade, while the rest of cells all remained stable. Expression of Foxp3, a Treg-related ge ne, was detected at the transcriptional level. We also used ELISA to detect the seru m protein concentration of IL-10, a Treg effector, and found that both Foxp3 and I L-10 were expressed more highly in the DEN group than the other two groups. Fi nally, we validated our results using clinical samples. RNA was extracted from tissu e samples of different stages of liver cancer for transcriptional analysis, which sho wed that TGF-β and IL-10 expressions were positively correlated with the develop ment of liver cancer. Moreover, we analyzed the correlation between expressions of TGF-P and IL-10, and found that the two were highly correlated in tissue samples from liver cancer patients.Conclusion:The study confirmed that SM-16 DEN induced liver cancer in mice model of TG F - beta has inhibitory effect, can be adjusted in the nucleic acid and protein level s TGF - the expression of beta, and inhibit the secretion of mice serum TGF-beta. Also confirm the SM - 16 can inhibit the progress of the liver cancer and maligna nt degree.Relevant testing found that SM-16 can reduce the proportion of Treg cells. Clinical studies confirmed that liver cancer tissues TGF-beta and IL10 show correlat ion.To sum up, TGF-beta can be easily induced by polarization of Treg cells and p romote the progress of liver cancer. |
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