Effect Of Thymosin β4on Hepatocytes Proliferation In Vitro And Serum Thymosin β4Level Of Patients With HCC

Objective Thymosin (34as an actin-sequestering peptide that can promote regeneration of heart after myocardial infarction, has the potential to be used to treat liver failure. It was also reported that thymosin (34upregulates hepatocyte growth factor in human hepatic stellate cells. In the earlier study, we found that serum thymosin (34which associates with MELD score can be used as an important potential predictor for liver failure caused by chronic HBV infection. The aim of this study is to observe the effect of thymosin β4in hepatocyte proliferation in vitro, and to investigate serum thymosin (34levels in patients with hepatocellular carcinoma.Methods1. Human normal hepatocytes (7702cells line) were cultured in vitro. Solutions with different concentration of thymosin P4and blank control solution were added in the culture media. Rates of growth were observed by MTT test at12h,24h,48h and60h after thymosin β4were added.(3-catenin as the key factor of Wnt signal pathway were detected by methods of immunocytochemistry and western blot. Cyclin Dl was detected by RT-PCR.2. Serum thymosin β4levels were measured in80patients with hepatocellular carcinoma,40patients with cirrhosis and40healthy controls. Serum thymosin (34levels were measured by ELISA. Levels of serum thymosin (34in different stages of HCC were compared. Patients with HCC were divided into groups according with or without interhepatic metastasis, portal vein tumor thrombus and distant metastasis and then compared.Results1. We found that thymosin β4stimulates proliferation of normal human hepatocytes. As concentration of thymosin β4is higher, the OD value of MTT is higher. There was statistically significant difference (P<0.01). We also found that thymosin β4upregulates expression of β-catenin. The mean IOD value in group1ng/ml, group100ng/ml, group1μg/ml vs control goup, group1ng/ml vs group1μg/ml and group100ng/ml vs1μg/ml were statistically significant different (P<0.01). But there were no statistically significant difference between group lng/ml and group100ng/ml (P=0.59). Meanwhile, thymosin β4downregulates E-cadherin expression, and there were statistically significant difference (P<0.05). Expression of Cyclin Dl was also upregulated by added of thymosin P4detected by RT-PCR.2. Compared with healthy controls, serum thymosin β4levels of patients with HCC and cirrhosis were lower and there were statistically significant difference(P<0.001),9.5306(5.2466-13.7666) μg/ml vs1.4972(1.0675-2.6932)μg/ml,1.8112(0.8367-2.3184) μg/ml. Patients with HCC were divided into group A, B, C and D according their BCLC stage. Levels of thymosin β4in these4groups were statistically significant different (P=0.034),1.0446(0.8911-1.6242)μg/ml、1.6667(1.1492-2.6533)μg/ml、2.1333(1.0425-3.1421) μg/ml and1.2748(1.1335-2.7301) μg/ml. Patients with HCC were divided into group0,1,2,3,4and5scores according CLIP score. Levels of thymosin (34in these6groups were statistically significant different(P=0.018),0.9841(0.8871-2.1787)μg/ml、1.5350(1.0821-2.6188) μg/ml、1.2783(1.0073-2.0041)μg/ml、2.7714(1.1805-3.1402) μg/ml、1.0627(0.9626-1.1814) μg/ml and2.0948(1.1582-4.0003)μg/ml. Meanwhile, Patients with HCC were also divided according interhepatic metastasis, portal vein tumor thrombus and distant metastasis. Serum thymosin (34levels were higher in patients with interhepatic metastasis than in whom without it (P=0.024),1.5872(1.1251-2.7407)μg/ml vs1.0632(0.9053-1.7015)μg/ml. Serum thymosin β4levels were higher in patients with portal vein tumor thrombus than in whom without it(P=0.010),2.4440(1.1424-3.1441)μg/ml vs1.1993(0.9991-1.9661)μg/ml. But serum thymosin β4levels in patients with distant metastasis had no statistically significant different with whom without it. Patients who were neither developed portal vein tumor thrombus nor distant metastasis were divided into group A, B and C according Child-pugh grade. Levels of thymosis β4were1.7228(1.0540-2.6878) μg/ml、1.3377(0.9934-1.8901)μg/ml and0.9820(0.7939-1.1842)μg/ml, and there were statistically significant difference (P=0.039).Conclusion1. In vitro, thymosin (34promote hepatocytes proliferation and upregulates expression of β-catenin and Cyclin D1.2. Serum thymosin (34levels in patients with HCC were lower than that in healthy controls. And serum thymosis β4levels in patients with interhepatic metastasis or portal vein tumor thrombus were higher than patients without them.