| Objectives: to investigate the effects of the shuganjianpi method (the Liver-soothing and Spleen-fortifying Method) on the proliferation and induced differentiation of HSCs, the promotion of liver regeneration and the method' s regulatory mechanism, to explore the mechanism of action on treatment of chronic hepatic diseases, and to offer a new insight into the furtherance of therapeutical principles and methods.Methods: 1: Effects of shuganjianpi method of of Chinese Traditional Medicine on hepatic stem like cell: all of the sera were divided into five groups, rat serum group, control group, shuganjianpi group which was subdivided into three subgroups according to the concentration of 5%, 10% and 20%, shugan group which was subdivided into three subgroups according to the concentration of 5%, 10% and 20% and jianpi group which was subdivided into three subgroups according to the concentration of 5%, 10% and 20%. MTT spectrometric assay was used to tested the effects of different concentration serum containing drug on proliferation of hepatic stem like cells. Toxicity of different concentration serum containing drug on hepatic stem like cells was evaluated by LDH activity in supernatant. 2. Study of molecular mechanism of effects of shuganjianpi method on expressions on HGF receptor, EGF receptor, FGF receptor, TGF-βreceptor and CTGF receptor in hepatic stem like cells, all of the sera divided into five groups, rat resum group, control group, shuganjiani group of concentration 10%, shugan group of concentration 10% and jianpi group of concentration . 10% The expressions on HGF receptor, EGF receptor, FGF receptor, TGF-βreceptor and CTGF receptor in hepatic stem like cells were assayed by western blot. 3. Effects of shuganjianpi method on differentiation of hepatic stem like cells: WB-F-344 cells were cultutres in differential system(DMEM, 10% FCS, , HGF50ng/ml, EGF20ng/ml, Insulin 1μg/ml and Dex 1μmol/L together with 10% serum containing Powder for Regulating Liver and Spleen combined with Decoction of Four Mild Drugs. Then the expressions of AFP and ALB mRNA were assayed by RT-PCR. 4: Effects of shuganjianpi method on apoptosis of hepatic stem like cells: all of the sera divided into five groups, rat resum group, control group, shuganjiani group of concentration 10%, shugan group of concentration 10% and jianpi group of concentration 101 Apoptosis of hepatic stem like cells was detected by flow cytometry. 5: Study of effects shuganjianpi method on hepatic stem cells' proliferation and hepatic regeneration: all of the sera divided into five groups, rat resum group, control group, shuganjiani group of concentration 10%, shugan group of concentration 10% and jianpi group of concentration 10%. The model of rat hepatic stem cells' proliferation was made by 2-AAF and 2/3PH method. Immunohistochemistry revealed CK, PCNA, Vimentin, AFP and ALB protein or their receptors in the liver.Results: 1. The effects of the shuganjianpi Method on HSC line WB-F-344: compared to contrast group, drug-containing serum group can stimulate WB-F-344 cells proliferation with remarkable deference (P<0.01). Drug-containing serum groups of different concentration can promote WB-F-344 cells proliferation in a concentration-dependent manner with significant difference in comparison with rat serum groups of different concentration (P<0. 05). activities in the supernatant of drug-containing serum groups of different concentration have no significant difference in comparison with rat serum groups of different concentration. 2. The expression of HGF, EGF and CTGF protein in WB-F-344 cells treated by different methods, is highest by the shuganjianpi Method, higher by the shugan (Liver-soothing) Method in method and high by the jianpi (Spleen-fortifying) Method. The expression of FGF receptor protein exhibits no significant difference in WB-F-344 cells treated by different methods, the expression level of TGF-βreceptor protein is highest in the shuganjianpi Method group in comparison with those in the other two groups, while the expression levels of TGF-βreceptor have no significant difference between shugan group and jianpi group. 3. AFP mRNA expression in WB-F-433 cells treated by the shuganjianpi Method during differentiation is down-regulated dramatically in comparison with that by shugan Method and jianpi Method respectively. And ALBmRNA expression in WB-F-433 cells treated by the shuganjianpi Method during differentiation increases remarkably in comparison with that by shugan Method and jianpi Method respectively.Conclusion: 1. HSC line, WB-F-433 cells can simulate proliferation on a dose-dependent manner by using the shuganjianpi Method (Powder for Regulating Liver and Spleen combined with Decoction of Four Mild Drugs, sinisanhesijunzitang in Chinese pinyin), the shugan Method (Powder for Regulating Liver and Spleen, sinisan in Chinese pinyin) and the jianpi Method (Decoction of Four Mild Drugs, sijunzitang in Chinese pinyin) without toxicity. 2. Compared with the shugan Method and the jianpi Method, the expression levels of HGFR, EGFR, CTGFR, TGF-βR and FGFR were up-regulated in WB-F-433 cells by using the shuganjianpi Method. 3. HSC lines WB-F-433 cells proliferation and differentiation were regulated by cytokine system when the shuganjianpi Method was used, and WB-F-433 cells might be promoted to differentiate into hepatocytes by the treatment method. 4. WB-F-433 cells apoptosis was inhibited significantly by using the shuganjianpi Method in comparison with the shugan and the jianpi method. 5. WB-F-433 cells proliferation and differentiation induced through the shuganjianpi Method are mediated by HGF, TGF, EGF, FGF and CTGF.
|
PDF Full Text Request