| Objective: Screening of rats' bone marrow mesenchymal stem cells by whole bone marrow adherent method and identifing of them by using flow cytometry instrument technologyby.To observe the effect of“Clean-heat,cool the blood,disintoxication and Removing blood stasis”combining with BMSC transplantation on the expression of TNF-a,iNOS gene and the expression of nuclear factor kappa B(NF-KB)in liver tissue of ALF rats.Method:1.The bilateral femurs and tibias of rats were taked out under the septic conditions,and flushing out bone marrow from marrow cavity in super clean bench.The bone marrow were administered in the culture medium containing10% fetal bovine serum.changing of the culture regularly and observing the cells morphology and growth under inverted microscope.By flow cytometry instrument to detect cell surface antigen CD29,CD44,CD34,CD45 expression for identification.2.Establish acute liver failure rats model and validate whether building successThe male SD rats,whose weight were about 180g-200 g were randomly divided into model group and control group.The model group rats were induced by gavage administration of TAA 400mg/(kg·d),interval repeat administration after 24 hours at a same time.The control group rats were administrated 2ml physiological saline gavage.Building after 48 hours,the abdominal aorta blood of living rats was taken.The levels of liver function index and plasma endotoxin were examined.Taking out part of liver tissue for pathology detection and observing the histopathological changes of liver under optical microscope.3.The experimental group and intervention treatmentSixty male SD rats,weighed for 180g-200 g,were randomly divided into the normal group(group A),thioacetamide group(TAA,group B),TAA +Compound glycyrrhizin glucoside piece group(group C),TAA + traditional Chinese medicine(group D),TAA + BMSCs(group E),TAA + traditional Chinese medicine + BMSCs(group F).There were 10 rats in Each group.The rats of group B?C?D?E?F were induced by gavage administration of TAA400mg/(kg·d),interval repeat administration after 24 hours at a same time,while group A was given 2ml saline by gavage.After the second gavage,group E and group F were injected BMSCs(about 1x107 cells every rat)by tail vein while group A?group B?group C?group D were injected equivalent BMSCs culture medium.24 h later,each group was injected the same liquid by tail vein repeatedly.When the first time for tail vein grafts,group D and F were induced by the gavage administration of tradition Chinese drugs pharmaceutics“Yin Chen Si Ling”every day,group C was gavaged compound glycyrrhizin glucoside piece,while group A?B?E were gavaged equivalent distilled water.All rats were put to death at 72 hour after treatment.The levels of liver function index and plasma endotoxin were measured.Detecting each group liver tissue of TNF-a and iNOS 's gene transcription by RT-PCR.Detecting the expression of NF-KB in liver tissue by SABC immunohistoehemical.Result:1.The adherent cells were successfully isolated.After three passage screening,the morphology of the cells gradually tended to be consistent,and the spindle shape was the main form of these cells.The distribution of cells was net-like,radial arrangement.The flow cytometry results showed that: cells high expression of CD44 and CD29,positive rate were 98.2% and 97.9%respectively;Low expression of CD34 and CD45,cell positive rate were 12.9%and 18.7%,which matched the characteristic of BMSCs.2.The validation of the model of acute liver failureCompared with control group,the model group ras' the levels of ALT?AST and plasma endotoxin were remarkbly increased,the difference between the two groups had significant statistical significance(P<0.01).The hematoxylin-eosin of model group's liver showed that the structure of hepatic cords were mostly destroyed,liver cell necrosis,apoptosis,inflammatory cell infiltration in liver.The hematoxylin-eosin of control group's liver showed that the rules and arrangement of liver cells with regular structure and normal cell morphology,no inflammatory cell infiltration.3.The prognosis of acute liver failure rats with different intervention treatment(1)ALT,AST and endotoxin level results: The treatment of the four groups(C,D,E,F)in the rat serum AST,ALT and plasma endotoxin levels significantly lower than the model group(group B),the difference was statistically significant(P < 0.01);But the treatment group compared with normal group(group A),AST,ALT,endotoxin level is still high,the difference was statistically significant(P < 0.01);Each treatment group compared with group F rats serum AST,ALT and plasma endotoxin level was lower than those of group C,D,E,the difference was statistically significant(P < 0.01);C,D,E comparison between three groups,there was no statistically significant difference(P > 0.05).(2)RT-PCR results : There was a small amount of TNF-a and iNOS 's gene transcription in the group A 's liver tissues.Compared with the group A,the mRNA levels of iNOS and TNF-a in groups(B?C?D?E?F)increased significantly,the difference was statistically significant(P < 0.01).The gene transcription level of the treatment groups(C?D?E?F)in rat liver tissue iNOS and TNF-a were significantly lower than model group(group B),the difference was statistically significant(P < 0.01);Comparison among different treatment group,group F iNOS and TNF-a gene transcription level was lower than those of C,D,E,the difference was statistically significant(P < 0.01);C,D,E,the three groups of iNOS and TNF-a gene transcription level,there was no statistically significant difference(P > 0.05).(3)Immunohistochemistry results: A small amount of cells with NF-KB expression in cytoplasm or nucleus could been seen in liver tissue of group A.Compared with group A,the number of cells expressing the NF-KB in the five groups whose rats was Lavageed were significantly increased,with significant difference(P < 0.01).After the intervention,the number of cells expressing the NF-KB in the treatment group(group C,D,E,F)were less than that of group B,the difference was statistically significant(P < 0.01),while compared with group A,the NF-KB expression were still high,the difference was statistically significant(P < 0.01).Comparison among treatment group,the cells which expressed the NF-KB of group F was decreased compared with group C?D?E,the difference was statistically significant(P < 0.05),and the number of cells expressed the NF-KB in groups C,E,F were similar,there was no statistically significant difference among them(P > 0.05).Conclusion:1.Bone marrow mesenchymal stem cells can be successfully screened out by whole marrow cultivate.It is an used Andy ideal method for in vitro culture.2.Acute Liver failure rats model can be successfully created by gavage administration of TAA 400mg/(kg·d),interval repeat administration after 24 hours at a same time.TAA 400mg/kg in two consecutive days,moreover,the model with high endotoxin.3.BMSCs transplaniation,Tradition Chinese drugs pharmaceutics“Yin Chen Si Ling”and Compound glycyrrhizin glucoside piece can Significantly reduce intrahepatic uncontrolled inflammatory reaction which was triggered by the endotoxin in acute liver failure.They can reduce the expression of NF-KB and downregulate the gene transcription of iNOS and TNF-a.It might be the mechanisms that they regulated the uncontrolling caseade of inflammation.4.The joint intervention of traditional Chinese Medicine and BMSCs transplantation has better effect than only with Chinese medicine treatment or only with BMSCs transplantation therapy.The two therapy-Traditional Chinese Medicine and BMSCs transplantation may has synergistic effect in intrahepatic uncontrolled inflammatory reaction which was triggered by the endotoxin in acute liver failure. |
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