Effects Of BMSCs Transplantation On Inflammatory Response And Tissue Repair After Acute Liver Injury Induced By RFA In Rats

Background: Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in our country.Currently,radiofrequency ablation(RFA)is one of the most effective methods for the treatment of HCC,especially for HCC less than5 cm.However,application of radiofrequency ablation in the treatment of HCC can cause certain thermal damage and inflammatory reactions on the liver tissue,thus,it is particularly important to investigate an effective treatment method that can reduce inflammation reactions and promote the recovery of liver injury.At present,the bone marrow mesenchymal stem cell(BMSCs)has attracted much attention because of its characteristics such as the self-renewal,multi-directional differentiation and immune-regulation.After serial passage and amplification,it still has strong multi-directional differentiation ability.Since BMSC can differentiate into liver cells,application of BMSC transplantation on the treatment of inflammatory reactions and tissue repair of liver injury after radiofrequency ablation still needs our further study.Objective:(1)To make an extraction,culture,purification and identification of BMSCs in rats so as to provide high-purity seeded cells for subsequent experiments.(2)To explore the method of establishment of a rat acute liver injury(ALI)model by using RFA so as to provide the guarantee for the following experiments.(3)To investigate the effect of BMSCs transplantation on the inflammatory response and tissue repair after acute liver injury induced by RFA in rats.Methods:(1)The BMSCs were extracted from the tibia and fibula of Wistar rats which were aged 4~6 weeks old and sacrificed after anesthesia,and then were cultured,extracted and purified by using the whole marrow adherent culture method.The second-generation BMSCs were used to count the number of passage cells and draw the growth curve of BMSCs.The flow cytometry was used for surface antigen marker identification of the third-generation BMSCs.The third-generation BMSCs were stained with alizarin red to perform osteogenic inducement and identification.(2)Firstly,42 Wistar rats were randomly divided into two groups: 30 rats in the first group and 12 rats in the second group;then,the first group was divided into 5groups(Group A,B,C,D and E)randomly,with 6 rats in each group;while,the second group was randomly divided into two group(RFA Group and Control Group),with 6 rats in each group.Group A,B,C,D and E in the first group were given radiofrequency ablation with the duration of 0min,2min,5min,8min and 11 min so as to observe the general condition and survival of the post-RFA rats and to determine the best duration for RFA.RFA Group in the second group was given radiofrequency ablation with the best duration already determined;while,Control Group was only inserted into the RFA electrode probe without booting up the RFA instrument for production of radio frequency.The serum ALT(Alanine Aminotransferase)and AST(Aspartate Aminotransferase)of RFA Group and Control Group at pre-and post-RFA6 hours were detected.After sacrifice of the rats,the liver injury area was observed by using pathological HE staining.(3)120 Wistar rats were randomly divided into 3 groups: Control Group(Group C),RFA Injury Group(RFA Group),RFA Damage +BMSCs Transplantation Group(RFA+BMSCs Group),with 40 rats in each group;and all the 3 groups were randomly divided into 4 observation time point: 1d,3d,7d and 14 d respectively,with10 rats in each group.The rat acute liver injury model of RFA Group and RFA+BMSCs Group were established by using RFA.RFA Group with RFA-induced liver injury was injected with 1 ml PBS solution in the tissue surrounding liver injury lesions;RFA+BMSCs Group with RFA-induced liver injury was injected with 1 ml PBS solution containing 1×106 BMSCs in the same position;group C only underwent laparotomy,and then was inserted with the RFA electrode probe instead of booting upthe RFA instrument and also injected with 1ml PBS solution at the same position.The general conditions and survival of the rats in each group were observed.The blood sample was drawn from 10 rats in each group at each time point(1d,3d,7d and 14 d,respectively)so as to detect the serum ALT and AST level by using blood biochemical analyzer,to detect the serum TNF-α and IL-10 level by using ELISA assay.The hepatic tissue homogenate TNF-α and IL-10 level in the same tissue of liver injury part were detected by using ELISA assay.The pathological observation of the same tissue in the remaining liver injury part was conducted.Results:(1)BMSCs grew well and the growth curve of BMSCs approximately showed in a S type,which belonged to the incubation phase at 1d~2d with relatively slow growth,belonged to the logarithmic growth phase at 3d~5d with rapid growth and belonged to the plateau phase at 6d~7d with slowing growth.High expression of CD29(99.2%),high expression of CD44(99.4%),high expression of CD90(99.6%),low expression of CD34(1.22%)and low expression of CD45(1.25%)were detected by using flow cytometry,which was consistent with the phenotypic identification of BMSCs.And the osteogenesis of BMSCs was induced and differentiated successfully.(2)The optimal RFA duration of the rat acute liver injury model established by using RFA was determined to be 5~8 minutes.After RFA Group was given radiofrequency ablation with the best duration already determined,the serum ALT and AST of RFA Group at 6h were significantly higher than those of Control Group(P<0.05).Pathological observation: According to the rough observation,the rat liver ablation damage region of RFA Group was rough,the central part of the probe canal was round defect,and the radiofrequency ablation necrosis area was distributed around the central part of the probe canal,with hard texture and gray white;and the junction zone of normal liver was hyperemia and hemorrhage area with a little hard texture and bright red.Under the light microscope,the necrotic cells in RFA Group were patchy,edematous and caryorrhexis.In the area of hyperemia and hemorrhage,there were hepatocyte edema,cell enlargement,deep staining of the nucleus,inflammatory cell infiltration and extensive hemorrhage.(3)General conditions and survival: The rats of Group C were in normal conditions and all rats survived;the rats of RFA+BMSCs Group were generally in acceptable conditions with the survival rate accounting for 95%;the rats of RFA Group were generally in poor conditions with the survival rate accounting for 75%.The serum ALT and AST levels: The serum ALT and AST levels of RFA Group at each time point and RFA+BMSCs Group at 1d and 3d were higher than those of Group C(P<0.05);while,no significant difference between RFA+BMSCs Group at7 d and 14 d and Group C in the serum ALT and AST levels was found(P>0.05);the ALT and AST of RFA Group at each time point was higher than that of RFA+BMSCs Group(P<0.05).Liver TNF-α and IL-10 levels: The liver TNF-α level of RFA Group at each time point and RFA+BMSCs Group at 1d and 3d was higher than that of Group C(P<0.05);no significant difference was found between RFA+BMSCs Group at 7d and 14 d and Group C in liver TNF-α levels(P>0.05);The liver TNF-α level of RFA Group at each time point was higher than that of RFA+BMSCs Group(P<0.05);the liver IL-10 of RFA+BMSCs Group at each time point and RFA Group at 1d and3 d was higher than that of Group C(P<0.05);no significant difference was found between RFA Group at 7d and 14 d and Group C in the liver IL-10 level(P>0.05);the liver IL-10 level of RFA+BMSCs Group at each time point was higher than that of RFA Group(P<0.05).Serum TNF-α and IL-10 levels: The serum TNF-α level of RFA Group at each time point and RFA+BMSCs Group at 1d and 3d was higher than that of Group C(P<0.05);no significant difference was found between RFA+BMSCs Group at 7d and 14 d and Group C in serum TNF-α levels(P>0.05);the serum TNF-αlevel of RFA Group at each time point was higher than that of Group RFA+BMSCs(P<0.05);the serum IL-10 level of RFA+BMSCs Group at each time point and RFA Group at 1d,3d and 7d was higher than that of Group C(P<0.05);RFA Group at 14 d and Group C showed no significant difference in the serum IL-10 level(P>0.05);the serum IL-10 level of RFA+BMSCs Group at each time point was higher than that of RFA Group(P<0.05).Pathological observation: Compared with RFA Group,RFA+BMSCs Group had less inflammatory cells,low degree of fibrosis and high degree of injury repair in the liver injury area.Conclusion:(1)It is easy to extract and cultivate a large number of high-purity Wistar rat BMSCs by using the whole marrow adherent culture method.The cultured BMSCs are featured with high purity,quick adherence and rapid amplification,which can be used for subsequent studies.(2)The rat acute liver injury model with the features of consistent damage,replicability and high survival rate can be established by using RFA,which provides a qualified animal model for further study on the mechanism of acute liver injury induced by RFA and the mechanism of liver injury caused by other factors.(3)BMSCs transplantation can reduce the inflammatory reaction afer acute liver injury induced by RFA in rats,and promote the repair of injury.