In Vitro Experiment Study On The Effect Of Clearing-heat And Detoxicating Combined With Cooling Blood And Removing Blood Stasis On Bone Marrow Mesenchymal Stem Cells In The Environment Of Hepatic Failure

Objective: Hepatic failure simulation environment was established by the rat serum,and the mesenchymal stem cells were cultured in vitro.To observe the mesenchymal stem cell proliferation,differentiation and immune regulation in liver failure environment,and the effect of Chinese medicine on mesenchymal stem cells in the environment of hepfatic failure.To further explore the detoxification method of cooling blood to remove stasis on liver failure in bone marrow mesenchymal stem cell and synergistic effect at the cellular level.Method:1.The screening of 1 rat BMSCs were isolated and cultured and passaged.Extraction,separation,from 3-4 week old SPF SD rats bone marrow culture BMSCs.Using the whole bone marrow culture method and passaged screening method for cell culture and purification.When the cell proliferation at 80~90% were passaged.For the detection and further transplant cells to the third generation.Through flow cytometry identified cells were used for BMSCs.2.the experimental serum preparation:30 rats were divided into model group and healthy group,21 rats in the model group model of liver failure made by intraperitoneal injection of D-GalN.After 48 h,3 rats were randomly selected from the model group and the healthy group,to collect liver tissue for pathological section,and the model was successful.Then 14 model rats were divided into two groups:Chinese medicine intervention group and liver failure group.Chinese medicine intervention group intervention group received intragastric administration of Ganduqing granule,liver failure group and healthy group were given equal dose of physiological saline.3 groups of rats were intragastric administration for 3 days,then abdominal aortic blood and serum separation,filtration sterilization,preparation of Chinese intervention serum and liver failure serum,serum of healthy,placed the serum in the refrigerator spare-20℃.3.the establishment of liver failure simulation environment of bone marrow mesenchymal cell culture system,acquisition and related indexesThe third generation of BMSCs were seeded in 96 hole plates,6 hole plates and 24 hole plates,the establishment of 3 experimental groups were added to healthy serum,the intervention of traditional Chinese medicine serum,liver failure serum for BMSCs culture.In 96 hole plates BMSCs training,with 3 wells in each group,respectively,in the culture of 1D,2D,3D,termination of culture,CCK-8was detected using enzyme,quantitative analysis standard test data.Observe the proliferation of BMSCs.6 well plate culture BMSCs,with 3 wells in each group,after 10 d,the termination of cultured cells was extracted,and the Western blot method for the detection of alpha fetoprotein(AFP),liver albumin(ALB),indoleamine 2,3 two oxidase(IDO),BMSCs analysis of hepatic differentiation and immune activity.24 well plate culture BMSCs,each group had 3 holes,climbing film processing,culture 10 d,culture and the production of CK18 termination,immunofluorescence slide.To differentiation was detected by immunofluo-rescence microscopy of liver BMSCs.Results:1.BMSCs in the serum in culture for 1 days and 2 days,the results of CCK-8 showed that there were no significant differences between the healthy group and liver failure group and traditional Chinese medicine group.On the third day,the results of CCK-8 showed that there were significant differences between the healthy group and liver failure group,the healthy group and Chinese medicine intervention group is the same.But no statistically significant between liver failure group and Chinese medicine intervention group.But after 10 day of cell culture,it was found that the cell growth of Chinese medicine intervention group was significantly better than that of the liver failure group.2.After 10 days of BMSCs culture,the cell were detected by CK18 immunofluorescence assay.The results showed the healthy group was negative,the liver failure group and the Chinese medicine intervention group was positive,the latter was strong,but it was significantly less than that of liver cells.To speculate that the cellular maturity of BMSCs hepatic differentiation was low.3.BMSCs after culture 10 days to detect by Western blot,ALB protein expression results showed: the health group,liver failure group and the traditional Chinese medicine group were expressed and gradually increase,between the groups have significant difference,but its expression quantity is less.AFP protein expression results showed: the health group,liver failure group and the traditional Chinese medicine group the were increased on expression intensity,and the differences between the groups were statistically significant.4.BMSCs after 10 days of culture,it was detected by Western blot anaylysis of IDO.No significant expression of IDO protein in healthy group.The expression of IDO protein in the traditional Chinese medicine intervention group was higher than that in the liver failure group,and the difference between the two groups was statistically significant.Conclusion:By means of the environment model of BMSCs transplanted liver failure in vitro,the proliferation activity of BMSCs in the environment ofliver failure was observed to decrease,with the trend of hepatic differentiation,and the immune regulation activity.Clearing heat and cooling blood,removing blood stasis and toxin of traditional Chinese medicine in liver failure environment in the proliferation of BMSCs in a short period of time had no obvious effect,but can promote cell differentiation,and significantly improve the cellular immune regulating activity.