| The liver has many functions such as protein synthesis,detoxification,and glycogen storage,and is the largest organ in mammals.The liver needs a high oxygen supply to maintain organ physiological functions and is highly susceptible to ischemia-reperfusion(I/R),an important factor causing liver injury in clinic.Therefore,finding a safe and effective method to enhance cell resistance and liver regeneration ability is the key to reduce liver I/R damage.Adipose-derived mesenchymal stem cells(ADSCs)are pluripotent stem cells that are abundant and readily available in tissues and have a high self-renewal capacity.ADSCs have the potential to differentiate into adipogenic,osteogenic,chondrogenic and myoblasts.Studies have shown that ADSCs have the ability to differentiate into hepatocytes,with hepatocyte functions such as urea formation,glycogen synthesis,cytochrome P450 enzyme activity and expression of hepatocyte-specific genes,and ADSCs can be used to treat liver diseases such as liver failure,hepatitis and liver fibrosis.ADSCs transplantation is an effective method to promote normal function and promote liver regeneration after extensive liver injury.ADSCs can secrete cytokines and growth factors related to liver regeneration.In the rodent models,differnet ADSCs transplantation methods after liver injury can show protective effects on the liver and can be stably integrated into the diseased liver tissues.However,in large experimental animals,the application of ADSCs in the treatment of I/R combined with partial hepatectomy has not been reported.Therefore,this study aims to provide a basis for research on liver disease treatment,organ transplantation and stem cell transplantation in the field of comparative medicine through the Bama miniature pig experimental model.Adipose tissues were obtained subcutaneously form the abdominal of Bama miniature pigs for isolation and culture of ADSCs,and the morphological characteristics of ADSCs were observed.The differentiation potential of porcine ADSCs was identified by using mesenchymal stem cell differentiation medium.The expression of surface stem cell-specific antigen in ADSCs was detected by flow cytomety.Eighteen Bama miniature pigs were randomly divided into Sham group,LLR group and ADSCs group,six in each group.In the Sham group,only CO2 pneumoperitoneum was established and the hepatic lobe were were turned over by laparoscopic instruments without hepatic blood flow interruption and hepatectomy.The LLR group and the ADSCs group were treated with laparoscopic left hemihepatectomy after 60 min for right hepatic ischemia.After resection,the ADSCs group was transplanted with ADSCs through the liver parenchyma immediately after completion of partial hepatectomy(1×106/kg),the LLR group was injected with the same amount of normal saline.And serum and liver tissue samples were collected for each index detection.Changes in inflammatory cells in peripheral blood and lvier biochemical parameters in serum were examined.Histopathology and ultrastructure of liver were observed.Antioxidant enzyme activities and lipid peroxidation level were detected by commercial kits.Detection of changes in serum inflammatory factors and regenerative factors by ELISA.The levels of inflammatory factors,autophagy,apoptosis-related genes and regeneration factors in liver tissues were detected by q RTPCR.Western Blot method was used to detect the changes of autophagy and apoptosis related proteins in liver tissues.Hepatic apoptotic positive cells were detected by TUNEL method,and the changes of autophagy,apoptotic and regeneration-related proteins were detected by immunohistochemistry.The results of this study are as follow:(1)Porcine ADSCs had a fibroblast-like spindle shape and proliferated rapidly in the culture medium.ADSCs positively expressed stem cell-specific antigens CD29,CD44 and CD105,but not express hematopoietic stem cell-specific antigen CD34.ADSCs can be differentiated into adipocytes,osteoblasts and hepatocytes by in vitro induction culture.(2)In the LLR group,the inflammatory cells WBC,NE,LY,serum enzymes ALT,AST,LDH ALP and T-BIL were significantly increased,and TP was significantly decreased.ADSCs treatment can reduce inflammatory cells in peripheral blood and accelerate liver function recovery,significantly inhibited the increase of NE,LY ALT,AST,LDH and T-BIL,however,there was no significant difference in the WBC,TP and ALP between the LLR group and the ADSCs group.(3)In the LLR group,the structure of hepatic lobule was destroyed,and there were multiple focal necrosis,severe vacuolar degeneration and a large amount of inflammatory cell infiltration were observed under light microscopy.Serious nuclear membrane shrinkage and endoplasmic reticulum dilatation were observed under electron microscopy.After ADSCs treatment,the pathological and ultrastructure changes of the liver were alleviated,the infiltration of inflammatory cells were reduced under light microscopy,and the vacuolar degeneration of hepatocytes,nuclear changes and endoplasmic reticulum expansion of hepatocytes were alleviated under electron microscopy.(4)The activities of antioxidant enzymes CAT,GSH-Px and SOD in serum in the LLR group were significantly decreased,MPO activity and MDA content were significantly increased.ADSCs treatment can enhance CAT and SOD activities,and significantly reduce MPO activity,alleviate the oxidative damage caused by I/R and partial hepatectomy,but there were no significant difference in the level of lipid peroxidation between the two groups.(5)The expression of pro-inflammatory factors IL-1?,IL-6,TNF-? and anti-inflammatory factor IL-10 protein and mRNA in serum and liver tissues in the LLR group and the ADSCs group were significantly increased,and serum CRP expression was significantly higher than the in the Sham group.ADSCs treatment significantly inhibited the inflammatory response induced by hepatic I/R and partial hepatectomy,reduced the expression of pro-inflammatory factors and mediators,and increased the expression of anti-inflammatory factor.(6)The expression of ATG5,ATG12 and Beclin1 mRNA in liver tissues in the LLR group and the ADSCs group were significantly increased,and the expression of P62 and m TOR mRNA were significantly decreased.ADSCs treatment significantly reduce the expression of ATG5 and ATG12,but there were no significant difference in the level of P62,Beclin1 and m TOR between the two groups.Compared with the Sham group,the expression of LC3 ? and Beclin1 protein increased significantly in the LLR group and the ADSCs group,and the expression of P62 protein decreased significantly.ADSCs treatment significantly increased P62 protein expression,decreased LC3 ? and Beclin1 protein expression,and inhibited autophagy progression caused by hepatic I/R and partial hepatectomy.(7)ADSCs treatment could inhibited the apoptotic process caused by hepatic I/R and partial hepatectomy.The number of TUNEL-positive cells and Caspase activites were significantly lower than those in the LLR group.ADSCs treatment significantly inhibited the expression of Fas,Fas L,Bax protein and mRNA in liver tissues,and increased the expression of Bcl-2 protein,but there was no significant difference in Bcl-2 mRNA expression in liver between the two groups.(8)ADSCs treatment can significantly enhance the process of liver regeneration after operation,increase the expression of angiogenic factors VEGF,ANG-1 and ANG-2 in serum,and decrease the level of serum HA.At the same time,ADSCs treatment enhanced the expression of growth factors HGF and Cyclin D1 mRNA in liver and inhibited the expression of TGF-?.The number of PCNApositive cells in the ADSCs group was significantly higher than that in the LLR group.In summary,porcine ADSCs express stem cell-specific antigens and has the potential to differentiate into adipocytes,osteoblasts and hepatocytes.After liver parenchymal transplantation,ADSCs can inhibit the increase of inflammatory cells in peripheral blood ater hepatic I/R and partial hepatectomy,and accelerate the recovery of liver function.ADSCs treatment reduce alleviate liver injury caused by I/R and partial hepatectomy by reducing oxidative stress,inhibiting inflammation,suppressing apoptosis and autophagy,and promoting the process of liver regeneration after surgery.In this study,ADSCs were successfully used to treat liver caused by hapatic I/R and partial hepatectomy,which proved that ADSCs had anti-oxidation,anti-inflammatory,anti-apoptotic,antiautophagy and promote liver regeneration in this model.This topic lays the foundation for the application of laparoscopic technology in the liver injury model of large experimental animals,and provides important theoretical basis for the research of liver disease treatment,organ transplantation and clinical application of stem cells. |
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