Studies On The Preparation And Therapeutic Effect For Experimental Liver Fibrosis Of Extracellular Vesicles Derived From Human Umbilical Cord Mesenchymal Stem Cells

Liver fibrosis is an over-repair process caused by liver damage and inflammation.It often accompanies most liver diseases,and can progress to cirrhosis or even liver cancer in severe cases.Hepatic stellate cells are the core of liver fibrosis and are mainly responsible for the production of extracellular matrix.They are in a static state in the normal liver.Once liver fibrosis occurs,hepatic stellate cells will transform into an activated state and form scars in the liver,resulting in changes in liver structure.At present,there is no especially targeted and effective treatment scheme for liver fibrosis.Stem cell transplantation is currently a popular choice in clinical trials.However,due to the incompleteness of stem cell therapy its clinical application has been greatly affected.Based on our previous research and literature support,we speculate that extracellular vesicles derived from human umbilical cord mesenchymal stem cells（hUC-MSC-EVs）can inhibit the activation of hepatic stellate cells and fiber synthesis,thereby exerting inhibition effect on liver fibrosis.The purpose of this study is to explore the preparation and therapeutic effect on experimental liver fibrosis of hUC-MSC-EVs.The main research contents and results of this thesis are as follows:（1）Preparation and characterization of hUC-MSC-EVsThe culture supernatant of human umbilical cord mesenchymal stem cells（hUC-MSCs）was collected and separated by ultra-high-speed centrifugation to obtain purified extracellular vesicles.Then the structure,particle size and surface markers of hUC-MSCs were analyzed.The results showed that the extracted extracellular vesicles had the characteristics as reported in the literature.（2）The effect of hUC-MSC-EVs on the metabolism and apoptosis of activated LX-2 cell modelAt the cellular level,a certain concentration of TGF-β1 was used to stimulate LX-2 cells and a activated human hepatic stellate cell line LX-2 model was constructed.The morphology of mitochondria was observed by ultra-high resolution microscope SIM,and the changes of mitochondrial membrane potential were analyzed by flow cytometry JC-1,and the lipid content in the cells was analyzed by ultra-high resolution microscope SIM.CCK-8,Annexin V-PI marked flow cytometry and 2’,7’-dichlorodihydrofluorescein diacetate（DCFH-DA）were used to detect the cell proliferation,apoptosis and reactive oxygen species production.The effect of extracellular vesicles on the expression of apoptosis-related proteins in activated LX-2 cells were verified by Western blot analysis.The experimental results showed that hUC-MSC-EVs could induce the deterioration of mitochondria in activated LX-2 cells,and promote the oxidative stress and apoptosis of the cells.（3）The effect of hUC-MSC-EVs on the synthesis and secretion of extracellular matrix,the TGF-β/SMAD signaling pathway and on rat liver fibrosis modelThe content of hydroxyproline in the cells was determined with colorimetric method,and the content of collagen-Iα1（Col-Iα1）in the culture supernatant was determined by ELISA.The protein expressions of fibrosis-related proteins α-SMA,Col-I,TIMP-1,MMP-1 and the protein expressions of TGF-β1,Smad2,Smad3,and Smad4 in the TGF-β/SMAD signaling pathway were analyzed by Western blot.The effects of hUC-MSC-EVs on rat liver function,morphology and collagen content were investigated using CCl4-induced rat liver fibrosis model.The results showed that hUC-MSC-EVs could inhibit collagen synthesis-related proteins,regulate extracellular matrix synthesis-related enzymes,and inhibit the degradation of extracellular matrix.hUC-MSC-EVs inhibited liver fibrosis depending on the TGF-β/SMAD signaling pathway.In the CCl4-induced rat liver fibrosis model,hUC-MSC-EVs could alleviate the liver fibrosis of the rat.Although there is no optimal therapy for the treatment of liver fibrosis,this study found that hUC-MSC-EVs treatment may be considered as a potential strategy to inhibit fibrosis,so as to be used for the inhibition of hepatic stellate cell activation and liver fibrosis.The hUC-MSC-EVs therapy may have good application prospects in the treatment of liver fibrosis.The innovations of this thesis:（1）This study confirmed that hUC-MSC-EVs have an inhibitory effect on hepatic stellate cells activated in liver fibrosis and can effectively alleviate liver fibrosis in rats.（2）It was found that hUC-MSC-EVs could induce mitochondria injury,promote oxidative stress,inhibit the metabolism of hepatic stellate cells,and promote the apoptosis of hepatic stellate cells.（3）It was discovered that hUC-MSC-EVs could inhibit the synthesis and secretion of extracellular matrix of hepatic stellate cells.（4）The study found that the inhibitory effect of hUC-MSC-EVs on liver fibrosis was achieved through the TGF-β/SMAD signaling pathway.