Animal Experimented Study On The Therapeutic Effect Of Adipose Derived Mesenchymal Stem Cell On Liver Diseases And The Therapeutic Safety

Objective ADSC has become the focus of tissue engineering and regenerative medicine research owing to their prominent advantage like injured sites homing instinct,immunological characterization,their capacity for self-renewal,multipotentiality for differentiation,the easy and repeatable to draw materials and the simple isolation procedures,and has been widely used on preclinical study in the digestive system,cardiovascular system and nervous system diseases.In our study,we first isolated SD rat adipose tissue to obtain ADSC and built the animal model of nonalcoholic fatty liver disease to investigate whether the ADSC transplantation can reverse the nonalcoholic fatty liver disease progression,then used SPION clusters @ PDA as MRI contrast agent to tracer ADSC homing ability for detecting and controlling the biological behavior of ADSC in vivo.Finally through three different culture methods of ADSC to investigate the relationship of ADSC and HCC,and to discuss the biological safety of ADSC,especially the tumorigenicity.In order to promote the clinical application of ADSC in the treatment of live diseases.Methods We first adopt the method of collagenase digestion combined with centrifugal separation to obtain ADSC from SD rat adipose tissue,the multipotentiality for differentiation,the cell morphology and the surface antigen was checked by special dyeing,inverted microscope and flow cytometric analysis to estimate that the cell is ADSC.The animal model of nonalcoholic fatty liver was made by high fat diet,and ADSC was transplanted into the animal model by portal vein.Then through biochemical analysis and pathological examination to analyze whether ADSC can improve liver function,lipid metabolism and its pathological features.Use SPION clusterss@PDA as MRI contrast agents to label ADSC,by CCK8 test,TUNEL test,flow cytometric analysis and special dyeing to investigate the effect of this nanoparticle on proliferation,apoptosis and stem cell biological characteristics of ADSC.Labeled ADSC transplanted into normal male ICR miceby caudal vein to investigated the organ toxicity,and transplanted into the animal model of acute liver injury and acute skin injury by caudal vein to investigate the homing of ADSC in vivo using MRI technology.Though the different culture conditions as conventional 2D culture,sphere culture and 3D culture to investigate the different effect of ADSC on the proliferation,apoptosis,adhesion,metastasis and invasion of hepatocellular carcinoma cells(Hep G2 and Hcclm3)in vitro,and by fluorescence quantitative RT-PCR and Western Blot to detect the changes of m RNA and protein of E-cadeherin,N-cadeherin,Vimentin,Zeb1 in the EMT signaling pathway in Hcclm3 and Hep G2 cells which were induced by different ADSC conditioned media.Finally though subcutaneous tumor formation test in nude mice to investigate the long-term toxicity of ADSC in vivo.Results Our cultured ADSC showed homogeneous distribution with a fibroblastic shape and were positive for CD29,CD44,CD73,CD90 and CD105,negative for CD31,CD34,CD45 and HLA-DR,and had the ability to differentiate into osteogenic,adipogenic and chondrogenic lineages.As showed the biochemical and pathological examination results,ADSC transplantation combining with dietary modification could further decrease the liver index,the serum levels of ALT,TBIL,TC,TG,FA,and the lipid accumulations to normal level,as well as reverse the hepatic pathological changes.SPION clusters@PDA labeling did not affect proliferation and apoptosis of ADSC cells,and had no effect on the stem cell biological characteristics of ADSC,there were no obvious pathological changes of the major organs and tissues from all groups.The labeled ADSC not only transported to the normal liver tissues,but also more gathered in the damaged liver ADSC,and no affect the therapeutic effect of ADSC on liver injures.Conditioned medium from ADSC in three different culture conditions suppressed the proliferation and motility of hepatoma cell,and 3D-ADSC-CM can enhance the inhibitory effect of ADSC on cell adhesion and movement,and promote the apoptosis of hepatoma cells.The levels of E-cadherin in the ADSC-CM treatment team were further increase to normal team,the levels of Vimentin,N-cadhenin andZeb1 could further decrease in the ADSC-CM treatment team compared with controlled team.No small nodules were formed in the subcutaneous inoculation site,there were not obvious pathological changes in major organs such as kidney,heart and liver from all groups.Conclusions The derived cells showed the typical characteristics of ADSC and conform to the MSC standards proposed by the international stem cell and tissue stem cell association.SPION clusters@PDA has a good biocompatibility and could be used as MRI contrast agent for ADSC tracking in vivo.It will provide a new thinking for the clinical efficacy and the quality control of ADSC clinical treatment.3D culture method can accelerate the inhibition of ADSC on the growth of hepatocellular carcinoma cells,and provide a new idea for the study of the relationship between ADSC and tumor cells.On the other hand,ADSC can effectively inhibit the growth of hepatocellular carcinoma cells,and inhibit the invasion and migration of hepatocellular carcinoma cells by down regulating EMT signaling pathway,and ADSC own good biological safety in vivo,show that ADSC therapy may be a potential therapeutic approach for HCC treatment.ADSC transplantation promotes NAFLD reversion through improving liver functions,promoting lipids metabolism and hepato-protective effects,showing that the application of ADSC in the treatment of liver disease is positive and effective.