| Acute liver failure (ALF) is a common clinical critical disease,but there is noeffective treatment for it.So fatality rate of ALF maintain60%-80%even now.Currentlyorthotopic liver transplantation represents the most suitable therapeutic option for patientswith hepatic failure.However,the limitation of liver transplantation,such as the shortage oforgan donors,high cost,complicated surgery,and life-long immunosuppressive regimenshave prompted the efforts to find the other alternative.Hepatocyte transplantation holdsgreat promise as an alternative to organ transplantation for the treatment of liverdiseases.Numerous studies indicate that transplants consisting of isolated hepatocyte cancorrect various metabolic deficiencies of the liver and can reverse hepatic failure.Butheterogeneity or varient hepatocyte transplantation common inevitably fail. The barriers tothe successful treatment of liver diseasee by hepatocyte transplantation are moreextensive.Immunological rejection is the major reason.The subcardinal is shortage ofsurvival time in vitro and limitation of proliferation or passage. Under thesecircumstances,many investigators have researched stem cells transplantation with the hopeof overcoming these limitations.Mesenchymal stem cells(MSCs) emerged as promising candidates for treatment ofacute liver injury.These cells have ability of self-renewal and a high degree ofplasticity,as they can transdifferentiate into multiple cell lineages.Under appropriateconditions,they can differentiate requisite cells and perform function. Contrasted withembryonic stem cells,MSCs are associated with fewer ethical concerns.More importantly,MSCs show immunomodulation, greater expansion capability and exhibit faster growth invitro. Adipose tissue has several advantage compared to other adult tissue as a source ofMSCs.Indeed,adipose-derived mesenchymal stem cells(AMSCs) have characteristic of lowimmunogenicity and abundant resource.Theses evidences suggest it maybe the most satisfactory candidate for treatment of end-stage liver diseases.But more trials need assessthe safety,feasibility and efficacy of AMSCs transplantation in acute liver failure.Limitationof only MSCs or hepatocyte transplantation hindered follow-up studies. To prevent thelimitation of single type of cells transplantation,we hypothesis cotransplantation AMSCsand hepatocyte for treatment of acute liver injury in rats. The interaction of cells functionand immunoregulation provided possibility that cotransplantation could be feasible.The first purpose of this study was to isolate and coculture of rat hapatocyte andhuman adipose derived stem cells(different resource of subcutaneous and orbital fat).Thephenotype of isolated human adiposed-derived stem cells was analyzed by flowcytometry.These stem cells were induced by osteogenic and adipogenic differentiationThen biological activity of different position of fat was compared. Direct or indirectcoculture of rat hepatocyte and human adipose derived mesenchymal stem cells(hAMSCs)stimulated by serum from acute liver failure for72h.Moreover, hAMSCs and rathepatocyte indirect cocultured in condition free of acute liver failure serum in a longertime.The interaction was discussed by gene expression,cell function and the level ofcytokines.At last of the study was carried out to evaluate the therapeutic potential andsuperiority of hAMSCs labled SPIO and rat hepatocyte cotransplantation in acuted liverfailure of rats,The major findings of the research are listed as follows:1. Isolation of hAMSCs from different site (subcutaneous and orbital fat):hAMSCswere harvested by a sharp separation method of type Ⅳcollagenase enzymedigestion..This method could obtain great quantity, stabilize stem cells.The surfacemarkers assessed by flow cytometry,suggesting their mesenchymal origin,rather thanhematopoietic or epithelial origin. These hAMSCs possessed multi-lineage differentiationpotential to become osteoblasts,and adipocytes.Compared biological activity of differentposition hAMSCs,including cell quantity of unit mass,cell adherence rate, cell growthkinetics and senescence β-Galactosidase staining of two sites(subcutaneous and orbitalfat).The higher cell quantity of unit mass and cell adherence rate assessed from orbitalfat,but there is no significant difference in other properties.2. To evaluate whether hAMSCs can support rat hepatocyte survival againstinflammation, both monoculture and direct coculture at different ratios(1:51:101:20 1:50)were exposed in ALF serum.There was no significant reduction of the percentage oflive cells in the coculture.After ALF serum stimulation,rat hepatocytes released asignificantly higher amount of albumin and urea when cocultured with hAMSCscompared to hepatocytes aloneIt validated that hAMSCs protected rat hepatocytes fromALF serum induced cytotoxicity. Among different ratios of hAMSCs and rat hepatocytes,the ratio of1:10exhibited the optimal rat hepatocyte preservation..Indirect or direct coculture hAMSCs and rat hepatocyte stimulated byserum,assessed the level of cytokines and hepatocyte function in supernate.The hepaticgenes expression of mesenchymal stem cells were measured in upper chamber. hAMSCsassisted rat hepatocytes to keep their cell viability, morphology and functions under thestimulation of ALF serum. Among the potential immunomodulatory factors in the coculturesystem, IL-6was below the detection limit in the monoculture of hepatocytes.Nevertheless,IL-6concentrations are dramatically high in the coculture both before and after the ALFserum treatment.To further confirm the role of IL-6, IL-6neutralizing antibody was addedin the coculture and the proportion of liver cells,as well as the hepatic fuctions decreasedobviously.It suggested IL-6might be the key bioactive molecule through which hAMSCsexert their protective effects.RT-PCR was performed to evaluate the liver-specific geneexpression of hAMSCs,none of the hepatic genes were expressed after coculture72hstimulated ALF serum. Also these cells did not acquired hepatocyte funcitons.Indirectcoculture hAMSCs and hepatocyte free of serum for a longer time, hAMSCs acquiredhepatic characteristics such as liver specific gene,a polygonal shape and hepatocyte fuctionfrom7days under coculture with hepatocyte. It indicated.the supportive effects on rathepatocyte function is through immunomodulation rather than differentiation during anacute phase response to ALF serum stimulation.So hAMSCs can be an alternative cellsource for the cell-based therapeutic strategies for maintenance of liver functions in vitro.3. Blue-stain iron particle could be seen in prussian blue staining labled mesenchymalstem cells, cytoactive reached over95%.The energy for growth,differentiation andproliferation uninfluenced after labled. It indicates SPIO successfully labeled hAMSCs.hAMSCs labeled SPIO were transplanted into rats with CCl4-induced liver injury.Thetransplantations were made via tail vein, femoral vein,portal vein injection.At sevenpost-transplantation days,serum biochemical parameters and liver specimens were evaluated. Markers of liver injury obviously decreased and liver specimens ameliorate ingroups of hAMSCs transplantation. Blue-stain iron particle could be seen in liverspecimens. But there is no statistics difference in various transplantation routes. Soundifferentiated hAMSCs have the ability to improve hepatic function in rats with acuteliver injury. We also found levels of IL-6,IL-10,IFN-γincreasing obviously in serum of ratsreceived hAMSCs transplantation.It indicate immunomodulation maybe one of thetherapeutic mechanisms for transplantation of hAMSCs in liver injury.To investigate the feasibility and effect of cotransplantation,hAMSCs labeled SPIOand rat hepatocyte were cotransplantated into acute liver injury. Cotransplantation has moreadvantages compared with transplantation of hAMSCs or hepatocyte,since it can decreasemarkers of liver injury and ameliorate liver specimens.In conclusion,this study take deep-going research of bionomics in hAMSCs obtainedfrom different sites of human,basing on successfully isolation. The interaction ofhAMSCs and rat hepatocyte was discussed by the level of cytokines,hepatocyte functionand gene expression. hAMSCs preserve rat hepatocyte functions in the setting ofinflammation.According to our data, the immunoregulatory effects of hAMSCs wereapparent. It seems that IL-6is the common effective cytokine in coculture of rathepatocytes with hAMSCs.Our study of transplantation supports the advantage ofhAMSCs and rat hepatocyte cotransplantation for acute liver injury. The transplantationroutes of tail vein, femoral vein,portal vein injection had the same effect on it.This studysupply more experimental data and a new alternative of stem cells transplantation oftherapeutic strategy of acute liver injury.
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