Effect Of Bone Marrow Mesenchymal Stem Cells On Experimental Liver Fibrosis In Rats And Its Potential Mechanisms

BackgroundLiver fibrosis is a pathological condition with excessive deposition of collagen and other components of extracellular matrix (ECM), which is a common characteristic of mutiple chronic and progressive hepatic diseases. At late phase, hepatic fibrosis may be developed into cirrhosis, leading to a variety of severe complications due to cirrhotic portal hypertension and liver dysfunction. The central event leading to liver fibrosis is an activation of hepatic stellate cells (HSC) or the activation of a variety of cell types to form a myofibroblastic phenotype, which results in increased synthesis of ECM while decreased degradation of ECM. There are a range of experimental studies to reverse liver fibrosis by targeting the activation of HSC. However, even though the majority of these approaches were effective in inhibiting differentiation of HSC into myofibroblasts in vitro, the effect of anti-fibrosis was very limited in vivo. Therefore, effective therapeutic strategies aimed at liver fibrosis are still lacking. Bone marrow mesenchymal stem cells (MSCs) are a kind of highly-conservative adult stem cells with characteristics of pluripotency. It has been reported that bone marrow MSCs transplantation reduced the degree of liver fibrosis in a mice model of hepatic fibrosis due to their potential to differentiate into hepatocytes. However, there are also discordant observations as they are one of the sources of myofibroblasts. Therefore, the current studies are designed to verify the effect of bone marrow MSCs transplantation on liver fibrosis.ObjectiveTo separate and purify bone marrow MSCs by whole bone marrow adherent culture method and identify of bone marrow MSCs. To search for the most effevtive concentration of DAPI for labeled bone marrow MSCs. To explore the effect of bone marrow MSCs transplantation on liver fibrosis by focusing on their hepatic localization, differentiation and potential mechanisms. Moreover, to explore whether bone marrow MSCs could differentiate into myofibroblasts induced by transforming growth factorβ1(TGFβ1) and dexamethasone (Dex).Methods1 Bone marrow MSCs were collected from bone marrow aspirate and were separated and purified by whole bone marrow adherent culture method. By flow cytometry their surface markers (CD29, CD90, CD34 and CD45) were identified in cultured. Bone marrow MSCs were evaluated for ability of adipogenesis and osteogenesis. Bone marrow MSCs were labeled with 4', 6-diamidino-2-phenylindole (DAPI). In addition, Type 1 collagen (Col 1), Vimentin (Vim) andα-smooth muscle actin (α-SMA) protein of bone marrow MSCs were examined by immunocytochemistry2 Liver fibrosis was induced by subcutaneous infusion of carbon tetrachloride (CCL4) for 9 weeks and administered 1×10⁶ bone marrow MSCs per rat by tail-vein injection after 8 weeks of CCL₄ infusion; bone marrow MSCs were labeled with 4', 6-diamidino-2-phenylindole (DAPI) before cell infusion. One week after bone marrow MSCs administration, animals were sacrificed and hepatic histopathology was examined by assays of morphology. In addition, expression of SRY mRNA levels was investigated with reverse transcription polymerase chain reaction (RT-PCR).Α-SMA expression in liver was examined by immunofluorescence. Col 1, Vim andα-SMA of bone marrow MSCs, which were induced by transforming growth factorβ₁ (TGFβ₁) and dexamethasone (Dex), were examined by immunocytochemistry and real time PCR.3. Liver fibrosis was induced by subcutaneous infusion of CCL₄ for 12 weeks and administered 1×10⁶ bone marrow MSCs per rat by tail-vein injection after 8 weeks of CCL₄ infusion. Four weeks after bone marrow MSCs administration, animals were sacrificed and hepatic histopathology, hepatic function were examined by assays of morphology and biochemistry. In addition, Col 1, Vim andα-SMA were examined by immunohistochemistry. TGFβ₁, Smad3 and Smad7 mRNAs were examined by real time PCR. Results1 Bone marrow MSCs had multiple differentiation potentials to be osteoblasts and adipocytes. CD29 and CD90, but not CD34 and CD45 antigens were specifically expressed in bone marrow MSCs. Bone marrow MSCs was labeled by 50ug/m1 DAPI without inhibiting proliferating ratio and viability of bone marrow MSCs. Col 1and Vim protein were detected in bone marrow MSCs withoutα-SMA protein.2 SRY mRNA was detected in MSCs transplantation group. DAPI positive bone marrow MSCs were found predominantly in the areas of scarring of liver in MSCs transplantation group. A large number ofα-SMA (+) MSCs were found in the areas of scarring of liver in MSCs transplantation group. DAPI fluorescence was highly distributed in the fiber areas and colocalized withα-SMA. Col 1, Vim andα-SMA mRNA and protein of bone marrow MSCs were increased after 48h induced by TGFβ₁. Moreover, Col 1, Vim andα-SMA mRNA and protein of bone marrow MSCs were significantly increased after 24h and 48h induced by TGFβ1 and Dex .3 Compared with model group,the liver fibrosis contents in MSCs transplantation group were not alleviated. There were no significant differences of ALT, AST and ALB between model group and MSCs transplantation group. Col 1, Vim andα-SMA protein were not decreased after MSCs transplantation compared with model control group. In addition, TGFβ1 and Smad3 mRNA expression was obviously up-regulated and Smad7 was obviously down-regulated in MSCs transplantation group.Conclusion1 Bone marrow MSCs had multiple differentiation potentials to be osteoblasts and adipocytes. Bone marrow MSCs were labeled by 50ug/m1 DAPI without inhibiting proliferating ratio and viability of bone marrow MSCs. Col 1and Vim protein were detected in bone marrow MSCs withoutα-SMA protein.2 The transplanted bone marrow MSCs might be differentiated into myofibroblasts in vivo. In vitro, bone marrow MSCs might be differentiated into myofibroblasts induced by TGFβ1 and Dex.3 The transplanted bone marrow MSCs might be differentiated into myofibroblasts in vivo. TGFβ1 and Smad3 mRNAs was obviously up-regulated and Smad7 mRNA was obviously down-regulated in MSCs transplantation rats for promoting homing of bone marrow MSCs into injured liver. Therefore,bone marrow MSCs were unable to reduce fibrosis in a rat model of chronic liver injury.