| Background and aim:Surgical resection is the preferred treatment for liver cancer. However, approximately80%of patients with liver malignancy are unsuitable for surgical resection due toinsufficient FLR to support postoperative function. At least40%of the total liver volumemust be preserved to minimize the morbidity of surgical resection in HCC patients with achronic liver disease. Portal vein embolization (PVE) is an important method for expandingthe remnant liver volume. Pre-existing cirrhosis usually leads to an inadequate and delayedregeneration of the future liver remnant (FLR) after portal vein embolization (PVE). Asignificant reduction in the degree of liver hyperplasia was observed after PVE in thepresence of cirrhosis, with an increase of only9.6to14%. The delayed regenerationsignificantly increased the waiting time for resection, which led to scheduled hepatectomycould not be performed on3–30%of PVE patients with cirrhosis due to tumor metastasis.However,80%of liver cancer patients are characterized with varying degrees ofcomplicated HBV-hepatic cirrhosis in China.Stem cells transplantation is a novel treatment concept for liver diseases, includingliver cancer and could provide a valuable tool to increase FLR regeneration after PVE.Bone mesenchymal stem cells (BMSCs) are a subpopulation of bone marrow stem cells andare able to differentiate into adipocytes, cartilage cells, bone cells, islet-like cells andhepatic cells in vitro. As described previously, BMSCs can decrease significantly livecirrhotic and increase liver function. BMSCs showed the highest potential for theregeneration of injured liver tissue compared with other subpopulations of the bone marrowstem cells. BMSCs hold great promise in the field of regenerative medicine and maybefirst-line choice for stem cell transplantation to treat some diseases. BMSCs transplantationcould provide a valuable tool to increase FLR regeneration after PVE. Transforming growth factor-beta (TGF-β)is one of the critical factors that promteliver cirrhosis and inhibi hepatocyte regeneration. There is a high expression in cirrhoticliver. Some reports showed the blocking TGF-β activity by a variety of strategies has beenshown to inhibit fibrosis in a series of experimental models. TGF-βeta1neutralizingantibody has been used to treat a variety of disease, including liver cirrhosis. Wnt/β-cateninand TGF-β/Smad signaling pathways has a cross-talk at β-catenin. Hepatocytedifferentiation of BMSCs will be promoted when Wnt/β-catenin signaling pathways isdown-regulated. So, I suppose that hepatocyte differentiation of BMSCs will be increasedwhen TGF-β is blocked by TGF-β1neutralizing antibody,and the efficacy of autologousBMSCs transplantation to promote FLR regeneration was increased too.In current study, Iinvestigated the efficacy of autologous BMSCs transplantation topromote FLR regeneration in a rat cirrhotic model induced by intraperitoneal injection ofcarbon tetrachloride, and investigated the potential mechanisms. Moreover, the promotingefficacy of autologous BMSCs transplantation were investigated furtherly when TGF-βeta1neutralizing antibody was used.Materials and Methods:1. Cirrhosis induction in rats Liver cirrhosis was induced in rats (weight between200and250g) by intraperitoneal injection of carbon tetrachloride (CCl4) mixed1-to-1witholive oil at a dose of3ml/kg body weight twice a week for8weeks as described previouslywith some modifications. These rats were allowed access to water containing phenobarbital(0.4g/L) as the only drink source to sensitize liver cells to carbon tetrachloride for four daysbefore the CCl4treatment. Ethanol (15%v/v) was added to the drinking water to enhancethe effect of the CCl4throughout the induction phase.2. Isolation and identification of autologous BMSCs: Bone marrow was aspirated fromboth femurs. BMSCs ware isolated and purified by cIensity gmdient centrifugationcombined with attchment culture method. By investigating their surface markers CD29,CD90and CD45using FACS Calibu and multiple differentiation potentials, BMSCs wereconfirmed.3. PKH26labeling of BMSCs. BMSCs were labeled by using a PKH26red fluorescentcell linker kit as a marker for tracing BMSCs in the liver.4. PVE. Portal veins were ligated in the left/medium hepatic lobes under a dissection microscope using a6-0silk suture, such that the embolized tissue accounted forapproximately70%of the volume of the whole liver. A1-mL aliquot of IodizedOi, diluted1:1with meglumine diatrizoate, was injected at the distal end of the main vessel trunk in theleft/medium hepatic lobes.5. Cell transplantation.2×106of BMSCs suspended in0.5mL serum-free DMEM-LG,or an equal-volume of serum-free DMEM, were injected into the FLR through the portalvein. Sham-operated animals without PVE were opened and closed again.6. FLR/total liver weight ratio，Ki-67labeling index and liver function were measured.a. FLR/total liver weight ratio=weight of right and caudate/total liver weight100%.b. Ki-67labeling index=the number of hepatocytes with Ki-67positive nuclei/totalnumber of hepatocytes×100%.c. Liver function. The serum levels of alanine aminotransferase (ALT), aspartateaminotransferase (AST), ALB, and total bilirubin (TBIL) were measured by using anautomatic biochemical analyzer.7. Potential mechanisms.a. Collagen deposition in FLR was analyzed by the Hyp conten, hematoxylin and eosin(H&E) and Masson Trichrome.b. To investigate the gen expression of HGF, IL-10, MMP-9and VEGF in FLR byRT-PCR.c. To investigate hepatocyte differentiation of BMSCs in FLR,the markers ofhepatocyte, AFP, CK18and ALB were investigated using confocal microscopy.8. Hepatic growth medium (HGF(10ng/Ml)+FGF-4(10ng/Ml))was added TGF-β(0ng/ml,5ng/ml and10ng/ml respectively). On7day after that, the gen expression of AFPwas invisteged by RT-PCR. On14day and28day, CK18and ALB gens were invistegedrespectively.9. DMEM was added TGF-β (0ng/ml,5ng/ml and10ng/ml respectively) when BMSCswere cultured in in vitro. The regeneration of BMSCs was measured by Am-Blue kit.10. TGF-βeta1neutralizing antibody is injected into caudal vein for4weeks,2times/week (1mlPBS+20ugTGF-β1). Six animals in every group were sacrificed on day7,14, and28respectively. FLR/total liver weight ratio was measured. Result:1. The FLR weight ratio to the total liver was significantly higher and total bilirubinlevels were significantly lower in the BMSCs group compared to the controls withoutBMSCs transplantation after14and28days post-PVE (67.05±8.13versus54.67±8.23,p=0.026,71.77±4.06versus63.63±7.03, p=0.043).2. There was a significant increase in the Ki-67index for the BMSCs group comparedto the controls at day14and28(27.50±3.78vs.21.00±2.37at day14, p=0.002;24.83±3.06vs.17.50±1.87at day28, p=0.001).3. After14and28days post-PVE, the ALB levels were significantly higher and theTBIL levels were significantly lower in the BMSCs group compared to the controls.4. The serum ALT levels were decreased at day7in the BMSCs group (p <0.01), butsignificantly increased at day28(p <0.05) in the BMSCs group compared to the controls.The serum AST levels showed no significant difference between these two groups at day7,but increased at day14and28in the BMSCs group compared to the controls.5. BMSCs significantly decreased the Hyp content and collagen accumulation after14and28days post-PVE.6. BMSCs significantly up-regulated the expressions of HGF, IL-10, VEGF andMMP-9after28days post-PVE.7. BMSCs expressed hepatocyte-specific markers, such as α-fetoprotein, cytokeratin18, and albumin in a time-dependent.8. The regeneration of BMSCs was inhibited by TGF-β.9. The Hepatic differation of BMSCs was inhibited by TGF-β.10. TGF-βeta1neutralizing antibody and MBSCs transplantation significantly couldincrease the FLR weight ratio to the total liver compared to the controls without TGF-βeta1neutralizing antibody after14and28days post-PVE.Conclusion:1. Autologous BMSCs can differentiate into hepatocyte in cirrhotic live.2. Autologous BMSCs can decrease the cirrhotic degree of live.3. Autologous BMSCs transplantation can promote FLR regeneration after PVE incirrhotic liver.4. The potential mechanisms for BMSCs transplantation to promote FLR regeneration maybe improving local microenvironment by decreasing cirrhosis, up-regulating the geneexpression of VEGF, HGF, IL-10and MMP-9.5. TGF-βeta1neutralizing antibody can not increase the promoting efficacy ofautologous BMSCs transplantation in liver regeneration after portal vein embolization.6. TGF-β was able to inhibit the regeneration of BMSCs.7. TGF-β was able to inhibit Hepatic differation of BMSCs.In a word, AutologousBMSCs are able to be obtained easily and manipulated, and there are no immunologicrejection and ethical issues concerning their use. Autologous BMSCs transplantation mayprovide a potential treatment for increasing successful hepatectomy rate in patients withcirrhosis bearing hepatocellular carcinoma. |
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