| Objective To establish a rat selective portal vein ligation model, and to evaluate the feasibility of this model by detecting the model indicators, such as the effect of intraoperative, postoperative results, changes in liver function, liver histopathological changes, et al. More, we study the serum-related cytokine changes. To establish the primary culture method of rat bone marrow mesenchymal stem cells in vitro and study its biological characteristics. Then study the effect of selective portal vein ligation model of rat on the homing bone marrow mesenchymal stem cells to the liver and its mechanism.Methods Selective ligation of the left lateral and middle lobes of rat liver, observed changes of blood supply and outward appearance of the ligated lobes and non-ligated lobes, every6rats were sacrificed after1,2,3,7and14days after surgery, and to observe the morphological changes, liver function indicators, including changes in alanine aminotransferase, total bilirubin, albumin of the rat liver, used HE staining to learn liver histopathological changes and collagen production, ELISA assay to study the serum related cytokines HGF, SDF-la expression level. Whole bone marrow adherent culture method for primary culture was used to get high purity of rat bone marrow mesenchymal stem cells, and to observe the morphological characteristics of it, used the flow cytometry to identify surface markers of BMSCs and observed its adipogenic, osteogenic differentiation. In vitro, studied the effects of serum at different time points of sPVL model on the chemotaxis of rat BMSCs by transwell migration assay, and by using the specific blockers AMD3100and to K252-a to block SDF-la/CXCR4axis and HGF/c-met axis to learn the effects of these two axis on the homing of BMSCs, and used western blotting experiments to further verify the effect.Results We have successfully established sPVL surgery in30rats. Over time, in the subgroup of the model, the weight of non-ligated lobes of the total weight of the liver gradually increase, while the ligated lobes decreased gradually, compared with the sham group, P<0.01. ALT peaked after2days, and falled back at day7after the surgery. Total bilirubin and serum albumin levels had no significant fluctuations. Compared with the sham group, HE staining showed that the volumes of hepatocytes in the non-ligated lobes enlarged, the hepatic sinusoid dilated and hepatocyte regeneration were observed. Masson staining showed that collagen deposited around the central veins and portal areas of the ligated lobes. After surgery, the serum level of HGF rapid increase while the level of SDF-1alpha did not change significantly. Transwell experiments confirmed that serum after sPVL had chemotaxis to BMSCs, this chemotactic effect has the consistency with the level of HGF, this effect can be blocked by HGF/c-met axis’s blocker K252-a, P<0.01. Serum chemotaxis of BMSCs is not due to SDF-1α/CXCR4axis, and by blocking it, there was no changes.western blotting showed that after co-incubation with the sPVL serum, the expression of c-met protein was up-regulated, and reduced by using the K252-a to block it.Conclusion We have successfully established a rat selective portal vein ligation model, and successfully isolated and culture of rat BMSCs, and further confirmed that the model can cause the change of serum HGF expression level and induced the homing ability of BMSCs, this is through the HGF/c-met axis. In this model, the key role of SDF-1α/CXCR4axis to the homing of BMSCs was not seen. |
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