| Abstract:Objective:Liver regeneration is a special, complex process of cell proliferation. With the original cells or stem cells to complete the regeneration process of general organization, the proliferation process of liver regeneration is different, it depends on the re-activation of all differentiation and maturation of remaining liver cells, proliferation activity was to start the follow-up of DNA replication, cell division and proliferation. Liver function index during the liver regeneration did not change significantly, the key proteins synthesis by the liver of plasma (albumin, clotting factors and anti-protease) is not significant changed. It is associated with the process of liver regeneration extracellular matrix re-configuration. Extracellular matrix (ECM) is not only a place for cell survival, but also of a only way and the buffer zone of autocrine and paracrine regulation between cells, which changes by the regulation of matrix metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs). The activation of MMP2, MMP9 plays an important role in liver regeneration. In this study, we tried to established rat animal model of 90% portal branch ligation, to research the mechanisms of MMP9, TIMP2 of liver regeneration in this model, to provide a theoretical basis of the clinical treatment in the remnant liver regeneration after hepatectomy and liver transplantation.Methods:96 male Sprague-Dawley rats were randomly divided into 2 groups:experimental group and control group of 48 rats, each group was randomly divided into eight sub-groups of 6 rats, respectively, POD0.5, POD1, POD3, POD5, POD7, POD14, POD21 and POD28. The rats are anaesthetized with 1.5% sodium pentobarbital intraperitoneal injection. Ligated the right portal vein branch supply right and caudate lobes and the portal vein branch supply the left lateral lobe and the median lobes, leaving only a small portal vein supply two mastoid lobes with 4-0 silk under a surgical microscope. Hepatic artery and bile ducts remained intact.Ligated side of the liver accounts for about 90% of the total hepatic mass.The rats of control group only free vein branch ligation, and the other operations is the same as the experimental group.Portal vein pressure and the weight of both ligated and unligated lobes of liver were measured. The morphological changes of the non-ligated liver lobes were observed by microscope.The expression of PCNA, MMP9 and TIMP2 of the non-ligated liver lobes were studied with immunohistochemistry. The apoptotic of the non-ligated liver lobes were studied with TUNEL method.To cultured HUVEC in the 6 well culture plates in vitro. Cells were randomly divided into 5 groups:conventional static group; 15mmHg (physiological state) 12h group; 15mmHg 24h group; 30mmHg (high pressure) 12h group; 30mmHg 24h group. The expression of MMP9 studied with immunohistochemistry. To detected the cell MMP9 mRNA expression using relative quantitative RT-PCR method and MMP9 protein expression using Western blot.Results:Animal experiments showed that:(1) 95.8% rats survived from the ligation of 90% portal branch. (Death in the POD21 and POD28 groups); (2)Hepatic lobes at the ligated side diminished progressively after ligation, whereas the lobes of the unligated side underwent compensatory regeneration. The ratio of non-ligated lobes weight to the whole liver increased slowly within 0.5d, speeded up significantly during 0.5-5d period, increased slowly after POD5,and got "the plateau stage"at POD7. The total mass of the whole liver only slightly lower than the 1d-3d after the first normal levels, the remaining time points were close to the normal levels, the difference was not statistically different.(3)Portal vein pressure increased immediately after the operation, it reached the peak point at 0.5d(P<0.01), and dropped to normal at POD7.(4)The control group showed the hepatocells arranged radially centered of the central vein. The experiment group showed that liver cells are swelling mildly, the Boundary of portal area, interlobular blood vessels and bile duct are clearly. The hepatocells started to proliferation obviously at POD1, there are many mitotic-phases, accompanied by the nuclear increased periportal areas, interlobular blood vessel dilatation and congestion.The hepatocell nuclei divided actively at POD3-5. The inflammatory cells are infiltrated in periportal area. There are more mitotic-phases existed at POD7-14, and the hepatocells are larger than before. Little inflammatory cells are infiltrated in interstitial area at POD21-28, and the small bile duct is hyperplasia, liver cord structure and sinusoidal is clear.(5)PCNA index were markedly increased within POD0.5-3(P<0.01).It reached the peak at POD5 and decreased slightly at POD7, but still higher than pre-operation level(P<0.01), then gradually return to normal lately. (6)It is hard to find any apoptotic cells from the pre-operation liver and the non-ligated lobes after 90% PBL at any time point.(7)The expression of MMP2,MMP9 and TIMP2 of the non-ligated liver lobes were markedly increased at 1d.It reached the peak at POD7 and gradually return to normal in POD7-28.(8)The expressions of PCNA in non-ligated lobes and portal vein pressure(PVP) had a positive correlation at POD1,3,5 and a negative correlation at POD14(P<0.05). The PCNA and MMP9 in liver had a positive correlation at POD0.5,1,7,21. The expressions of PCNA and TIMP2 had a positive correlation at POD1,7,14,21.The experiments in vitro showed that:with the increase of pressure and time, the rate of MMP9 positive cells is gradually raised. MMP9 mRNA expression were significantly increase in 15mmHg 12h group,15mmHg 24h group and 30mmHg 12h group, and not changed significantly in 30mmHg 24h group. The expression of MMP9 were significantly increased in 15mmHg 12h and 30mmHg 12h group, and slightly increase in 15mmHg 24h and 30mmHg 24h group.Conclusion:Animal experiments showed that:ligation of 90% portal branch can induce active regeneration of hepatic cell of non-ligated liver lobes in rats and restore to previous liver weight.90% PBL of rat is a comfortable model of liver regeneration. The portal vein pressure is closely related to liver regeneration of 90% PBL. MMP9 and TIMP2 proteins play an important role in this process. The experiments in vitro showed that:mechanical stress can stimulate MMP9 expression of vascular endothelial cells, and provide a theoretical basis for the important role of MMP9 in the portal vein pressure leaded to liver regeneration. |
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