Reconstruction Of Hepatic Sinusoid During Regeneration After Partial Hepatectomy In Rats

Objective:The aim of this study was to observe the process of hepatic sinusoid reconstruction in morphology and hepatic portal hemodynamics changes, detect the expression of VEGF and TGF-β1and their relationship with the hepatic sinusoid reconstruction during liver regeneration after70%hepatectomy in rats and establish a new method to separate HSECs with high-efficiency and high-purity in rats.Methods:We allocated thirty-five male healthy rats with experimental group (n=28) and sham group (n=7) randomly. These28rats were randomly assigned to four time points (24,72,120,168h after70%hepatectomy), seven rats at each time point. These rats were used to build the liver regeneration model after70%hepatectomy. We used abdominal ultrasound to detect the blood flow of the portal vein of the rats from each group at the corresponding time points, and then we took specimens and calculated the liver regeneration rate. Hepatic sinusoid density was calculated using immunohistochemical staining for CD32, and the expression of PCNA in the residual liver was detected by lmmunohistochemistry. The level of VEGF mRNA and TGF-β1mRNA in the remnant liver were determined by qRT-PCR. The expression of VEGF and TGF-β1in the remnant liver were determined by immunohistochemistry and Western-blot. We allocated another five male healthy rats to build the In situ perfusion model. HSECs was isolated by flow cytometry cell sorting, based on collagenase perfusion in combination Percoll density gradient centrifugation. Morphological changes of HSECs was decected by Light microscopy, and ultrastructural of HSECs was detected by scanning electron microscopy.Results:Blood flow in hepatic portal vein fell to a minimum24h after partial resection of liver, and did not reach a peak until120h after that. Liver regeneration rate increases throughout the process of liver regeneration. The number of PCNA-positive liver cells increased0-24h and peaked at24h, while PCNA-positive HSECs reached maximum at72h after hepatectomy. CD32was only expressed on HSECs membrane in rats. Liver sinusoid density in the remnant liver reduced to a minimum24h after hepatectomy, and reached maximum at168h after hepatectomy. The levels of VEGF mRNA and TGF-β1mRNA peaked at72h and120h respectivly detected in the residual liver by qRT-PCR after partial hepatectomy. The expression of VEGF increased between0h and72h and peaked at72h after hepatectomy, while the expression of TGF-β1rised slowly between0h and120h, reaching the peak at the120h after hepatectomy detected by immunohistochemistry and Western-blot. Flow cytometry recovery was63.30±1.39%, and cell viability was94.48±2.07%. HSECs was "cobblestone"-like arranged in Light microscope. The structural characteristics of the window hole could be observed by SEM.Conclusions:With the start-up and development of regeneration of hepatocytes, hepatic sinusoid rebuild the normal structure gradually after hepatectomy. Portal vein blood flow decreased first, and then gradually returning to normal with hepatic sinusoid reconstruction. In the regeneration process induced by liver resection, VEGF and TGF-β1may regulate hepatic sinusoid reconstruction by the regeneration of liver cells. Flow cytometry cell sorting is a new method to separate HSECs with high-efficiency and high-purity.