兰州大学机构库 >学院待认领
大鼠肝切除术后肝血窦重建的实验观察及初步机制研究
Alternative TitleReconstruction of hepatic sinusoid during regeneration after partial hepatectomy in rats
张冬红
Thesis Advisor焦作义
2014-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword肝切除 肝再生 血窦重建 肝窦内皮细胞 流式细胞术 细胞分选
Abstract目的:观察大鼠肝切除术后肝血窦重建的形态学过程和门静脉血流动力学改变规律;探讨肝切除术后,肝再生过程中VEGF和TGF-β1的表达;建立一种高效率、高纯度分选HSECs的新方法。 方法:按照随机的办法将35只大鼠分为实验组(n=28)和对照组(n=7)。将实验组随机分配到术后24、72、120和168 h四个时间点。对各组大鼠在相应的时间点测门静脉血流量,计算肝再生率;免疫组化染色测标本CD32、PCNA表达;QRT-PCR测残肝VEGF和TGF-β1 mRNA;免疫组化和Western-blot测残肝VEGF和TGF-β1表达。另取5只大鼠用于HSECs的分选。 结果:肝切除术后24 h门静脉血流下降到最小值,至120 h达高峰;肝再生率逐渐增大,168 h达最大值;免疫组化示:PCNA阳性肝细胞数和阳性HSECs数分别在术后24 h和72 h达最大值;CD32只在HSECs胞膜表达,残肝血窦密度于术后24 h降到最低,168 h达最大值。QRT-PCR测VEGF和TGF-β1 mRNA分别在术后72 h和120 h达高峰。免疫组化和Western-blot结果示:VEGF在术后72 h达到峰值;TGF-β1在术后120 h达到高峰。流式细胞分选回收率为63.30 ± 1.39%。 结论:在肝再生过程中,随着肝细胞再生,肝血窦逐步重建。门静脉主干血流量呈现先降低后恢复;VEGF和TGF-β1可能通过再生肝细胞调控肝血窦重建;流式细胞分选是一种高效率、高纯度分选HSECs的新方法。
Other AbstractObjective: The aim of this study was to observe the process of hepatic sinusoid reconstruction and hepatic portal hemodynamics changes, detect the expression of VEGF and TGF-β1 during liver regeneration after 70% hepatectomy and establish a new method to separate HSECs with high-efficiency and high-purity in rats. Methods: We allocated thirty-five rats with experimental group (n = 28) and sham group(n = 7) randomly. These 28 rats were randomly assigned to four time points (24, 72, 120, 168 h after 70% hepatectomy), seven rats at each time point. We detected the blood flow of the portal vein and calculated the liver regeneration rate of the rats from each group. The expression of PCNA and CD32 in the residual liver was detected by immunohistochemistry. The level of VEGF and TGF-β1 mRNA in the remnant liver were determined by qRT-PCR. The expression of VEGF and TGF-β1 in the remnant liver were determined by immunohistochemistry and Western-blot. We allocated another five rats to isolate HSECs by flow cytometry cell sorting. Results: Blood flow in hepatic portal vein fell to a minimum 24 h after partial resection of liver, and peaked at 120 h. Liver regeneration rate increases throughout the process of liver regeneration. The number of PCNA-positive liver cells peaked at 24 h, while PCNA-positive HSECs reached maximum at 72 h. CD32 was only expressed on HSECs membrane in rats. Liver sinusoid density in the remnant liver reduced to a minimum 24 h after hepatectomy, and reached maximum at 168 h after hepatectomy. The levels of VEGF and TGF-β1 mRNA peaked at 72h and 120h respectivly. The expression of VEGF peaked at 72 h after hepatectomy, while the expression of TGF-β1 reached the peak at the 120 h. Flow cytometry recovery was 63.30 ± 1.39% Conclusions: Hepatic sinusoid rebuild the normal structure gradually after hepatectomy. Portal vein blood flow decreased first, and then gradually returning to normal. VEGF and TGF-β1 may 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.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223689
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
张冬红. 大鼠肝切除术后肝血窦重建的实验观察及初步机制研究[D]. 兰州. 兰州大学,2014.
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