Notch-Hif-1-alpha信号通路参与调控大鼠肝再生过程的研究

目的：探讨使用酌分泌酶抑制剂Fli-06 特异性阻断Notch 信号通路后，大鼠肝部分切除术后肝再生的情况并初步阐明 Notch-Hif-1-alpha信号通路调控肝再生的可能机制。方法：取SD 大鼠分为对照（生理盐水注射组，n=24）和抑制剂组（酌分泌酶抑制剂 注射组，n=24）。给予药物处理后，两组分别施行大鼠肝部分切除术，术后0 d，1 d，3 d，5 d，每个时间点分别留取对照组（n=6）及抑 制剂组（n=6）再生的肝组织，并检测相应肝重体重比，免疫组化法检测再生肝PCNA（增殖细胞核抗原，Proliferation Cell Nuclear Antigen）表达，RT-PCR 检测再生肝组织中的Notch1、Hes1、VEGF mRNA的变化，Western-Blot 法检测NICD（Notch 胞内段，Notch Intracellular Domain）、Hif-1-alpha（低氧诱导因子-1-alpha，Hypoxia Inducible Factor-1琢)蛋白在肝再生过程的变化情况。结果：1、肝部分切除 术后3 d和5 d，抑制剂组肝重体重比明显低于对照组差异有统计学意义（P<0.05）；2、免疫组织化学染色结果提示：抑制剂组再生 肝PCNA阳性细胞率在术后1 d，3 d，5 d 均明显低于对照组（P<0.05）；3、Western blot 结果表明：NICD 和Hif-1-alpha蛋白水平明显低 于对照组（P<0.05）；同时RT-PCR 结果提示：抑制剂组Hes1 的mRNA表达量术后1 d，3 d明显低于对照组，差异具有统计学意义 （P<0.05）。同时，抑制剂组VEGF mRNA 水平在术后3 d，5 d明显低于对照组，差异具有统计学意义（P<0.05）。结论：在大鼠肝部分 切除术后肝再生过程中，使用酌分泌酶抑制剂Fli-06 抑制Notch 信号通路后，大鼠的肝再生能力明显降低，Notch-Hif-1alpha信号通 路可能参与调控了大鼠肝部分切除术后肝再生过程。

Study on the Notch-Hif-1-alpha Signaling Pathway in Liver Regeneration

Objective:To investigate whether gamma secretase inhibitor Fli-06 could have an impact on the liver regeneration after liver resection in a rat model. With this in vivo model, we explore the underlying mechanism of notch signal pathway.Methods:SD rats were divided into control group (with intraperitoneal injection of normal saline, n=24) and experimental group (with intraperitoneal injection of gamma secretase inhibitor, n=24). The two groups were treated by partial hepatectomy, and each group was divided into four subgroups according to the time point when remnant liver tissues were preserved, namely, 0 d, 1 d, 3 d and 5 d. The liver weight / body weight ratio was detected, the expression of PCNA in regenerating liver was detected by immunohistochemistry method, and the Notch1, Hes1 and VEGF mRNA in remnant liver tissues was detected by RT-PCR method. NICD and Hif-1 protein of the liver tissues during the liver regeneration process were analyzed and compared by Western-Blot methods.Results:1. The liver weight/body weight ratio in inhibitor group was significantly lower than the control group. There was a significant difference between the two groups (P<0.05). 2. Immunohistochemical results suggested that the percentage of PCNA positive cells in the regenerating liver in the inhibitor group after 1 d, 3 d, 5 d were significantly lower than the control group (P<0.05). 3. Western Blot results suggested that the expression levels of NICD and Hif-1 alpha in 1 d, 3 d,5 d were significantly lower than the control group (P<0.05). RT-PCR results indicated that Hes1 mRNA in inhibitor group were lower than the control group after 1 d, 3 d (P<0.05). VEGF mRNA in inhibitor group were lower than the control group after 3 d, 5d (P<0.05).Conclusion:The ability of the liver regeneration of rats after hepatectomy decreased significantly after gamma secretase inhibitors inhibited Notch signaling pathway. The results showed that the Notch-Hif-1 alpha signaling pathway may be involved in the regulation of rat liver regeneration after partial hepatectomy.