PUMA抑制剂对小鼠肝脏缺血—再灌注损伤的保护作用初探

发布时间：2019-05-22 03:17

【摘要】：目的：p53正向细胞凋亡调控因子(p53upregulated modulator of apoptos is, PUMA)是Bcl-2家族唯BH3结构域亚家族成员,位于线粒体内,正常情况下表达很低,可被多种损伤因素如射线、病毒、化疗药物等诱导激活。PUMA通过其特有的BH3结构域与Bcl-2或Bcl-xL等抗凋亡蛋白结合后抑制其抗凋亡作用。PUMA抑制剂模拟BH3结构域与抗凋亡蛋白结合,阻碍PUMA与Bcl-2样蛋白结合,凋亡被抑制。我们想观察PUMA在两种不同的模型下表达情况以及PUMA抑制剂能否对细胞或组织起到保护作用。方法：(1)选取怀孕13.5天的ICR小鼠,提取小鼠胚胎成纤维细胞(mouse embryo fibroblast, MEFs)(2)用不同浓度阿霉素(Doxorubicin, DOX)刺激MEFs观察PUMA表达及细胞损伤情况,并用PUMA抑制剂观察对细胞的保护作用。(3)用C57BL/6小鼠建立缺血-再灌注模型,分为假手术组、手术组和PUMA抑制剂组。(4)采取不同缺血和再灌注时间观察血清ALT水平,做Western Blot检测PUMA蛋白水平表达,做反转录-实时定量PCR检测PUMA mRNA水平表达,做肝脏HE切片看损伤情况,TUNEL染色看肝脏细胞凋亡情况。结果：当阿霉素浓度在0.5-5mg/L时,可以刺激MEFs PUMA表达升高,加药浓度再升高PUMA表达反而降低；用1μMPUMA抑制剂可以抑制阿霉素低浓度刺激的PUMA表达,且能保护MEFs抵抗阿霉素导致的损伤。对于小鼠缺血-再灌注损伤模型,根据缺血、再灌注时间不同,损伤情况各异。当缺血时间为60分钟,再灌注1小时,肝脏损伤较轻,PUMA表达升高,PUMA抑制剂可保护肝脏抵抗损伤；当缺血时间长至90分钟,再灌注一小时,肝脏损伤较严重时,PUMA表达升高不明显,PUMA抑制剂无法起到保护作用。结论：PUMA在一定损伤范围内可被诱导激活,此时凋亡是主要损伤途径,应用PUMA抑制剂可以降低PUMA蛋白水平和转录水平表达,细胞凋亡被抑制,保护细胞或组织抵抗损伤。当刺激因素过强,凋亡不是损伤的主要表现途径,PUMA表达升高不明显。PUMA抑制剂在体内体外均可发挥作用,说明其应用可能会很广泛。我们今后将会用PUMA KO小鼠验证我们的结果并建立肝脏冷缺血-再灌注模型或器官移植模型观察PUMA缺失或被抑制后能否对器官产生保护作。
[Abstract]:Objective: p53 positive apoptosis regulator (p53upregulated modulator of apoptos is, PUMA) is a member of the BH3 domain subfamily of Bcl-2 family, which is located in the plasmid. The expression of p53 is very low under normal conditions, and can be damaged by a variety of damage factors such as rays and viruses. Puma can inhibit the anti-apoptosis effect of Bcl-2 or Bcl-xL through its unique Bcl-2 or Bcl-xL domain. Puma inhibitor mimics the binding of BH3 domain to anti-apoptotic protein. Apoptosis was inhibited by blocking the binding of PUMA to Bcl-2-like proteins. We want to observe the expression of PUMA in two different models and whether PUMA inhibitors can protect cells or tissues. Methods: (1) the mouse embryonic fibroblasts (mouse embryo fibroblast, MEFs) (2 were extracted from ICR mice on the 13th day of pregnancy. MEFs expression and cell injury were observed by different concentrations of doxorubicin (Doxorubicin, DOX) (doxorubicin (Doxorubicin, DOX). The protective effect of PUMA inhibitor on cells was observed. (3) the model of ischemia-reperfusion in C57BL/6 mice was established and divided into three groups: sham operation group, operation group and PUMA inhibitor group. (4) the level of serum ALT was observed at different time of ischemia and reperfusion. The expression of PUMA protein was detected by Western Blot, the expression of PUMA mRNA was detected by reverse transcription-real-time quantitative PCR, the injury was observed by HE section of liver, and the apoptosis of liver cells was observed by TUNEL staining. Results: when the concentration of doxorubicin was in 0.5-5mg/L, the expression of MEFs PUMA was increased, and the expression of PUMA was decreased when the concentration of doxorubicin was increased and then increased. 1 渭 MPUMA inhibitor could inhibit the expression of PUMA stimulated by low concentration of doxorubicin and protect MEFs from doxorubicin induced damage. For the model of ischemia-reperfusion injury in mice, the injury was different according to the time of ischemia and reperfusion. When the ischemia time was 60 minutes and the reperfusion time was 1 hour, the liver injury was mild and the expression of PUMA was increased. PUMA inhibitor could protect the liver from injury. When the ischemia time was as long as 90 minutes and the liver injury was serious for one hour, the expression of PUMA did not increase significantly, and PUMA inhibitor could not play a protective role. Conclusion: PUMA can be induced and activated in a certain range of injury. Apoptosis is the main pathway of injury. PUMA inhibitor can reduce the expression of PUMA protein and transcription, inhibit apoptosis and protect cells or tissues from injury. When the stimulator is too strong, apoptosis is not the main way of injury, and the expression of PUMA is not significantly increased. PUMA inhibitors can play a role in vivo and in vitro, indicating that PUMA inhibitor may be widely used. In the future, we will use PUMA KO mice to verify our results and establish a cold ischemia-reperfusion model or organ transplantation model to observe whether PUMA deletion or inhibition can protect the organs.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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