【作者】 郭晓芳；

【导师】 顾勤；

【作者基本信息】 南京大学 ， 急诊医学， 2011， 硕士

【摘要】 实验一P53在缺血再灌注导致急性肾损伤小鼠肾脏的表达目的：探讨缺血再灌注致急性肾损伤(AKI)小鼠肾脏不同部位p53的表达及细胞凋亡的变化。方法：采用随机对照动物实验方法,将12只小鼠随机分为两组：假手术组和AKI组,每组6只。采用双侧肾蒂夹闭45 min后松开动脉夹的方法建立小鼠AKI模型,于建模后48 h采血检测尿素氮、肌酐,取肾脏组织行HE染色观察肾组织病理学变化,采用Western Blot法测定p53蛋白含量,免疫荧光法确定肾脏不同部位p53的表达,TUNEL法检测肾脏细胞凋亡,免疫组化方法检测TNFR、Caspase-3及Bcl-2蛋白水平。结果：(1)AKI组小鼠血尿素氮和肌酐水平均明显高于假手术组(P<0.01)；HE染色显示假手术组肾组织细胞形态完整,排列整齐,无明显病理改变,AKI组则表现为肾小管上皮细胞刷状缘脱落、空泡及滴状变性,皮髓质间有明显淤血带；(2)p53在假手术组小鼠肾脏无表达,而AKI组小鼠缺血再灌注48h后p53蛋白则明显增加(P<0.05),并且主要在肾皮质表达；(3)假手术组小鼠肾脏未检测到凋亡细胞,AKI组小鼠肾脏凋亡细胞指数明显增加(P<0.01)；TNFR蛋白及Caspase-3蛋白水平升高(P<0.01),Bcl-2蛋白水平下降(P<0.01)。结论：急性肾损伤时,肾组织p53表达增加,主要定位于皮质,并可能参与介导细胞凋亡实验二急性肾损伤小鼠肾脏P53表达与细胞凋亡的关系目的：探讨缺血再灌注致AKI小鼠肾脏p53的表达与细胞凋亡的关系。方法：采用随机对照动物实验方法,将18只小鼠随机分为三组：假手术组、AKI组及PFT-α组,每组6只。采用双侧肾蒂夹闭45 min后松开动脉夹的方法建立小鼠AKI模型,PFT-α组于建模前5 min腹腔注射p53抑制剂(PFT-α)2.2mg/kg,并于建模后48 h采血检测尿素氮、肌酐,取肾脏组织行HE染色观察肾组织病理学变化,采用Western Blot法测定p53蛋白含量,TUNEL法检测肾脏细胞凋亡免疫组化方法检测(?)TNFR、Caspase-3及Bcl-2蛋白水平。结果：(1)PFT-α组和AKI组小鼠血尿素氮、肌酐水平均明显高于假手术组,而PFT-α组与AKI组比较血尿素氮、肌酐水平明显降低(P<0.01)；肾组织HE染色显示AKI组肾小管上皮细胞刷状缘脱落、空泡及滴状变性,皮髓质间有明显淤血带；PFT-α组肾小管上皮部分刷状缘脱落消失,空泡及滴状变性减轻,皮髓质间无明显淤血带；(2)与AKI组相比,PFT-α组小鼠肾脏p53蛋白含量减少(P<0.01),凋亡细胞指数也明显减少(P<0.01); TNFR蛋白及Caspase-3蛋白水平也明显降低(P<0.01), Bcl-2蛋白水平升高(P<0.01)。结论：急性肾损伤时,p53可能通过调控凋亡蛋白Bcl-2.、TNFR蛋白水平,促进Caspase-3的释放,从而介导细胞凋亡。更多还原

【Abstract】 PartⅠThe expression of P53 protein in kidney following ischemia/reperfusion injury in mice【Abstract】Objective:To investigate the expression of p53 protein in different renal regions and cell apoptosis in mice after renal ischemia/reperfusion injury. Methods: Twelve mice were randomly divided into two groups:sham-operated group(n=6) and AKI group(n=6). The models were established by clamping bilateral renal arteries for 45min and then reperfusion. The changes of content of serum creatinine and urea value were determined and renal pathologic changes were observed at 48 hours after establishing models. The p53 expression in kidney was evaluated by Western Blot and immumofluorescence methods. Apoptosis of renal cells was observed by TUNEL assay. Immunohistochemical methods were used to detect the changes of expression of NFR、Caspase-3 and Bcl-2 proteins. Results:(1) Compared to sham operated group, the contents of serum creatinine and urea nitrogen were increased significantly in AKI group(P<0.01);As compared with those of sham operated group, some changes such as renal tubular epithelial cell brush border loss, cavition and droplet degeneration were observed; (2) P53 protein increased significantly in AKI group and were negatively expressed in the sham operated group (P<0.05), P53 protein mainly expressed in cortical; (3) Apoptotic cells increased significantly in AKI group and were negatively expressed in the sham operated group(P<0.01); After ischemic/reperfusion injury, the expression level of TNFR and Caspase-3 increased (P<0.01)and a decrease for Bcl-2 (P<0.01). Conclusion:These results suggest that renal ischemia/reperfusion can induce apoptosis in rat kidneys and p53 protein may be involved in the process of renal ischemia/reperfusion injury.PartⅡRelation of cell apoptosis in kidney after ischemia/reperfusion injury and p53 protein【Abstract】Objective:To investigate relationship among renal cell apoptosis induced by ischemic/reperfusion injury and activation of p53. Methods:Eighteen mice were randomly divided into three groups:sham-operated group、AKI group and PFT-αgroup (n=6). The models were established by clamping bilateral renal arteries for 45min and then reperfusion. PFT-αgroup models were established by intraperitoneal injection PFT-α2.2mg.kg at five minutes before AKI models. The changes of content of serum creatinine and urea value were determined and renal pathologic changes were observed at 48 hours after establishing models. The p53 expression in kidney was evaluated by Western Blot and immumofluorescence methods. Apoptosis of renal cells was oberserved by TUNEL assay. Immunohistochemical methods were used to detect the changes of expression of TNFR、Caspase-3 and Bcl-2 proteins.. Results:(1) Compared to AKI group, the contents of serum creatinine and urea nitrogen were decreased significantly in PFT-αgroup (P<0.01); As compared with those of AKI group, some changes such as renal fewer tubular epithelial cell brush border loss, cavition and droplet degeneration were observed. (2) P53 protein and apoptotic cells decreased significantly in PFT-αgroup compared to AKI group (P<0.01); As compared to AKI group, the expression level of TNFR and Caspase-3 decreased (P<0.01)and a increase for Bcl-2 (P<0.01). Conclusion:P53 protein induced apoptosis through decreased the expression of caspase-3、TNFR, and increased the expession of Bcl-2 proteins in kidney following ischemia/reperfusion injury.更多还原