摘要
目的探讨缺血预处理（IPC）对大鼠缺血再灌注（IR）肾损伤引起肾小管上皮细胞凋亡的保护作用及其与Bcl-2、Bax表达变化的关系。方法采用夹闭小鼠双侧肾蒂30min的方法建立小鼠IR肾损伤模型；微型动脉夹夹闭小鼠双侧肾蒂15rain的方法进行预缺血，4d后再进行IR。实验共分为5个大组：对照组（C组），假手术组（S组），IR组，IPC组（IPC＋IR组），IPC对照组（S＋IR组）；除C组外，其他4组根据再灌注的时间分亚组（再灌注后0h，3h，6h，12h，24h，48h，3d，5d，7d）；反转录聚合酶链反应（RT—PCR）方法检测小鼠肾组织Bax、Bcl-2mRNA表达水平；免疫组织化学方法检测小鼠肾组织Bax、Bcl-2的分布及蛋白质表达水平。原位末端转移酶标记（TUNEL）法免疫荧光染色检测。肾小管上皮细胞凋亡。结果1．与S组、S＋IR组比较，IR组IR后血肌酐、血尿素氮、肾脏病理损害评分逐渐加重，再灌注24h达到峰值，各亚组之间差异均有统计学意义（P均〈0．01）；肾组织Bax、Bcl-2mRNA表达急剧上升，分别于6h、24h到达峰值，相对表达水平分别为2．66±0．12、2．70±0．10，各亚组间比较差异均有统计学意义（P均〈0．01）；肾组织Bax、Bcl-2蛋白质的表达水平，各亚组间比较差异均有统计学意义（P均〈0．05），TUNEL免疫荧光染色C组、S组小鼠肾小管上皮未见明显的凋亡细胞；IR组在IR后肾小管上皮细胞凋亡数逐渐增多，再灌注后24h达到峰值[（25．07±2．29）％]。2．IPC＋IR组肾脏病理损害明显减轻，Bax、Bcl-2mRNA及蛋白质表达水平、细胞凋亡明屁低于IR组及S＋IR组（P均〈0．05）。结论Bax／Bcl-2与IR肾损伤密切相关，IPC町能通过调控Bax／Bcl-2参与急性肾损伤的调控。
Objective To investigate the protective effect of ischemia preconditioning（IPC） on apoptosis in- duced by renal ischemia - reperfusion （IR） and relations to the changing expressions of Bcl - 2, Bax in rat kidney. Methods Ischemia models were induced by elipping bilateral renal pedicles for 30 min by using the arteU clamp ; IPC group was induced by clipping bilateral renal pedicles for 15 miu,4 days later IR was performed again by clipping bila- teral renal pedicle for 30 rain. Rats were randomly divided into 5 groups with 5 animals in each group：control group（ C group ）, sham - operation group （ S group）, IR group, IPC group （ IPC ＋ IR group ）, sham IPC group （ S ＋ IR group ）, all groups were randondy divided into 9 sub groups （0 h,3 h,6 h,12 h ,24 h,48 h,3 d,5 d,7 d） except C group according to the time points after reperfusion. Occurrence of apoptosis was detected by terminal deoxynuleotidyl transferase media- ted dUTP nick end and labeling（TUNEL） method;the mRNA expression and protein levels of Bax and Bcl -2 were de- tected by reverse transeriptase - polymerase chain reaction and quantitave immunohistoehemisty. Results （ 1 ） Com- pared with S group and S ＋ IR group, serum ereatinine, blood urea nitrogen, kidney pathological damage scores in IR group gradually increased alter IR,and peak point was 24 h after repeffusion;among all the subgroups there was a sig- nificant difference （all P 〈 0.01 ）. The expression of Bax, Bcl- 2 mRNA raised sharply in IR group after reperfusion, peaking at 6 h ,24 h of reperfusion respectively,2.66± 0.12,2.70 ± 0.10, and among all the subgroups there was a sig- nificant difference （ all P 〈 0.01 ） ; the expression of Bax, Bel - 2 protein had significant difference （ all P 〈 0.05 ）. TUNEL immunofluoreseenee staining showed C group and S group had no obvious apoptosis cells in renal tubular epi- thelium;epithelial cell apoptosis after IR gradually increased in IR group, peaking at 24 h of reperfusion [ （25.07 ± 2.29） % ]. （2） Compared with IR group and S ＋ IR group, pathological injury was significantly decreased in IPC ＋ IR group ; the expression of Bax, Bcl - 2 mRNA and protein, apnptosis cells were significantly decreased in IPC ＋ IR group （ all P 〈 0.05 ）. Conclusions Bax, Bel - 2 are closely assoeiated with kidney injury induced by IR. IPC may regulate acute kidney injuries by regulating Bax/Bcl -2.
出处
《中华实用儿科临床杂志》
CSCD
北大核心
2016年第5期354-358,共5页
Chinese Journal of Applied Clinical Pediatrics
基金
湖南省科技厅重点科研基金（2014SK2008）
湖南省高层次卫生人才“225”工程资助
