摘要
目的 探讨正、反义组织金属蛋白酶抑制剂 1(TIMP 1)对肾小球系膜细胞凋亡的影响。方法 利用前期工作中克隆出的人TIMP 1全长cDNA ,分别构建出表达正义及反义TIMP 1的重组真核表达载体pcDNA3 TIMP 1(PTs)、pcDNA3 ATIMP 1(PTas) ,并将上述重组质粒转染至大鼠的肾小球系膜细胞中 ,Northern杂交检测正、反义TIMP 1的表达。无血清诱导大鼠系膜细胞凋亡 ,利用DNA缺口末端标记 (TUNEL)分析、DNA电泳技术在无血清后的不同时间点检测细胞凋亡 ,RT PCR的方法检测凋亡相关基因的表达。结果 转染PTas的大鼠系膜细胞可以高效表达反义TIMP 1,于无血清诱导 12h开始发生凋亡 ;无外源基因转染及空载体转染的大鼠系膜细胞在无血清 48h发生凋亡 ;而转染正义TIMP 1的大鼠系膜细胞可高效表达正义TIMP 1,在无血清 4d时才发生凋亡。TIMP 1的高表达或低表达可引起大鼠系膜细胞bax的下调或上调 ,但并不影响bcl 2的表达。结论 TIMP 1可以抑制无血清诱导的大鼠系膜细胞的凋亡 ,反义TIMP 1对之则有促进作用。这提示TIMP 1在肾脏除了抑制细胞外基质的作用外还具有新的功能。
Objective To investigate the role of tissue inhibitor of metalloproteinase 1 (TIMP 1) in apoptosis of rat glomerular mesangial cells (RMC) in vitro. Methods TIMP 1 full length cDNA cloned in previous work was used to construct two recombinant plasmids, eukaryotic expression rector, pcDNA3 TIMP 1 (PTs) encoding sense TIMP 1 and pcDNA3 ATIMP 1 (PTas) encoding antisense TIMP 1. These two vectors were then transfected into RMC with lipofectin DOTAP. Northern blotting was used to detect the expression of sense and antisense TIMP 1. Apoptosis was induced in normal RMC, RMC transfected with pCDNA3, PTs and PTas by serum deprivation. Those cells were monitored for apoptosis over extended periods of time (12 h～4 d) by TUNEL and DNA electrophoressis. Furthermore, the expression of apoptosis related genes was also investigated by RT PCR. Results Northern blot analysis and RT PCR demonstrated that cells transfected with exogenous genes effectively expressed sense or antisense TIMP 1 mRNA. RMC transfected with PTas underwent apoptosis after 12 h of serum deprivation, normal RMC and RMC transfected with pcDNA3 underwent apoptosis after 48 hours in serum free medium, while RMC transfected with PTs did not undergo apoptosis until 4 days after serum deprivation. Furthermore, TIMP 1 upregulation inhibited expression of bax but had no effect on the expression of bcl 2. Conclusion TIMP 1 suppressed apoptosis of RMC, which suggests a novel activity of TIMP 1 in kidney. Antisense TIMP 1 promotes apoptosis in RMC. The effects of TIMP 1 on RMC apoptosis perhaps correlate with inhibition of bax expression.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2001年第7期411-414,共4页
National Medical Journal of China
基金
国家自然科学基金!资助项目 (30 0 0 0 0 80 )
