摘要
目的探讨金属蛋白酶组织抑制剂1(TIMP-1)在高糖诱导的肾小球系膜细胞(MC)凋亡中的作用及机制。方法用流式AnnexinV/PI双染法及吖啶橙染色检测大鼠MC经不同浓度葡萄糖作用后的凋亡率。用脂质体法将正义、反义人TIMP(hTIMP)-1转染到MC中。采用PCR及RT-PCR检测hTIMP-1的整合及大鼠内源性TIMP-1(rTIMP-1)、bax和bcl-2表达。用CaspACETMAssaySystem检测半胱氨酸天胱氨酸蛋白酶(caspase-3)的蛋白活性。结果高糖以剂量及时间依赖的方式诱导凋亡,相关系数r分别为0.925、0.9867,P均<0.01。在葡萄糖浓度为30mmol/L培养条件下,hTIMP-1正义转染组的MC24h凋亡率为(4.30±1.11)%,48h为(7.78±0.92)%,与正常对照组[24h(14.95±1.60)%,48h(43.03±4.20)%]及空载体组[24h(16.50±0.83)%,48h(40.82±2.46)%]相比,细胞凋亡显著减少(P<0.01);hTIMP-1反义转染组MC24h凋亡率为(22.5±1.60)%,48h为(53.68±3.40)%,与正常对照组和空载体组相比,细胞凋亡显著增加(P<0.01)。高糖作用后,正义TIMP-1下调MCbaxmRNA的表达,反义hTIMP-1使之表达上调。与对照组(A值0.1407±0.007)相比,正义hTIMP-1组caspse-3活性(A值0.086±0.009)显著降低,(P<0.01),反义hTIMP-1组caspase-3活性(A值0.186±0.02)显著增高,(P<0.01)。TIMP-1转染不影响bcl-2mRNA水平的表达。结论TIMP-1能够抑制高糖诱导的MC凋亡,这种作用部分是通过bax及easpase-3途径来实现的。
Objective To investigate the effects and mechanisms of tissue inhibitor of metailoproteinase 1 （TIMP-1） on the apoptosis of rat mesangial cells （MC） induced by high glucose. Methods Rat mesangial ceils were cultured in medium 1640 with different glucose concentrations for 24 h, 48 h and 72 h respectively. The apoptosis ratio was tested by both flow cytometry AnnexinV/PI double stains and AO stains. MCs were transfected with empty pcDNA3.1 vector, TIMP-1S-pcDNA3.1 （PCT）, and TIMP-1AS-pcDNA3.1 （PCA） by hposomal transfection reagent. The expression of human TIMP-1, endogenous rat TIMP-1, bax and bcl-2 were examined by PCR and RT-PCR. Caspase-3 activity was analysized by CaspACETM Assay System. Results High glucose could induce apoptosis in a dose- and time-dependent manner. The coefficient of determination was r=0.925 and r=0.9867 respectively（P 〈 0.01 ）. Exposed to high glucose （30 mmol/L）, the apoptosis ratio of PCT group was 4.3%±1.11% at 24 h,and 7.78%±0.92% at 48 h, respectively. As compared to control（14.95%±1.6% for 24 h, 43.03%±4.2% for 48 h） and empty vector groups, fewer MCs underwent apoptosis （P〈 0.01） in PCT group. The apoptosis ratio of PCA group was 22.5%±1.6% at 24 h, and 53.68%±3.4% at 48 h, which suggested much more MCs in PCA group experienced apoptotic changes （P 〈0.01 ）. Under the influence of high glucose, PCT transfection could down-regulate the expression of bax, whereas PCA transfection could up-regulate the expression of bax by contrast. Compared with control group （the value of A was 0.1407±0.007）, the activity of caspase 3 in PCT group decreased obviously （the value of A was 0.086±0.009, P 〈 0.01 ）, while its activity increased gready （the value of A was 0.186±0.02, P〈 0.01） in PCA group. The exogenous human TIMP-1 seemed to have little effect on the expression of bcl-2 mRNA. Conclusions TIMP-1 can inhibit the apoptosis of MCs induced by high glucose, which partly involves in lowering the expression of bax and attenuating
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2005年第8期477-482,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金(30270617
30477621)
