脂氧素A4通过上调Smac表达诱导肾间质成纤维细胞凋亡  

Lipoxin A4 induces apoptosis of rat renal interstitial fibroblasts via up regulation of Smac expression

作　　者：吴升华[1] 陆超[1] 董玲[1] 姜新猷[1] 

机构地区：[1]南京医科大学第一附属医院儿科,210029

出　　处：《中华肾脏病杂志》2004年第5期339-342,共4页Chinese Journal of Nephrology

基　　金：江苏省135医学重点人才工程基金(2002-45)

摘　　要：目的验证脂氧素A4(LXA4)是否诱导肾间质成纤维细胞凋亡,并探讨其作用机制是否与半胱氨酸天冬氨酸蛋白酶(caapase)第2个线粒体激活因子(Smac)表达上调有关。方法将大鼠肾间质成纤维细胞(NRK-49F)培养于含5%胎牛血清的培养液中,用较高浓度的LXA4刺激后,应用吖啶橙/溴化乙锭双荧光染色观察细胞凋亡形态,应用碘化丙锭/Annexin双染色及流式细胞仪计数凋亡的细胞,应用对硝基苯胺法测定caspase-3酶活性。应用Western印迹方法测定Smac表达,对NRK-49F细胞用脂质体转染Smac反义寡核苷酸,再观察LXA4处理后Smac表达、细胞凋亡的改变。结果LXA4在100nmol/L、1μmol/L的高浓度时,分别引起9.83%、33.82%的NRK-49F细胞发生凋亡、caspase-3活性升高、Smac表达上调。应用Smac反义寡核苷酸处理细胞后,可抑制LXA4所致的Smac表达与细胞凋亡。结论较高浓度的LXA4能够引起大鼠肾间质成纤维细胞发生凋亡,其机制与上调Smac表达有关。Objective To examine whether lipoxin A4(LXA4) induces apoptosis of rat renal interstitial fibroblasts and explore the mechanism concerned.Methods Rat renal interstitial fibroblasts (NRK 49F cells)were incubated in RPMI 1640 medium supplemented with 5%fetal calf serum and exposed to LXA4 at the concentration of 10 nmol/L, 100 nmol/L or 1 μmol/L for 24 hours. Prior to experiment,some NRK 49F cells were transfected with Smac antisense oligodeoxynucleotide. Apoptosis of NRK 49F cells was recognized by double staining using fluorescent dye acridine orange and ethidium bromide,and observed under laser scanning confocal microscopy and counted by flow cytometry following propidium iodide and annexin staining. Activity of caspase 3 was measured by colorimetric assay. The expression of Smac was determined by Western blotting analysis.Results LXA4 at the concentration of 100 nmol/L or 1 μmol/L induced apoptosis of 9 83%or 33 82%of NRK 49F cells respectively, and reduced the cells of S and G2～M phase and increased the cells of G0～G1 phase in a dose dependent manner. Treatment of NRK 49F cells with LXA4 up regulated the expression of Smac protein and increased the activity of caspase 3. The transfection with Smac antisense oligodeoxynucleotide inhibited the LXA4 induced apoptosis and expression of Smac in NRK 49F cells. Conclusion LXA4 at high concentration can induce apoptosis of rat renal interstitial fibroblasts via the up regulation of Smac expression.

关 键 词：表达 肾间质成纤维细胞 细胞凋亡 上调 脂氧素 诱导 反义寡核苷酸 溴化乙锭 脂质体转染 线粒体 

分 类 号：R692[医药卫生—泌尿科学]