摘要
目的:检测脂氧素A4(LXA4)是否诱导大鼠肾间质成纤维细胞凋亡,并探讨其作用机制是否与钙激活中性蛋白酶10(calpain10)基因表达有关。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DEVD对硝基苯胺法测定半胱天冬蛋白酶3(caspase-3)酶活性。应用RT-PCR方法测定calpain10基因表达,对NRK-49F细胞用脂质体转染calpain10反义寡核苷酸,再观察LXA4处理后calpain10表达、细胞凋亡、caspase-3酶活性的改变。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、calpain10基因表达上调。应用calpain10反义寡核苷酸转染细胞后,可抑制LXA4所致的calpain10基因表达、细胞凋亡与caspase-3酶活性。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,其机制可能与上调calpain10基因表达有关。
AIM： To examine whether lipoxin A4（LXA4） induces apoptosis of rat renal interstitial fibroblasts and explore the mechanisms. 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, nor to experiment, some cells were transfected with calpain 10 antisense oligodeoxynucleotide. Apoptosis of cells was recog nized by double staining using fluorescent dye acridine orange and ethidium bromide, 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 calpain 10 mRNA was determined by RT - PCR. RESULTS： LXA4 at the concentration of 100 nmol/L or 1 μmol/L induced 9.83% or 33.82% apoptosis of cells, respectively. Treatment of cells with LXA4 upregulated the expression of calpain 10 mRNA and increased the activity of caspase - 3. The transfection of the cells with calpain 10 antisense oligodeoxynucleotide inhibited the LXA4 - induced apoptosis, activity of caspase - 3 and expression of calpain 10. CONCLU- SION： LXA4 at high concentration induceds apoptosis in rat renal interstitial fibroblasts via up - reguting of calpain 10 mRNA expression.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2006年第1期143-147,共5页
Chinese Journal of Pathophysiology
基金
江苏省135医学重点人才工程资助项目(No.2002-45)
