中性粒细胞弹性蛋白酶抑制剂对U937细胞增殖和凋亡的影响

Neutrophil elastase inhibitor on proliferation and apoptosis of U937 cells

目的比较两种中性粒细胞弹性蛋白酶（NE）抑制剂GW311616A和西维来司钠（sive—lestat）对U937细胞增殖和调亡的影响。方法采用四唑蓝比色试验（MTT）检测细胞增殖抑制率；用透射电镜观察细胞形态学变化；用流式细胞术检测细胞凋亡[膜联蛋白V／碘化丙锭（AnnexinV／PI）双标记法]及细胞周期变化；应用间接免疫荧光法观察细胞内NE表达水平；同时利用ELISA法和比色法检测细胞内NE含量和活性的变化。使用SPSS13．0软件对所得数据进行统计分析，采用完全随机设计资料的方差分析和Dunnett—t检验对各处理组数据进行比较。结果MTr法检测结果显示两种药物均能抑制U937细胞的增殖，且具有一定的剂量依赖效应，GW311616A和西维来司钠的Ic50值分别为150、214μmol／L。GW311616A对U937细胞的增殖抑制作用高于西维来司钠（P〈0．01）；电镜结果显示两种药物处理的U937细胞均能看到典型的细胞凋亡形态；AnnexinV／PI双标记法结果表明两种药物均能引起U937细胞的早期凋亡，GW311616A（150μmol／L）组的凋亡率为13．60％，西维来司钠组（150μmol／L）为3．69％，GW311616A对其凋亡的影响比西维来司钠更明显（P〈0．01）；流式细胞术检测结果显示GW311616A（150μmol／L）组的凋亡率为14．61％，细胞周期主要阻滞于G2／M期，西维来司钠（150μmol／L）组的凋亡率为4．25％，细胞周期主要阻滞于S期；间接免疫荧光法检测结果显示GW311616A组荧光强度明显减弱，而西维来司钠组荧光强度未明显减弱；ELISA和比色法结果显示两种药物均能使U937细胞内NE含量和活性降低，但GW311616A对他们的影响大于西维来司钠（P〈0．01）。结论GW311616A和西维来司钠均能抑制U937细胞的增殖，引起其凋亡，但前者比后者更有效，更能造成细胞的损伤。

Objective To study and compare the effect of neutrophil elastase inhibitors （GW311616A and sivelestat） on the proliferation and apoptosis of U937 cells. Methods Inhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTY assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by An- nexin V-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The ex- pression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and ac- tivity of NE in U937 cells were measured through ELISA assay and colorimetric method. Results M3T showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 bLmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat （P 〈0.01 ）. Typical apoptosis morphological changes of U937 cells was observed through electron microscope. Annexin V-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150μmol/L GW311616A group （ 13.60% ） was significantly higher than that of 150μmo]/L sivelestat group （3.69%） （P 〈 0.01 ）. The result of flow cytometry indicated that the apoptosis ratio of 150 μmoL/L GW311616A group was 14.61% , U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 μmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat（ P 〈0.01 ）. Conclusion GW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.