PBEF对HPMEC和A549细胞的影响及对ARDS发病机制的研究  

Effects of PBEF on HPMEC and A549 cells and the pathogenesis of ARDS

作　　者：王熙宇[1] 周发春[1] 罗子国[2] 刘欣[1] 刘畅[1] Wang Xiyu;Zhou Fachun;Luo Ziguo;Liu Xin;Liu Chang(Department of Critical Care Medicine, The First Affiliated Hospital of Chongqing Medical University;Institute of Life Science ,Chongqing Medical University)

机构地区：[1]重庆医科大学附属第一医院重症医学科,重庆400016 [2]重庆医科大学生命科学研究院,重庆400016

出　　处：《重庆医科大学学报》2018年第7期899-906,共8页Journal of Chongqing Medical University

基　　金：重庆市卫生局重点资助项目(编号:2013-1-007)

摘　　要：目的:探讨前B细胞克隆增强因子(pre-B cell colony enhancing factor,PBEF)在急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)发病机制中的作用。方法:体外培养人肺微血管内皮细胞株(HPMEC)和人肺泡Ⅱ型上皮细胞株(A549),采用不同浓度(0、50、100、500、1 000、2 000 ng/mL)重组人PBEF(r PBEF)分别作用于细胞,采用四甲基偶氮唑(methyl thiazolyl tetrazolium,MTT)法检测细胞活性。选用100和1 000 ng/mL r PBEF刺激细胞,并设置空白对照组,采用流式细胞术法检测细胞周期和细胞凋亡率;采用透射电子显微镜观察细胞凋亡的形态学变化;采用蛋白印迹法检测半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、B细胞淋巴瘤/白血病-2基因(B-cell lymphoma/leukemia-2 gene,Bcl-2)的蛋白表达水平;采用实时荧光定量PCR法和蛋白印迹法检测水通道蛋白-1(aquaporin 1,AQP1)、白细胞介素-8(interleukin-8,IL-8)、白细胞介素-1β(interleukin-1β,IL-1β)的mRNA和蛋白表达情况。结果:100、500、1 000、2 000 ng/mL r PBEF组相比空白对照组明显抑制了细胞活性(P<0.05)。流式细胞仪检测可见,与对照组[HPMEC:(19.347±2.052)%;A549:(17.297±0.800)%]比较,100和1 000 ng/mL r PBEF组细胞S期百分比[HPMEC:(43.847±1.272)%、(63.300±2.102)%;A549:(36.247±2.045)%、(46.400±1.346)%]明显增多(P=0.000);与对照组[HPMEC:(8.433±0.600)%;A549:(3.877±0.666)%]相比,100和1 000 ng/mL r PBEF组细胞凋亡率[HPMEC:(11.317±0.533)%、(15.227±0.637)%;A549:(6.120±0.439)%、(8.633±0.497)%]明显升高(PHPMEC=0.001,PHPMEC=0.000;P_(A549)=0.002,P_(A549)=0.000)。1 000 ng/mL r PBEF处理细胞后透射电子显微镜下可见典型凋亡细胞和凋亡小体。同时,与对照组[HPMEC:(1.279±0.077);A549:(2.824±0.129)]相比,100和1 000 ng/mL r PBEF组Bcl-2蛋白表达水平[HPMEC:(0.418±0.043)、(0.190±0.012);A549:(1.276±0.212)、(0.601±0.164)]明显下调(均P=0.000),而caspase-3蛋白表达水平[HPMEC:(0.763±0.030)、(1.170±0.056);A549:(0.217±0.010Objective：To investigate the role of pre-B-cell colony enhancing factor（PBEF） in the pathogenesis of acute respiratory distress syndrome （ARDS）. Methods：Human pulmonary microvascular endothelial cell line （HPMEC） and human type H alveolar epithelial cell line（A549） were constructed by increasing human recombinant PBEF（rPBEF） concentrations（0,50,100,500,1 000,2 000 ng/mL） in a stepwise manner. The cell viabilities to rPBEF were tested by MTT assay. And then the cells were stimulated with 100 and 1 000 ng/mL rPBEF while the blank control group was set up. The cell cycle and cell apoptosis were analyzed using flow cytometry（FCM）： Morphological changes of apoptosis were observed by transmission electron microscopy. The expressions of caspase-3, B-cell lymphoma/leukemia-2 gene were detected by Western blot. The expressions of aquaporin 1,interleukin- 8 and interleukin-1β were detected using RT-PCR and Western blot. Results：The cell viabilities were significantly inhibited in the rPBEF group with 100,500,1 000,2 000 ng/mL rPBEF compared with those in blank control group（P〈0.05）. FCM showed that the percentage of S phase in 100 and 1 000 ng/mL rPBEF groups[HPMEC ： （43.847 ± 1.272） %, （63.300 ±2.102） %; A549 ： （36.247± 2.045 ）%, （46.400 ± 1.346）%）] were significantly higher than those in the blank control group[HPMEC ： （ 19.347 ± 2.052）% ;A549 ： （ 17.297 ± 0.800） %] （all P=0.000） and the apoptic rate in 100 and 1 000 ng/mL rPBEF groups [HPMEC ： （ 11.317 ± 0.533 ） %, （15.227 ± 0.637）%;A549：（6.120 ± 0.439）%, （8.633 ± 0.497）%] were significantly higher than those in the control group[HPMEC ： （8.433 ± 0.600）%;A549：（3.877 ± 0.666）%）] （P HPMEC=0.001 ,P HPMEC=0.000;P A549=0.002,P A549=0.000）. Transmission electron microscopy showed typical apoptosis, such as heterochromatin concentrated which was set in the nuclear membrane and visible apoptotic bodies in 1 000 ng/mL rPBEF group. The expressions of

关 键 词：前B细胞克隆增强因子 急性呼吸窘迫综合征 细胞凋亡 水通道蛋白-1 

分 类 号：R563[医药卫生—呼吸系统]