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±0.010)、(0.375±0.032)]较对照组[HPMEC:(0.459±0.032);A549:(0.114±0.007)]明显升高(PHPMEC=0.000,PHPMEC=0.000;P_(A549)=0.001,P_(A549)=0.000);AQP1 mRNA和蛋白表达水平明显下降[mRNA:HPMEC(0.543±0.113)、(0.287±0.093)vs.(1.050±0.155)(P=0.002,P=0.000);A549(0.823±0.104)、(0.463±0.184)vs.(1.317±0.215)(P=0.013,P=0.001);蛋白:HPMEC(0.494±0.038)、(0.233±0.030)vs.(0.824±0.067)(均P=0.000);A549(0.850±0.157)、(0.484±0.118)vs.(1.344±0.136)(P=0.005,P=0.000)],而IL-8、IL-1β基因和蛋白表达水平明显升高(P<0.05)。结论:PBEF可能通过下调Bcl-2基因表达,使caspase-3表达水平增加,从而诱导HPMEC和A549细胞凋亡,并通过下调AQP1表达参与急性呼吸窘迫综合征的发生发展。
Objective: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 Bcl-2 protein in 100 and 1 000 ng/mL rPBEF groups[HPMEC : (0.418 ± 0.043), (0.190 ± 0.012) ;A549: ( 1.276 ± 0.212), (0.601 ± 0.164)] were significantly decreased (all P=0.000) and the expressions of caspase-3 protein were significantly increased in 100 and 1 000 ng/mL rPBEF groups[HPMEC: (0.763 ± 0.030), ( 1.170 ±0.056) ; A549 : (0.217 ± 0.010), (0.375 ± 0.032)], respectively, compared with those of blank control group (P HPMEC= 0.000, P HPMEC=0.000;P A549=0.001 ,P A549=0.000). The expressions of AQP1 gene and protein in 100 and 1 000 ng/mL rPBEF groups were statistically lower than blank control group[mRNA : HPMEC (0.543 ± 0.113 ), (0.287 ± 0.093 ) vs. ( 1.050 ± 0.155 ) ( P=0.002, P=0.000), A549 (0.823 ± 0.104), (0.463 ± 0.184) vs. (1.317 ± 0.215) (P=0.013,P=0.001) ;protein:HPMEC(0.494 ± 0.038), (0.233 ± 0.030) vs. (0.824 ± 0.067)(all P=0.000),A549(0.850 ± 0.157), (0.484 ± 0.118) vs. (1.344 ± 0.136)(P=0.005 ,P=0.000)] while the expressions of IL-8 and IL-1β gene and protein were statistically higher than blank control group in 100 and 1 000 ng/mL rPBEF groups(P〈0.05). Conclusion:PBEF may induce the apoptosis of HPMEC and A549 by down-regulating the expression of Bcl-2 and up-regulating the expression of caspase-3 and PBEF could induce low expression of AQP1 which suggests that PBEF may play an critical role in the development of ARDS.
作者
王熙宇
周发春
罗子国
刘欣
刘畅
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)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
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)