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

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.