摘要
目的探索沉默气道血管平滑肌细胞(Vascular smooth muscle cells,VSMCs)的去整合素金属蛋白酶33(adisintegrin and metalloproteinase-33,ADAM33)基因表达对共培养体系中气道血管内皮细胞(Vascular endothelial cell,VECs)增殖、管腔形成的影响和作用机制。方法构建人主动脉平滑肌细胞(Human aortic smooth muscle cells,HASMCs)和人肺微血管内皮细胞(Human pulmonary microvascular endothelial cells,HPMECs)的共培养体系。通过慢病毒转染技术沉默ADAM33基因表达,将实验对象分为内皮细胞空白组、共培养组、共培养+shRNA阴性对照组、共培养+ADAM33-shRNA组。ELISA方法检测共培养体系sADAM33、VEGFA、VEGER2、ang-1和ang-2的表达;CCK-8、Transwell实验观察HPMECs的增殖、管腔形成能力;Western-blotting方法检测Tie2/PI3K/Akt/mTOR信号通路关键分子Tie2、PI3K、AKT、mTOR的蛋白表达和AKT、mTOR磷酸化水平。结果:(1)与共培养组(0.851±0.036)和共培养+shRNA阴性对照组(0.828±0.047)相比,共培养+ADAM33shRNA组OD值(0.699±0.038)显著减低(P<0.05);(2)与共培养组(159.169±15.740)和共培养+shRNA阴性对照组(157.357±21.612)相比,共培养+ADAM33shRNA组的成管长度(120.812±2.791)也显著减低(P<0.05);(3)沉默共培养体系中HASMCs的ADAM33基因表达后,VEGFA、VEGFR2、ang-1和ang-2等促血管生成因子表达含量明显减低(P<0.05),同时Tie2、PI3K、p-Akt、p-mTOR的表达水平下降(P<0.05)。结论沉默ADAM33的基因表达能够减少sADAM33从气道VSMCs的细胞膜上脱落释放,通过降低VEGF/VEGFR的表达、抑制Tie2/PI3K/Akt/mTOR通路活性而调控气道VECs的增殖和管腔形成能力,进而参与哮喘气道血管重塑。
Objective To investigate the effect of ADAM33 gene silencing in VSMCs on the proliferation and lumen formation of airway vascular endothelial cells(VECs)in a co-culture system and the possible regulatory mechanism.Methods The Human aortic smooth muscle cells(HASMCs)and human pulmonary microvascular endothelial cells(HPMECs)were used to construct a cell co-culture system.ADAM33 gene expression was silenced by lentivirus transfection technique,and the subjects were divided into endothelial cell blank group,co-culture group,coculture+shRNA negative control group,and co-culture+ADAM33-SHRNA group.The expressions of sADAM33,VEGFA,VEGER2,ang-1 and ang-2 in co-culture system were detected by ELISA.The proliferation and lumen formation of HPMECs were observed by CCK-8 and Transwell experiments.The protein expression of Tie2,PI3K,Akt,and mTOR key molecules in Tie2/PI3K/Akt/mTOR signaling pathway and the phosphorylation levels of AKT and mTOR were detected by Western-blotting method.Results①Compared with the co-culture group(0.851±0.036)and the co-culture+shRNA negative control group(0.828±0.047),the OD value of the co-culture+ADAM33shRNA group(0.699±0.038)was significantly decreased(P<0.05).②Compared with the co-culture group(159.169±15.740)and the co-culture+shRNA negative control group(157.357±21.612),the tube length of the co-culture+ADAM33shRNA group(120.812±2.791)was also significantly decreased(P<0.05).③After ADAM33 gene expression of HASMCs was silted in co-culture system,the expression levels of VEGFA,VEGFR2,ang-1 and ang-2 were significantly decreased(P<0.05),while the expression levels of Tie2,PI3K,P-Akt and P-mtor were decreased(P<0.05).Conclusions Silencing the expression of the ADAM33 gene could reduce the release of sADAM33 from the membrane of the airway VSMCs,regulate the proliferation and lumen formation of airway VECs by reducing the expression of VEGF/VEGFR and inhibiting the activities of the Tie2/PI3K/Akt/mTOR signaling pathways,and then participate in airway vascular remodeling in asthma.
作者
胡小娟
曼古努·石那别克
胡昕
于敏杰
闫芳
HU Xiaojuan;MANGUNNU Shi Nabek;HU Xin;YU Minjie;YAN Fang(The first ward of Respiratory and respiratory Critical Care Center,the First Affiliated Hospital of Xinjiang Medical University,Urumuqi Xinjiang 810000,P.R.China)
出处
《中国呼吸与危重监护杂志》
CAS
CSCD
2024年第8期569-574,共6页
Chinese Journal of Respiratory and Critical Care Medicine
基金
国家自然科学基金(81760005)
