摘要：
Objective To study the role of CAV1 in hypoxia associated proliferation of human pulmonary ar-tery fibroblasts ( PAFs) . Methods The PAFs cultured in vitro were divided into four groups, the control group ( C:21%O2), the 10% oxygen group (10%O2), the 5% oxygen group (5%O2) and the 2% oxygen group (2%O2), and then they were given cells proliferation test and the maximal proliferation group was selected as the hypoxia group (H) for further study. The high expression of CAV1 plasmid (pCAV1) was constructed. After that, the PAFs were divided into the control group ( C:21%O2 ) , the hypoxia group ( H) , the blank control group ( NC: hypoxia+null plasmid group) and the high expression of CAV1 plasmid group (pCAV1), and then the cells proliferation was detec-ted by MTT and PCNA immunohistochemistry, and the expression of CAV1, cyclinD1 and c-IAP2 by western-blot in each group respectively. Results Hypoxia induced the proliferation of PAFs in dose dependent manner, and the maximal PAFs proliferation were observed in the 2%O2 group within 48 h while compared with the group C (1. 20+0. 02 vs 0. 54+0. 04, P<0. 01). In the hypoxia group, the expression of CAV1 decreased (1. 23 ± 0. 04 vs 0. 90 ± 0. 02, P<0. 01), while the expression of cyclinD1 (0. 19 ± 0. 03 vs 1. 15 ± 0. 06, P<0. 01) and c-IAP2 (0. 63 ± 0. 04 vs 0. 78 ± 0. 09, P<0. 01) increased, and PAFs proliferation increased (MTT:0. 78 ± 0. 04 vs 1. 20 ± 0. 02, P<0. 01;PCNA:0. 29 ± 0. 03 vs 0. 54 ± 0. 03, P<0. 01). When CAV1 was high expressed in PAFs (0. 55 ± 0. 03 vs 0. 90 ± 0. 03, P<0. 01), the expression of cyclinD1 (0. 88 ± 0. 02 vs 0. 52 ± 0. 02, P<0. 01) and c-IAP2 (0. 87 ± 0. 02 vs 0. 72 ± 0. 02, P<0. 01) decreased, and PAFs proliferation decreased, too (MTT:1. 20 ± 0. 02 vs 1. 00 ± 0. 06, P<0. 01;PCNA:0. 52 ± 0. 03 vs 0. 38 ± 0. 03, P<0. 01). Conclusion Hypoxia can down-regulate CAV1 expression in PAFs, promote PAFs proliferation and inhibit PAFs apoptosis, and cyclinD1 and c-IAP2 may be the downstream targets of CAV1 in the regulation of PAFs proliferation and apoptosis.%目的 研究小窝蛋白1(CAV1)在缺氧相关肺动脉成纤维细胞(PAFs)增殖调控中的作用.方法 将体外培养的人PAFs分为:对照组(C:21%O2);10%氧浓度组(10%O2);5%氧浓度组(5%O2)及2%氧浓度组(2%O2)进行细胞增殖实验,选取细胞增殖最明显组为缺氧组(H)进行后续实验.并构建CAV1高表达质粒(pCAV1),然后再将PAFs分为对照组(C:21%O2),缺氧组(H),空白对照组(NC:缺氧+空质粒转染组)和CAV1高表达组(pCAV1:缺氧+pCAV1转染组),采用Western-blot法检测各组细胞中CAV1、细胞周期蛋白D1(cyclinD1)和细胞凋亡抑制蛋白2(c-IAP2)含量,四甲基偶氮唑蓝(MTT)及增殖细胞核抗原(PC-NA)免疫组化法检测细胞增殖情况.结果 缺氧能刺激PAFs增殖,并呈浓度依赖性,于2%氧浓度刺激48小时PAFs增殖达峰值,与对照组相比差异有统计学意义(1.20+0.02 vs 0.54+0.04,P<0.01);缺氧组PAFs中CAV1表达下调(1.23±0.04 vs 0.90±0.02,P<0.01),cyclinD1(0.19±0.03 vs 1.15±0.06,P<0.01)和c-IAP2(0.63±0.04 vs 0.78±0.09,P<0.01)表达上调,细胞增殖增加(MTT:0.78±0.04 vs 1.20±0.02,P<0.01;PCNA:0.29±0.03 vs 0.54±0.03,P<0.01);CAV1在PAFs中高表达后(0.55±0.03 vs 0.90±0.03,P<0.01),cyclinD1(0.88±0.02 vs 0.52±0.02,P<0.01)和c-IAP2(0.87±0.02 vs 0.72±0.02,P<0.01)表达下调,PAFs增殖减少(MTT:1.20±0.02 vs 1.00±0.06,P<0.01;PCNA:0.52±0.03 vs 0.38±0.03,P<0.01).结论 缺氧能通过下调CAV-1在PAFs中的表达,促进PAFs的增殖、抑制其凋亡.cyclinD1和c-IAP2可能是CAV1调控PAFs增殖和凋亡的下游靶点.
