Xu L, Tian G Y, Wang L H, Liu Y B, Gao Z F, Li G H, Fu X H
Department of Respiratory and Critical Care Medicine, the Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2017 Feb 12;40(2):118-122. doi: 10.3760/cma.j.issn.1001-0939.2017.02.008.
In this study, a primary culture system for the rat distal pulmonary arterial smooth muscle cell (PASMC) was established to observe the effect of Bortezomib a treatment on the basal intracellular calcium concentration (Ca(2+) ), store operated calcium entry (SOCE) and Orai-1 expression in rat PASMC. We employed the primary culture method for the rat distal PASMC including the enzymatically dissociation of PASMC from the freshly isolated distal pulmonary artery and the culture of PASMC. The In Cyte system was used to measure the basal Ca(2+) and SOCE after substantial treatment.Orai-1 protein expression in rat pulmonary artery smooth muscle was detected by Western blot. Compared with Hypoxia group, the basal Ca(2+) were significantly reduced in Hypoxia+ BTZ group(<0.01). The basal Ca(2+) 340/380 ratio of Normoxia group was(1.07±0.02). The basal Ca(2+) of Hypoxia group was(1.49±0.03); The Hypoxia+ BTZ group was(1.17±0.03). Compared with Hypoxia group, the store operated calcium entry were significantly reduced in Hypoxia+ BTZ group(<0.01). The SOCE 340/380 ratio of Normoxia group was(0.56±0.02). The SOCE of Hypoxia group was(0.84±0.02); The Hypoxia+ BTZ group was(0.66±0.02). The level of Orail-1 protein in pulmonary artery smooth muscle of Hypoxia group was (181.5±12.7)% higher than control group which was(100±0)%, (<0.05). In the Hypoxia+ BTZ group Orai-1 protein expression was recovered(146.7±15.1)%, (<0.05). Bortezomib inhibit chronically hypoxic enhancement of Orail-1 protein expression, basal Ca(2+) and SOCE in rat distal pulmonary arterial smooth muscle cells.
在本研究中，建立了大鼠远端肺动脉平滑肌细胞（PASMC）的原代培养体系，以观察硼替佐米治疗对大鼠PASMC基础细胞内钙浓度（Ca(2+)）、钙库操纵性钙内流（SOCE）及Orai-1表达的影响。我们采用大鼠远端PASMC的原代培养方法，包括从新鲜分离的远端肺动脉中酶解PASMC以及PASMC的培养。使用In Cyte系统在大量处理后测量基础Ca(2+)和SOCE。通过蛋白质免疫印迹法检测大鼠肺动脉平滑肌中Orai-1蛋白表达。与缺氧组相比，缺氧+硼替佐米组的基础Ca(2+)显著降低（<0.01）。常氧组基础Ca(2+) 340/380比值为(1.07±0.02)。缺氧组基础Ca(2+)为(1.49±0.03)；缺氧+硼替佐米组为(1.17±0.03)。与缺氧组相比，缺氧+硼替佐米组的钙库操纵性钙内流显著降低（<0.01）。常氧组SOCE 340/380比值为(0.56±0.02)。缺氧组SOCE为(0.84±0.02)；缺氧+硼替佐米组为(0.66±0.02)。缺氧组肺动脉平滑肌中Orail-1蛋白水平比对照组高(181.5±12.7)%，对照组为(100±0)%，（<0.05）。在缺氧+硼替佐米组中，Orai-1蛋白表达恢复至(146.7±15.1)%，（<0.05）。硼替佐米可抑制大鼠远端肺动脉平滑肌细胞中慢性缺氧对Orail-1蛋白表达、基础Ca(2+)和SOCE的增强作用。
