低氧对大鼠不同级别肺动脉平滑肌细胞功能的影响

Effects of hypoxia on the function of smooth muscle cells from different orders of pulmonary arteries

目的培养鉴定大鼠不同级别肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs),探讨其对缺氧刺激反应性的不同。方法酶消化法分离大鼠不同级别PASMCs并分为3组:大肺动脉组,包括肺动脉干及肺内一级主干;中动脉组,包括肺内主干的第二级和第三级分支;小肺动脉组,包括肺内主干四级分支后的更细小分支。分别给予常氧(DMEM+2%FBS、5%CO2、21%O2、37℃)和低氧(DMEM+2%FBS、5%CO2、3%O2、37℃)培养48 h,用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)比色法和Western blotting检测增生细胞核抗原(proliferating cell nuclear antigen,PCNA)的蛋白表达水平的方法检测细胞的增生能力;应用伤口愈合实验检测细胞的迁移能力。结果倒置显微镜下PASMCs呈现长梭形,免疫荧光法检测平滑肌细胞标志物即平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)为阳性,生理情况下(常氧),3组肺动脉平滑肌细胞增生及迁移能力均较一致;低氧培养48 h后,中小肺动脉平滑肌细胞的增生与迁移能力均显著高于大动脉,PCNA结果与MTT结果一致,差异具有统计学意义(P<0.05)。结论在低氧条件刺激下,不同级别肺动脉平滑肌细胞反应性不同,中小肺动脉平滑肌细胞的增生及迁移能力均显著高于大肺动脉平滑肌细胞。

Objective To explore the effects of hypoxia on the proliferation and migration of rat pulmonary artery smooth muscle cells （ PASMCs） isolated and cultured from different orders of pulmonary arteries. Methods The pulmonary artery branches were gently isolated from male Sprague-Dawley rats（250-350 g） from Capital Medical University and eventually cut into three groups according to the vascular grading：the proximal pulmonary arteries （ pulmonary trunk and the first order branches of the intrapulmonary arteries ） , the middle pulmonary arteries （ second to third order intrapulmonary arteries ） , and the distal pulmonary arteries （ forth to fifth order intrapulmonary arteries）. Primary PASMCs were cultured from the pulmonary arteries and α-smooth muscle actin（α-SMA） in the cells was identified by immunofluorescence. PASMCs were cultured under normoxia（ DMEM＋2%FBS, 5%CO2 , 21%O2 , 37 ℃） and hypoxia （DMEM＋2%FBS, 5%CO2, 3%O2, 37 ℃）. Cell proliferation was quantified by using 0. 5 g/L methyl thiazolyl tetrazolium（MTT） assay. Western blotting was used for detecting the protein expression level of proliferating cell nuclear antigen （ PCNA ） in PASMCs. Wound healing test was explored to assess the migration function of the cells. Results Cells isolated from different orders of pulmonary arteries from rats tended to be long spindle and grew in the “peak-valley” mode under microscope. Immunofluorescence results showed that all of the three groups of cells are positively expressα-SMA which is a marker of smooth muscle cells. Under physiological condition （normoxic）, three groups of PASMCs have the similar ability of proliferation and migration. While after incubation under hypoxia（3%O2 , 48 h） , all cells from the three groups showed increased proliferation ability（ assessed by MTT and PCNA expression） as well as the migration ability. Moreover, compared to the proximal pulmonary arteries, PASMCs from the middle and distal pulmonary arteries showed much more sensitive to hypoxia stimulus. Conclusion These results suggest that PASMCs from middle and distal pulmonary arteries have higher ability of proliferation and migration than those from proximal under hypoxia condition. This will help to give us some information about the possible mechanism of different responses of different orders of pulmonary arteries under the same pathophysiological stimulus.