CAJ | 학술논문

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益)和低氧( DMEM+2%FBS、5%CO2、3%O2、37益)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。
목적배양감정대서불동급별폐동맥평활기세포( pulmonary artery smooth muscle cells,PASMCs),탐토기대결양자격반응성적불동。방법매소화법분리대서불동급별PASMCs병분위3조：대폐동맥조,포괄폐동맥간급폐내일급주간；중동맥조,포괄폐내주간적제이급화제삼급분지；소폐동맥조,포괄폐내주간사급분지후적경세소분지。분별급여상양( DMEM+2%FBS、5%CO2、21%O2、37익)화저양( DMEM+2%FBS、5%CO2、3%O2、37익)배양48 h,용사갑기우담서염( methyl thiazolyl tetrazolium, MTT)비색법화Western blotting검측증생세포핵항원( proliferating cell nuclear antigen,PCNA)적단백표체수평적방법검측세포적증생능력；응용상구유합실험검측세포적천이능력。결과도치현미경하PASMCs정현장사형,면역형광법검측평활기세포표지물즉평활기기동단백(α-smooth muscle actin,α-SMA)위양성,생리정황하(상양),3조폐동맥평활기세포증생급천이능력균교일치；저양배양48 h후,중소폐동맥평활기세포적증생여천이능력균현저고우대동맥,PCNA결과여MTT결과일치,차이구유통계학의의(P<0.05)。결론재저양조건자격하,불동급별폐동맥평활기세포반응성불동,중소폐동맥평활기세포적증생급천이능력균현저고우대폐동맥평활기세포。
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.