模拟失重对肺微血管内皮细胞屏障功能的影响

Effect of simulated agravity on barrier function of pulmonary micro-vascular endothelial cells

目的研究模拟失重对肺微血管内皮细胞屏障功能的影响,为防御失重所导致的不良影响寻找新的治疗靶点。方法采用回转器模拟失重效应干预肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVEC),回转培养72h,同时设1g对照静置培养组。之后用免疫荧光染色标记细胞骨架肌动蛋白F-actin,并在激光共聚焦显微镜下观察细胞骨架的改变;光镜下观察单核细胞(THP-1)与PMVEC的黏附作用,并计算黏附率;绿色荧光标记的腺病毒感染细胞,荧光显微镜下观察,并在流式细胞仪检测感染效率。结果回转72h PMVEC胞质内F-actin的表达减少,微丝的拉丝感和方向感明显弱化,排列松散,细胞伸展不好,体积变小;模拟失重抑制PMVEC对单核细胞的黏附,导致黏附率(37.69%±6.5% vs 51.25%±8.2%,P<0.05)下降;模拟失重组的腺病毒感染率更高(84.55%±5.31%vs 66.32%±5.74%,P<0.05),更容易受到腺病毒感染。结论模拟失重可以损伤PMVECs的屏障功能。

Objective To find the novel treatment target of agravity-induced adverse reactions by studying the effect of simulated agravity on the barrier function of pulmonary micro-vascular endothelial cells（PMVEC）. Methods Effect of agravity on PMVECs was simulated with a clinostat. PMVEC were gyratorily cultured for 72h and lg of PMVEC was statically cultured as controls. Cytoskeletal F-actin was marked with immunofluorescence staining and changes of cytoskeleton were observed under laser confocal microscope. Adhesion of monocytes（THP-1） to PMVEC was observed under light microscope and their adhesion rate was calculated. Green fluorescence-labeled adenovirus-infected PMVEC were observed under fluorescence microscope and detected by flow cytometry. Results After 72h of gyratory culture, the expression level of F-actin in PMVEC was lower, the drawing and direction feelings of microfilament were significantly weaker, the arrangement of PMVEC was looser and their extension was poorer, and the volume of PMVEC was smaller. Stimulated agravity suppressed the adhesion of PMVEC to THP-1, leading to their adhesion rate decreased from 51.25% ± 8.2% to 37.69% ±6.5%（P〈0.05） and the adenovirus infection rate of PMVEC increased from 66.32% ±5.74% to 84.55% ± 5.31%（P〈0.05）. Conclusion Simulated agravity can injure the barrier function of PMVEC.