全反式维A酸对胎鼠肺泡Ⅱ型上皮细胞增殖和肺表面活性蛋白C及水通道蛋白5表达的影响

Effect of all - trans retinoic acid on proliferation of fetal alveolar epithelial type H cells and expression of pul- monary surfactant C and aquaporin 5

目的 探讨全反式维A酸（at-RA）对胎鼠肺泡Ⅱ型上皮细胞（fAECⅡs）增殖和肺表面活性蛋白C（SPC）、水通道蛋白5（AQP5）表达的影响。方法 分离、纯化孕19 d的胎鼠肺组织，得到fAECⅡs。细胞培养1 d后，以at-RA作为干预方式，在at-RA作用1、2、3 d后，使用噻唑蓝（MTT）法检测细胞增殖情况和活力，应用倒置显微镜观察细胞生长状况，采用实时荧光定量PCR（RT-PCR）法检测 SPC mRNA、AQP5 mRNA 的表达，W estern blot法检测SPC、AQP5蛋白的表达。结果 1.at-RA作用1 d对细胞增殖和活力无影响（P 〉0.05）；而作用2 d后，at-RA明显促进细胞增殖、增强细胞活力（P 〈0.05），且促进作用在第3天最显著（P 〈0.05）。2.与对照组相比，at-RA使得细胞状态更佳，细胞贴壁更紧，折光性更好。3.与对照组相比，at-RA在第1、2、3天均可上调AQP5 mRNA及AQP5蛋白的表达（t= -19.58、-10.44、-16.01、46.25、12.79、-27.96，P均 〈0.05），分别为对照组的281.07% 、766.67% 、1163.33% 和792.65% 、1310.52% 、1561.56% 。4.与对照组相比，at-RA在第1、3天上调 SPC mRNA、SPC 蛋白的表达（蛋白为对照组的615.480% 、369.450% ；mRNA 为对照组的728.33% 、400.83%）（t= -26.34、-25.26、-25.25、-31.71，P均〈0.05），但在第2天，下调 SPC mRNA、SPC蛋白（对照组的66.57% ，11.269%）的表达（t=9.12、13.80，P均〈0.05）。结论 at-RA可促进 fAECⅡs增殖，增强细胞活力；促进SPC和AQP5的表达。

Objective To investigate the effect of all -trans retinoic acid （at- RA） on fetal alveolar epithelial type Ⅱ cells （fAEC Ⅱ s） proliferation and the expression of pulmonary surfactant C （SPC） as well as aquaporin 5 （ AQP5 ）. Methods fAEC Ⅱ s were isolated and purified from fetal lung of pregnant SD rats （ 19 days）. After being cultured for 1 day, and the fAEC Ⅱ s were interfered by at - RA for 1,2 and 3 days. Cell proliferation, viability as well as growth state, expressions of SPC mRNA as well as AQP5 mRNA and expressions of protein SPC as well as AQP5 were respectively detected by using 4,5 - dimethylthiazol - 2 - yl - 2,5 - diphenyhetrazolium bromide （MTT） , inverted microscope, real - time fluorescence quantitative PCR （ RT - PCR ） and Western blot. Results （ 1 ） When fAEC Ⅱ s were treated with at - RA for 1 day, and the cell proliferation and viability did not change （ P 〉 0.05 ）, while the proliferation and viability were significantly improved in 2 days （ P 〈 0.05 ）, and the difference was the most obvious （ P 〈 0.05 ） at 3 days. （2） Compared with the control group, the cell growth state was better, and the cell adherence was tighter and the refraction was higher in at - RA group. （3） Compared with the control group, at - RA up - regulated the expressions of AQP5 mRNA and AQP5 protein（ t = - 19. 58, - 10. 44, - 16. 01, - 46. 25, - 12. 79, - 27.96, all P 〈 0.05 ）, and the percentages of control group were 281.07% ,766.67% , 1 163.33% and 792.65% , 1 310.52% , 1 561.56% in 1,2 and 3 days respectively. （4） Compared with control group, the expressions of SP - C mRNA and SPC protein were up - regulated when ceils were exposed to at - RA for 1 and 3 d, but while they were down - regulated at 2 days （ protein ： the percentages of control group were 615. 480%, 369. 450% and 11. 269% , respectively ; mRNA ：728.33% ,400.83%, 66.57%,respectively） （t = -26.34, -25.26,13.80, -25.25, -31.71,9.12,all P〈0.05）.Conclusions at-RA can promote the proliferation and differentiation of fAEC Ⅱ s, enhance the fAEC Ⅱs viability, and improve the expression of SPC and AQP5.