弹性蛋白酶肺气肿表面活性物质蛋白A、B的基因表达

Change of expression of surfactant protein A and B in elastase-induced emphysema

目的研究肺气肿发生、发展过程中表面活性物质蛋白A、B(SP-A、B)的基因表达变化。方法金黄地鼠气道一次性注入弹性蛋白酶,分别于注酶后的7、14、21、30、60和90天处死。动物右肺组织福尔马林灌注固定后,石蜡包埋、切片,分别行HE和抗SP-A、B的免疫组化染色,并进行显微—微机形态定量。左肺液氮冷冻后,保存于-70℃,异硫氰酸胍一步法提取总RNA,甲醛变性胶电泳,转移至尼龙膜。扩增、酶切质粒,并回收、^(32)P标记SP-A、B DNA片断。最后,行Northern Blot,放射自显影后,测定光密度。结果 1.免疫组化显示:注酶7～30天SP-A、B阳性细胞百分率逐步降低,其中,30天SP-A、B阳性细胞百分率与正常组有明显差异(P<0.01),由30～90天SP—A、B阳性细胞百分率明显回升,但仍低于正常组。2.Northern Blot结果:注酶30天SP-A和B mRNA量明显低于正常组,但是,到达90天后SP-A、B mRNA量基本与正常组无明显差异。结论肺气肿发病过程中SP-A、B基因表达的动态变化提示,表面活性物质蛋白A、B变化可能参与肺气肿的发生和发展。

Objective To investigate the change of expression of surfactant A and B in genesis and development of emphysema. Methods Emphysema was induced in hamsters by the intratracheal administration of porcine pancreatic elastase (PPE) .The hamsters were killed respectively in 7, 14, 21, 30, 60 and 90 days after elastase administration. Right lungs were perfusion - fixed, praffin - embeded, HE and anti - SP - A and B immunohistochemistry stain and make a microscope - computer morphometric study.left kings were frozen in liquid nitrogen and stored in - 70℃, Total RNA was extracted with one step method of gunidinc isothiocyanate, frationated on formaldehyde gel and transferred to nylon membranes. SP- A and B DNA fragments were cloned, obtained by digestion and labeled by 32 P. Einally, performed Northern Blot, exposed to Kodak film at - 70℃ and quantitated by scanning densitometry of the autoradiagtams. Results 1 Im-munohistochemistry: Percentage of SP- A and B positive cells was gradually decreased during 7 ~ 30 day after PPE administration, there was obvious decrease compared with normal group (P< 0.01) .The percentage significantly increase during 30~90 days, but still less than one of normal group.2 Northern Blot: SP - A and B mRNA was significantly decreased compared with normal group, but there was no distinct difference with normal group in 90 days. Conclusion Change of expression of SP - A and B gene represented that change of SP - A and B might be related to the genesis and development of emphysema.