摘要
【目的】探讨胎鼠肺泡Ⅱ型细胞原代培养及鉴定方法,建立肺泡Ⅱ型细胞培养模型,为体外研究肺泡Ⅱ型细胞及胎儿肺发育提供实验手段。【方法】采用低浓度胰酶消化20d胎鼠肺组织,经差速离心和特异性免疫吸附后,分离、纯化胎鼠肺泡Ⅱ型细胞,进行原代培养;根据细胞生长特点、形态特征、细胞表型和肺表面活性特异蛋白A的分泌,用免疫细胞化学染色、透射电镜及酶联免疫吸附法对分离的细胞进行形态学和功能鉴定。【结果】原代培养的肺Ⅱ型细胞12h开始贴壁,外观呈多边形,岛状生长。培养的24～48h,细胞连接成单层,胞浆内颗粒明显,培养的6￣7d细胞内颗粒大量减少;免疫细胞化学鉴定细胞肺表面活性特异蛋白A(surfactantproteinA,SP-A)染色阳性,透射电镜可见观察到细胞内板层小体,培养液中SP-A含量随培养时间延长逐渐增多,于培养36h达峰值,为80ng/mL;细胞纯度达(92±2)%,产量为(18.6±7)×106。【结论】差速离心和免疫黏附法是一种高效的分离和纯化胎肺Ⅱ型细胞的方法,所得细胞产量大、纯度高,可以用于体外研究肺泡Ⅱ型细胞和晚期胎肺发育的研究。
[Objective ]The aims of this study were to develop the primary culture technology and identification method for alveolar type Ⅱ ceils （AEC Ⅱ ） . Providing experiment means for investigating AEC Ⅱ and fetal lung development in vitro by estabhshing AEC Ⅱ culture modeL[Methods]Low density trypsin was taken to digest lung tissues, which were obtained from 20-day fetal rats. Differential centrifugation and specific immunosorption were used to demesh and purify AEC Ⅱ . Immunocyto-chemistry, electron micrograph, and enzyme - linked immunoserbent assay （ELISA） were used to identify the morphology and function of the cultured ceils, according to the ceils growth feature, morphologic characteristics, phenotypic characteristics and the secretion of surfactant protein A.[Results]AEC Ⅱ adhered after cultured for 12 h, the shape of ceil was polygon under microscope. And ceils connected like islands. Following 24-48 h in culture, ceils formed confluent monolayers. There were fine particles in intracytoplasm. On days 6-7 in culture, fine particles decreased. The expression of SP-A in AEC Ⅱ was positive and lamellarbodies were revealed under electron micrography. The quantity of SP-A in culture solution was 80 ng/mL when cultured for 36 hours. The cells purify was （92±2） % and quantity was （18.6±7） ×10^6 every fetal lung. [Conclusions] Differential centrifugation and specific immunosorption were the efficient methods to isolate, purify, and culture rat AECⅡ cells. Cells with high quantity and purity make use to study functions of AEC Ⅱ ceils and the development of fetal lung.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2006年第5期496-499,505,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金资助项目(2003B30505)
关键词
胎鼠
肺泡Ⅱ型细胞
原代培养
分离
鉴定
fetal rat
type Ⅱ alveolar epithelial ceil
primary cell culture
isolation
identification
