大鼠肺泡Ⅱ型上皮细胞的分离、纯化和鉴定

Isolation,Puritication and Identification of Alveolar Type Ⅱ cells of Rats

目的:探讨大鼠肺泡Ⅱ型上皮细胞(ATⅡ)分离、培养及鉴定的方法。方法:采用Dobbs法提取ATⅡ。肺动脉灌洗减少肺内红细胞,气管灌洗除去肺泡腔内的白细胞,将胰蛋白酶、胶原酶灌入肺内消化分离细胞;采用免疫贴附法纯化细胞,将细胞悬液培养于覆被IgG的平皿中,有Fc段受体的细胞被黏附,而无Fc段受体的ATⅡ得以纯化。ATⅡ的鉴定采用电镜和肺表面活性物质相关蛋白A(SP-A)免疫组化染色,其在电镜下有特征性板层小体,免疫组化染色见胞浆有SP-A表达。结果:纯化后台盼蓝染色显示细胞活力为95%以上。通过SP-A免疫组化染色判定,纯化后ATⅡ纯度可达92%。结论:分离、纯化大鼠ATⅡ时,合适的胰蛋白酶浓度及作用时间对细胞活性有重要作用,大鼠IgG黏附纯化可以得到高纯度的ATⅡ,通过电子镜观察板层小体和免疫组化染色检测细胞SP-A表达可用于鉴定ATⅡ。

Objective To study the method of primary culture of alveolar type II （AT II） cel1s.Methods AT II cells were isolated by Dobbs＇s method.The cells were purified by using rat IgG coated dishes and were characterized by using electronic microscopy and SP-A immunohistochemical staining.The cellular ultra-structure of AT II cells was observed by electronic microscopy.Results The trypan blue staining showed the cell viability over 95 percent.Immunohistochemical staining using anti-SP-A demonstrated that the purity of AT II cells was 92%.Conclusion In AT II cells isolation,purification and identification procedures,optimizing the concentration of digest enzyme and time can improve the cell vitality.Purification of AT II cells with IgG coated plate is a better method than other reported methods.AT II cells could be characterized through detection of the lamellar bodies with electronic microscopy and SP-A Immunohistochemical staining.