摘要
目的提取新生小鼠的原代肺泡Ⅱ型上皮细胞(AECⅡ),并进行培养和鉴定。方法应用酶消化和免疫磁珠分选方法分离并纯化C57B/L6新生小鼠AECⅡ。根据AECⅡ特异的细胞形态,利用透射电镜进行鉴定。正置荧光显微镜观察AECⅡ特异性肺泡表面活性蛋白-C前体(proSP-C)及表面活性蛋白-B(SP-B)的表达。免疫印记技术检测新生小鼠AECⅡ中特异性肺泡表面活性蛋白-A(SP-A)及proSP-C的表达。结果每只新生小鼠可获得(2±0.5)×10^5AECⅡ,纯度>90%。电镜下可见丰富的AECⅡ及其特异结构板层小体。正置免疫荧光显微镜观测AECⅡ中proSP-C和SP-B蛋白的表达,免疫印迹结果显示细胞内SP-A和proSP-C蛋白表达呈阳性。结论利用酶消化和免疫磁珠分选的方法可成功分离出高产量、高纯度的新生小鼠AECⅡ。
Objective Primary alveolar type Ⅱ epithelial cells(AECⅡ)of neonatal mice were extracted,cultured,and further identified.Methods AECⅡ from newborn C57B/L6 mice were isolated and purified by enzymatic digestion and immunomagnetic bead sorting.AECⅡ can be identified with transmission electron microscopy owing to their cell-specific morphology.The expression of specific alveolar prosurfactant protein C(proSP-C)and surfactant protein B(SP-B)in AECⅡ cytoplasm was observed with upright fluorescence microscopy.The expression of surfactant protein A(SP-A)and proSP-C in AECⅡ of newborn mice was detected with western blotting.Results We were able to obtain(2±0.5)×10^5 AECⅡ from each neonatal mouse,with a purity of more than 90%.Under the electron microscope,large numbers of AECⅡ and their unique lamellar body structures could be seen.The expression of proSP-C and SP-B in AECⅡ was observed with immunofluorescence microscopy,and positive expression of SP-A and proSP-C in cells was detected with western blotting.Conclusion Newborn mouse AECⅡ can be successfully isolated,with high yields and purity,using enzymatic digestion combined with immunomagnetic bead sorting.
作者
国敏
郝佳琳
朱庆锋
李丹妮
李小曼
GUO Min;HAO Jialin;ZHU Qingfeng;LI Danni;LI Xiaoman(Institute of Translational Medicine,China Medical University,Shenyang 110122,China;Department of Medical Oncology,The First Hospital,China Medical University,Shenyang 110001,China)
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2020年第12期1057-1060,共4页
Journal of China Medical University
基金
国家自然科学基金(81300800)。
