大鼠Ⅱ型肺泡上皮细胞传代培养可行性研究

Feasibility study for rat alveolar type Ⅱepithelial cells subculture

目的通过对离体培养的原代细胞和第3代Ⅱ型肺泡上皮细胞（alveolartypeⅡepithelialcells，AECⅡ）进行分化表型鉴定，探索AECⅡ离体传代培养的可行性。方法联合应用肺循环灌注、支气管肺泡灌洗、双酶联合螯合剂灌注消化、物理过滤、差速离心、贴壁纯化、免疫吸附等方法分离和纯化大鼠AECⅡ。纯化的AECⅡ接种于基质胶包被的培养皿，在含10μg／L角质细胞生长因子、5％胎牛血清的DMEM／F12培养基中进行离体培养。收集原代和第3培养细胞，透射电镜观察细胞超微结构，免疫细胞化学染色检测肺泡表面活性蛋白A（surfactantproteinA，SP-A）表达情况，并进行碱性磷酸酶染色鉴定细胞分化表型。结果AECⅡ产量为（21．3±5．8）×10^5／鼠，活细胞比例为（96．5±0．8）％。细胞培养至第3代，其形态、生长特征与原代培养细胞无明显差异。透射电镜观察证实原代细胞及第3代细胞均具备AECⅡ的特征性结构，SPA免疫细胞化学染色显示原代细胞及第3代细胞阳性比例均超过95％，碱性磷酸酶染色显示原代细胞及第3代细胞阳性比例分别为（85．2±3．7）％和（90．4±4．2）％。结论在适宜的培养条件下，离体培养的第3代AECⅡ能够维持相对稳定的分化表型，可以用于离体实验。

Objective To exploration the feasibility of alveolar type Ⅱepithelial cells （AEC Ⅱ ） in vitro subculture by identification the differentiation phenotype of primary and 3rd generation cells. Methods Rat AEC U were isolated and purified by sequent processes composed with lung circulation perfusion, bronehoalveolar lavage, dual-enzyme and chelator infusion digestion, physical filtration, differential eentrifugation, adherent purification, immunoadsorption. Purified AEC Ⅱ then were inoculated into Matrigel-coated culture dishes, and in vitro cultured by DMEM/F12 medium containing 10μg/L keratinocyte growth factor and 5% fetal bovine serum. The primary and the 3rd generation cells were collected for differentiation phenotype identification by transmission electron microscope observe cell uhrastructure, immunocytochemical detection surfactant protein A （SP-A） expression and the alkaline phosphatase staining appraisement. Results The total yields of AEC Ⅱ were （21.3±5.8） × 10^6 cells per rat, and the proportion of living cells reached to （96.5± 0.8）%. The morphology and growth characteristics of the 3rd generation cells were not obvioursly different from the primary cultured cells. Transmission electron microscope confirmed that the 3rd generation cells possess AEC Ⅱ characteristic ultrastructure as well as the primary cells. SP-A immunocytochemical staining demonstrated that the proportions of positive cells for the primary and the 3rd generation exceeded 95%, alkaline phosphatase staining positive rates of primary cells and 3rd generation cell were （85.2±3.7）% and （90.4±4.2） respectively. Conclusions As supportted with appropriate in vitro culture conditions, the 3rd generation AEC Ⅱ is capable to maintain relatively stable diferentiation phenotype, and could be be used in in vitroexperiments.