人Ⅱ型肺泡上皮细胞的分离培养及表型维持研究

Isolation and characterization of human alveolar type Ⅱ cells and phenotypes maintaining study

目的建立人Ⅱ型肺泡上皮细胞（ATⅡ）分离、纯化、原代培养及鉴定的方法，探索体外培养的表型维持情况。方法取手术切除的边缘肺组织，用胰酶、弹性蛋白酶联合法消化分离人ATⅡ细胞粗悬液，经过滤、差速贴壁、密度梯度分离、磁珠分选纯化。用肺表面活性蛋白C前体（pro-SP-C）免疫荧光、溶酶体绿色荧光染料（Green DND-26）荧光探针染色以及透射电镜鉴定人ATⅡ细胞；用pro-SP-C免疫荧光法和Green DND-26荧光探针染色法评估细胞纯度；锥虫蓝染色法评估细胞活性；并运用逆转录-聚合酶链反应（RT-PCR）检测体外培养过程中肺表面活性蛋白（SP-A、SP-B、SP-C、SP-D）的表达情况。结果人ATⅡ细胞产量为（5～10）×10^5／g；锥虫蓝染色细胞活性为（93±2）％；pro-SP-C免疫荧光、Green DND-26荧光探针鉴定、评估细胞纯度一致，为98％左右，透射电镜下观察ATⅡ细胞特有的板层小体结构清晰可见。SP-A、SP-C表达持续时间在24d左右（SP-A16d：0．52±0．03，20d：0．35±0．02，24d：0．26±0．01，28d：0．10±0．08；SP-C16d：0．68±0．16．20d：0．31±0．04，24d：0．18±0．06，28d：0．14±0．09），SP-B、SP-D表达持续时间可在28d左右（SP-B16d：1．05±0．17．20d：0．76±0．35．24d：0．55±0．15，28d：0．36±0．19；SP-D16d：0．52±0．19，20d：0．33±0．12，24d：0131±0．04，28d：0．23±0．02）。结论成功建立了一种分离、纯化及鉴定人ATⅡ细胞的方法，此方法能较持久维持ATⅡ细胞表型。

Objective To establish a method of isolate, purify, primary culture and identify human alveolar type Ⅱ cells （AT Ⅱ ） in vitro, as well as its possible maintaining phenotype characteristics. Methods The marginal lung tissue was collected. AT Ⅱ cells were isolated with trypsin and elastase, purified by a series of steps, such as, cell sieve filtration, differential adhesion, gradient separation and anti-CD14 beads separation. AT Ⅱ cells were identified with immunofluoreseence of human pro-surfactant-assoeiated protein C （pro-SP-C）, Green DND-26 probe and electron microscope. The purity of AT n cells was measured by immunofluorescenee of human pro-SP-C and Green DND-26 probe. The viability of AT Ⅱ cells was measured by trypan blue staining. The phenotypes （SP-A, SP-B, SP-C, SP-D ） were monitored with reverse transcription-polymerase chain reaction （RT-PCR） at different time points. Results The output of AT Ⅱ cells from lung tissue was （5-10）×10^5/g, and the cell viability was （93±2）% with trypan blue staining, the cell purity was about 98% with pro-SP-C immunofluorescenee and Green DND-26 fluorescent probe, the lamellar bodies were clearly observed with transmission electron microscope. In the aspect of phenotypes maintaining, the time of surfactaut expression was about 24 days [ SP-A： 0.52 ± 0.03 （day 16）, 0.35 ＋ 0.02 （day 20）, 0.26 ± 0.01 （day 24）, 0.10±0.08 （day 28）; SP-C： 0.68 ± 0.16 （dayl6）, 0.31±0.04 （day 20）, 0.18 ±0.06 （day 24 ）, 0.14 ± 0.09 （day 28 ）], and the longest one was more than 28 days [ SP-B： 1.05 ± 0.17 （day 16 ）, 0.76±0.35 （day 20）, 0.55 ± 0.15 （day 24）, 0.36 ± 0.19 （day 28）; SP-D： 0.52 ± 0.19 （day 16）, 0.33 ± 0.12 （day 20）, 0.31±0.04 （day 24）, 0.23 ± 0.02 （day 28 ）]. Conclusion We successfully established a procedure to separate, purify, identify of AT Ⅱ cells, which retain primary phenotypic characteristics over long period.