促炎症细胞因子对新生猪肺泡Ⅱ型上皮细胞相关生长因子表达的影响

Effect of proinflammatory cytokines on growth factor expression of type II alveolar epithelial cells from neonate piglet lungs

下载PDF阅读器目的 建立出生后1～3 d新生猪肺泡Ⅱ型上皮细胞（AEC-Ⅱ）体外分离纯化及鉴定方法,探讨促炎症细胞因子对AEC-Ⅱ长因子（GFs）的影响,比较不同消化酶溶液、纯化方法获得细胞产量、活力及AEC-Ⅱ纯度的差异.方法 原代培养AEC-Ⅱ24 h给予不同浓度IL-1β、IL-6刺激48 h,观察AEC-Ⅱ增殖变化,RT-PCR检测胰岛素样生长因子-1（IGF-Ⅰ）、血小板源性生长因子（PDGF）、表面活性物质蛋白（SP）-A及-B mRNA的表达情况,并检测IGF-Ⅰ抗体对AEC-Ⅱ增殖及SP-A、SP-B mRNA表达的影响.结果 30000 U/L弹力蛋白酶/0.1%胰酶在37℃下消化新生猪肺组织20 min获得细胞产量为（5.33±0.54）×106/g（肺重＋心重）,显著高于其他各组（P〈0.01）.免疫黏附法纯化AEC-Ⅱ产量明显高于Percoll法,为（38.0±28.0）×106/头新生猪.AEC-Ⅱ原代培养24～96 h状态最佳.随IL-1β、IL-6刺激浓度升高,AEC-Ⅱ增殖能力及IGF-Ⅰ及SP-A mRNA表达水平降低,但PDGF、SP-B mRNA的表达无明显变化.IGF-Ⅰ抗体刺激下,AEC-Ⅱ增殖能力及SP-A、SP-B mRNA表达水平均降低.结论 IL-1B、IL-6可能通过调节AEC-Ⅱ中IGF-Ⅰ mRNA的表达影响细胞的增殖及功能.

Objective To establish a method of isolation, purification and identification of type II alveolar epithelial cells （ AEC- II ） from neonate piglet lungs of 1 - 3 days old and to investigate effects of proinflammatory cytokines on expression of growth factors （GFs）. The yield,viability and purity of AEC-II obtained using different enzyme digestion and purifying methods were compared. Methods After the first 24-hour culture of AEC-II ,the media containing interleukin （IL）-113, IL-6 and IGF- I at different concentrations were used to culture AEC- II for another 48 hours. And then the ceils were counted and the expressions of insulin-like growth factor （ IGF- I ）, platelet-derived growth factor （ PDGF）, surfactant proteins （SP） -A and SP-B mRNA were determined by real time PCR. Results A significantly higher yield of AEC- II was achieved by digesting the lung with 30 unit/ml elastase and 0. 1% trypsin at 37 ℃ for 20 min, the yield was （5. 33 ±0. 54） × 10^6 after adjusted by the weight of lung and heart （P 〈0. 01 ）. The number of purified AEC- ii obtained by immune adherence method was （38.0 ±28.0） x 106perpiglet which was higher than by the method of percoll. The optimal phenotype maintenance time of AEC- ii was the first 24- 96 hours in the primary culture. With increasing concentrations of IL-1β and IL-6 ,there were decreased proliferation and expression of SP-A and IGF- I mRNA in the cultured AEC- II ,but SP-B mRNA expression was not affected. Both AEC- II proliferation and expression of SP-A, SP-B mRNA decreased significantly after cultured with anti-IGF- I. Conclusion In a new model of cultured AEC- II from neonate piglets, IL-1β and IL-6 inhibited AEC- II proliferation and SP-A mRNA expression through IGF- I -dependent mechanisms.