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

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.

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

Objective To establish a method of isolation, purification and identification of type Ⅱ alveolar epithelial cells (AEC- K ) 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)-1β,IL-6 and IGF-Ⅰ at different concentrations were used to culture AEC-Ⅱ for another 48 hours. And then the cells 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 t for 20 min, the yield was (5.33 ±0.54) × 106 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) × 106 perpiglet 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-1 β 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.