TLR4及MD2反义基因对内毒素诱导的肺Ⅱ型细胞活化的影响.docVIP

TLR4及MD2反义基因对内毒素诱导的肺Ⅱ型细胞活化的影响( 李永旺1 麻莉3 张德明2 钱桂生2 第三军医大学，1.麻醉科，2.呼吸科，重庆 400037 3.第二炮兵总医院妇产科，北京 100088 摘要：目的:观察TLR4及MD2反义基因对内毒素（LPS）诱导的肺Ⅱ型细胞NF-κB活化的影响。方法：培养的肺Ⅱ型细胞随机分为正常细胞（对照组）组、LPS组、LPS+转染空载体组、LPS+转染TLR4反义基因组，LPS+转染MD2反义基因组，LPS+转染TLR4及MD2反义基因组。分别通过Northern blot检测转染细胞的TLR4 及MD2 mRNA表达，Western blot检测转染细胞TLR4及MD2蛋白表达，电泳迁移率改变法（EMSA）检测 NF-κΒ的活性，ELISA测定细胞培养上清中TNF-α和IL-6含量。结果：与对照组相比，LPS刺激后的细胞TLR4 及MD2 mRNA和蛋白表达、NF-κB活性、TNF-α和IL-6的生成均显著增加（P＜0.01）NF-κB活性及TNF-α和IL-6的含量均明显降低（P＜0.01gynaecology and obstetrics, The General Hospital of Second Artillery Army, Beijing 100088, China Abstract：Objective: To observe effects of TLR4 and MD2 antisense gene on activation of NF-κB in alveolar typeⅡepithelial cells(ATⅡcells) induced by lipopolysaccharide (LPS). Methods: Cultural ATⅡcells were randomized to normal cells (control) groups, LPS groups, LPS+empty vector groups, LPS+ TLR4 antisense gene groups, LPS+MD2 antisense gene groups, LPS+TLR4-MD2 antisense gene groups. The expressions of TLR4 and MD2 mRNA and protein in ATⅡcells were detected by Northern blot and Western blot respectively．The activity of NF-κB in ATⅡcells was measured with e1ectrophoretic mobility shift assay(EMSA)．The TNF-α and IL-6 concentrations in the supernatant of cultured ATⅡcells were tested with ELISA．Results: Compared with control groups, in other each groups, the expressions of TLR4 and MD2 mRNA and protein, the NF-κΒ activity, the levels of TNF-( and IL-6 increased remarkably (P0.01). After stimulating with LPS, compared with others groups, in the antisense gene groups, the expressions of TLR4 and MD2 mRNA and protein, the NF-κΒ activity, the levels of TNF-( and IL-6 decreased remarkably (P0.01). Conclusion：The antisense gene of TLR4 and MD2 can inhibit activation of ATⅡcells induced by LPS. Keywords：alveolar typeⅡepithelial cell；toll like receptor; MD2; 我们的前期研究[1]发现，在LPS所致肺急性炎症反应过程中，肺泡Ⅱ型上皮细胞被激活并且表达有TLR4及MD2受体，显示TLR4及MD2可能参与了LPS介导的肺泡Ⅱ型上皮细胞活化，为进一步研究TLR4及MD2在LPS激活肺泡Ⅱ型上皮细胞的过程中的作用，我们用构建的TLR4及MD2反义表达载体转染了肺泡Ⅱ型上皮细胞，