SiRNA靶向抑制哮喘小鼠T细胞非受体酪氨酸蛋白激酶Lck的体内研究

In vivo study of tyrosine protein kinase Lck inhibited by siRNA in T cells of asthmatic mice

目的 通过siRNA的方法体内靶向抑制哮喘小鼠T细胞非受体酪氨酸蛋白激酶Lck的基因表达,研究Lck特异性siRNA对哮喘小鼠的治疗作用.方法 化学法合成小鼠T细胞Lck基因特异的siRNA片段,以in vivo-jetPEITM作为转染试剂,将合成的RNA片段与转染试剂混合后通过尾静脉注射到哮喘小鼠体内,48 h后处死小鼠,ELISA法检测肺泡灌洗液（BALF）中细胞因子IL-4、IL-17的含量；HE染色观察肺组织病理改变情况；免疫组织化学法检测肺组织中Lck表达的变化；Western Blot法检测肺组织匀浆中Lck蛋白的含量.结果 SiRNA干扰组哮喘小鼠BALF中IL-4、IL-17[（ 142.62±15.57） pg/ml,（74.18 ±5.36）pg/ml]的水平较哮喘组[（234.68±11.15） pg/ml,（96.76±8.28） pg/ml]明显下降（P＜0.01）,肺组织炎症病理改变有所减轻,免疫组化提示肺组织中Lck蛋白表达下降,肺组织匀浆中Lck蛋白的表达下降（P＜0.05）.结论 Lck特异性siRNA可以降低哮喘小鼠肺组织中炎症因子IL-4、IL-17的水平,减轻肺部炎症反应,具有一定的治疗作用.

Objective Using the technology of siRNA to inhibit the gene expression of no-receptor tyrosine protein kinase Lck in T cells of asthmatic mice,and to study the therapeutic effect of Lck specific siRNA in asthmatic mice.Methods Receptor tyrosine protein kinase Lck specific siRNA fragments were taken from chemosynthesis.In vivo-jetPEITM was used to transfect the siRNA into mice body through tail vein injection.The mice were killed 48 hours later,and the levels of IL-4,IL-17 in bronchoalveolar lavage fluid （BALF） were detected with respondent ELISA kits.The change of inflammatory histopathology in lung was observed with H.E.staining.The expression of Lck in lung was detected with immunohistochemistry （IHC）,and the level of Lck in lung tissue homogenate was detected with Western Blot.Results Compared with asthmatic group[ （234.68 ± 11.15 ） pg/ml,（ 96.76 ± 8.28 ） pg/ml],the levels of IL-4,IL-17 [ （234.68 ± 11.15）pg/ml,（96.76 ±8.28） pg/ml] in the BALF of siRNA interference group decreased, and the inflammation in the lung relieved.IHC indicated that the expression of Lck in lung decreased and the level of Lck in lung tissue homogenate decreased （ P 〈 0.05 ）.Conclusions Lck specific siRNA could reduce the level of IL-4,IL-17 in the lung tissues of asthmatic mice,and relieve the inflammatory reaction in lung.