脂质体介导HSP27干扰质粒转染人肺泡Ⅱ型上皮细胞对胶原合成的影响  

HSP27 RNAi plasmid tranfected by liposome involves in collagen synthesis in human alveolar type Ⅱ epithelial cells

作　　者：邓海静[1,2] 高学敏[1,2] 薛新新 杜世璞 孙月[1,2] 徐洪[1,2] 杨方[1,2] 

机构地区：[1]河北联合大学 医学实验研究中心,唐山063000 [2]河北联合大学老年医学国际科技合作基地,唐山063000

出　　处：《解剖学杂志》2015年第1期22-26,共5页Chinese Journal of Anatomy

基　　金：国家自然科学基金(81072254,81302395);河北省高等学校科学技术研究重点项目(ZD20131035)

摘　　要：目的：用脂质体Lepo 2000介导热休克蛋白27（HSP27）干扰质粒转染A549人肺泡Ⅱ型上皮细胞株,检测转染后对转化生长因子β1 （TGF-β1）诱导的A549人肺泡Ⅱ型上皮细胞向肌成纤维细胞转化中对胶原合成的影响.方法：流式细胞仪检测不同配比的Lepo 2000与HSP27质粒转染效率;激光共聚焦检测最优配比的干扰质粒的转染效率;Real-time PCR检测HSP27基因的表达并筛选最佳干扰HSP27质粒;免疫印迹检测最佳干扰HSP27质粒转染效果及胶原表达的变化.结果：8 μg∶20μl的配比细胞转染效率可达到83％,为最优脂质体与HSP27质粒的配比;干扰HSP27质粒4个不同的基因干扰片段中,干扰HSP27质粒第4个干扰片段基因沉默效果最好,能达到70％的HSP27基因沉默效果.成功转染HSP27的干扰质粒能明显下调TGF-β1诱导的A549人肺泡Ⅱ型上皮细胞向肌成纤维细胞转化中Ⅰ型胶原和Ⅲ型胶原蛋白的表达.结论：HSP27干扰质粒能明显抑制TGF-β1诱导的A549人肺泡Ⅱ型上皮细胞Ⅰ型和Ⅲ型胶原的合成.Objective： To detect the effect of TGF-β1 on the collagen synthesis when inducing A549 human alveolar type Ⅲ epithelial cells to myofibroblasts after successful transfection of HSP27 interference plasmids with Lepo 2000 into A549 human alveolar type II epithelial cells. Methods： The transfection efficiency of different ratios of Lepo 2000 and HSP27 interference plasmid was detected by flow cytometry. The optimal ratio of transfection efficiency was measured by confocal microscopy. The expression of HSP27 mRNA was detected by real-time PCR and screened out the best HSP27 interference plasmid to use in the followed experiments. The expressions of the best HSP27 interference plasmid and collagen type Ⅰ and Ⅲwere further measured by Western blotting. Results： The optimal ratio of transfection efficiency was 8 μg ： 20μl which could reach 83 G. Among the four different HSP27 interference plasmid, the fourth plasmid showed the best interference effects which could block the expression of seventy percent of HSP27 at mRNA level. The expressions of collagen type I and UI were down- regulated on TGF-β1-induced A549 human alveolar type Ⅲ epithelial cells to myofibroblasts after successful transfection of HSP27 interference plasmids with Lepo 2000 in（6 A549 human a!veolar type II epithelial cells. Conclusion： The HSP27 interference plasmid could significantly inhibit TGF-β1-induced collagen synthesis in A549 cells.

关 键 词：肺泡Ⅱ型上皮细胞株 Lepo 2000转染试剂 热休克蛋白27质粒 Ⅰ型和Ⅲ型胶原蛋白 TGF-β1 

分 类 号：R563[医药卫生—呼吸系统]