MMP9、S100A10基因沉默稳转小鼠细胞系的构建及验证

Construction and verification of stable down-regulated MMP9,S100A10 mouse brain microvascular endothelial cell lines

目的分别构建小鼠MMP9、S100A10沉默的稳转细胞系,并验证功能。方法构建能有效干扰S100A10、MMP-9表达的慢病毒LV-musMMP9-shRNA、LV-musS100A10-shRNA,将其转染293T细胞进行包装、扩增及质量检测。将包装好的慢病毒分别转染b.End3,嘌呤霉素筛选稳转细胞系,通过荧光显微镜、RT-qPCR检测对应目的基因表达量,构建稳转细胞系。结果成功构建有效干扰S100A10、MMP-9表达的稳转细胞系,其滴度分别为1×10~8 TU/mL、3×10~8 TU/mL。荧光显微镜检测稳转细胞的荧光表达接近100%;RT-qPCR分别检测MMP9、S100A10基因表达下调率分别为78%、72%。结论构建有效干扰S100A10、MMP-9表达的稳转细胞系,为控制单一变量直观探究S100A10、MMP-9影响隐球菌穿过血脑屏障的能力等研究奠定了重要基础,为隐球菌性脑炎/脑膜炎的临床治疗靶点研究开辟新的思路。

Objective To construct stable down-regulated MMP9, S100A10 mouse brain microvascular endothelial cell lines,then verify the reduction level of gene expression.Methods By transfecting LV-musMMPg-shRNA,LV-musS100A10- shRNA into b.End3 respectively, then the very cell lines were screened by puromyein.The obtained differential b. End3 cell lines were observed by fluorescence microscope and varified by RT-qPCR.Results The study successfully constructed stable transfected cell lines which could effectively interference S100A10, MMP-9 expression of 3 ×10^8 TU/mL and 1 × 10^8 TU/mL LV-musS100A10-shRNA respectively.Conclusions The targeted down-regulation b.End3 cell lines are useful for further ex- ploring the role of molecule S100A10 or MMP-9 in host blood-brain barrier by control variables and the possible target of treatment exploration of cryptococcal encephalitis/meningitis.