MMP9、S100A10基因沉默稳转小鼠细胞系的构建及验证

中国真菌学杂志　 2017, Vol. 12 　Issue (1): 19-22,18.

MMP9、S100A10基因沉默稳转小鼠细胞系的构建及验证

陈雪雯, 朱红梅, 温海

上海市长征医院皮肤科全军真菌病重点实验室上海市医学真菌分子生物学重点实验室, 上海 200003

收稿日期:2016-10-16 出版日期:2017-02-28 发布日期:2017-02-28
通讯作者: 温海,E-mail:wenhaipfk@126.com;朱红梅,E-mail:hmzhu_cn@163.com E-mail:wenhaipfk@126.com;hmzhu_cn@163.com
作者简介:陈雪雯,女（汉族）,硕士研究生在读.E-mail：snowflychen@163.com
基金资助:
国家自然科学基金（31470252，81271800，31270181）

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

CHEN Xue-wen, ZHU Hong-mei, WEN Hai

PLA Key Laboratory of Fungal Diseases and Shanghai Key Laboratory of Molecular Medical Mycology, Department of Dermatology, Changzheng Hospi tal, Second Military Medical University, Shanghai 200003, China

Received:2016-10-16 Online:2017-02-28 Published:2017-02-28

摘要/Abstract

摘要：

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1×10⁸ TU/mL、3×10⁸ 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。

关键词: 慢病毒载体, 基质金属蛋白酶9（MMP9）, S100A10, b.End3

Abstract:

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-musMMP9-shRNA,LV-musS100A10-shRNA into b.End3 respectively,then the very cell lines were screened by puromycin.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 3×10⁸ TU/mL and 1×10⁸ TU/mL LV-musS100A10-shRNA respectively.Conclusions The targeted down-regulation b.End3 cell lines are useful for further exploring 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.

Key words: Lentivirus vector, MMP9, S100A10, b.End3

中图分类号:

R392.1

陈雪雯, 朱红梅, 温海. MMP9、S100A10基因沉默稳转小鼠细胞系的构建及验证[J]. 中国真菌学杂志, 2017, 12(1): 19-22,18.

CHEN Xue-wen, ZHU Hong-mei, WEN Hai. Construction and verification of stable down-regulated MMP9,S100A10 mouse brain microvascular endothelial cell lines[J]. Chinese Journal of Mycology, 2017, 12(1): 19-22,18.

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