一氧化氮合酶基因转染小鼠内皮祖细胞的实验研究

发布时间：2018-02-08 10:26

本文关键词： 内皮祖细胞内皮型一氧化氮合酶基因一氧化氮氧自由基　出处：《广东医学》2014年22期 　论文类型：期刊论文

【摘要】：目的探讨利用脂质体介导内皮型一氧化氮合酶(eNOS)基因体外转染小鼠骨髓来源的内皮祖细胞(EPC)的可行性。方法密度梯度离心法分离ICR小鼠骨髓来源单个核细胞培养制备小鼠EPC并鉴定;构建、扩增、提取并纯化pcDNA3.0/eNOS基因质粒;用EPC培养基配成2×105·mL-1细胞悬液,按0.6 mL/孔重新铺6孔板,待细胞生长至80%左右融合时,用脂质体LipofectamineTM2000体外转染eNOS基因至EPC。转染48 h后,采用RT-PCR检测EPC中eNOS的表达,硝酸还原酶法检测EPC中一氧化氮(NO)的含量,荧光探针DCFH-DA活性氧检测氧自由基(ROS)的含量。结果成功培养EPC,成功构建pcDNA3.0/eNOS基因载体。转染48 h后,EPC中eNOS表达量明增加(P0.01),NO含量明显升高(P0.01),ROS含量明显降低。结论脂质体介导eNOS基因能够有效转染小鼠EPC,并能在EPC中有效表达。
[Abstract]:Objective to investigate the feasibility of transfection of endothelial nitric oxide synthase (Enos) gene into murine bone marrow-derived endothelial progenitor cells (EPCs) by liposome in vitro. Methods Mononuclear cells derived from bone marrow of ICR mice were isolated by density gradient centrifugation. Mouse EPC was prepared and identified. The plasmid of pcDNA3.0/eNOS gene was constructed, amplified, extracted and purified, the suspension of 2 脳 10 5 路mL-1 cells was prepared by EPC medium, and the 6 well plate was re-laid according to 0.6 mL / well. When the cell grew to about 80%, the eNOS gene was transfected into EPCs by liposome LipofectamineTM2000 in vitro. After 48 h of transfection, RT-PCR was used to detect the expression of eNOS in EPC, and nitrate reductase method was used to detect the content of nitric oxide (no) in EPC. Results the pcDNA3.0/eNOS gene vector was successfully cultured and constructed successfully. After 48 h of transfection, the expression of eNOS in EPC was increased significantly, and the content of P0.01N was significantly increased. Conclusion Liposome can significantly decrease the content of Ros. ENOS gene can be effectively transfected into mouse EPCs and expressed in EPC.
【作者单位】：江苏大学附属医院普外科;
【基金】：国家自然科学基金资助项目(编号:81070287) 江苏省自然科学基金资助项目(编号:BK2011484) 江苏省博士后科研资助计划项目(编号:1302096B)
【分类号】：R329.2

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