一氧化氮合酶基因转染小鼠内皮祖细胞的实验研究

Experimental study on endothelial nitric oxide synthase gene transfecting in murine endothelial progenitor cells

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(P<0.01),NO含量明显升高(P<0.01),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。

Objective To establish an effective method to transfect murine bone marrow- derived endothelial progenitor cells （EPCs） with liposome- mediated endothelial nitric oxide synthase （eNOS） gene. Methods Mononu- clear cells from ICR murine bone marrow were isolated by density gradient centrifugation, and murine EPCs were prepared and identified. The pcDNA3.0/eNOS gene plasmid was constructed, amplified, extracted and purified. The eNOS gene was transfected into EPCs in vitro using LipofectamineTM 2000. Forty -eight hours after transfection, eNOS expression of EPCs was detected by PT -PCR, NO content of EPCs was determined in nitrate reduetase method, and reactive oxygen species （ROS） content of EPCs was detected by DCFH - DA fluorescent probe. Results EPCs were cultured and pcD- NA3.0/eNOS gene plasmid was constructed successfully. The expression of eNOS was significantly up - regulated （P 〈 0. 01 ）, the content of NO was raised significantly （P 〈0. 01 ） , while the content of ROS was reduced significantly all in EPCs. Conclusion The eNOS gene can be successfully transfected and expressed in murine EPCs by liposome -media- ted method.