中国抗生素杂志
中國抗生素雜誌
중국항생소잡지
CHINESE JOURNAL OF ANTIBIOTICS
2010年
4期
305-310
,共6页
朱勇军%翁玄%陈婷梅%张健
硃勇軍%翁玄%陳婷梅%張健
주용군%옹현%진정매%장건
抗菌肽%PR-39%慢病毒载体%抗菌活性%骨髓间充质干细胞
抗菌肽%PR-39%慢病毒載體%抗菌活性%骨髓間充質榦細胞
항균태%PR-39%만병독재체%항균활성%골수간충질간세포
Antimicrobial peptides%PR-39%Lentiviral vector%Antibacterial activity%Bone mesenchymal stem cells
目的 构建脯氨酸精氨酸抗菌肽(proline-arginine rich 39-amino acid peptide,PR-39)慢病毒载体,并转染兔骨髓间充质干细胞(bone mesenchymal stem cells,BMSC),检测其在BMSC中的表达及抗菌活性.方法 PCR扩增目的 克隆,并连接入慢病毒载体质粒pGC-FU中,与包装质粒pHelper1.0、pHelper2.0在脂质体2000介导下共转染293T细胞生产慢病毒颗粒,Real-Time PCR法检测其滴度;分离培养BMSC,BMSC转染PR-39慢病毒,荧光显微镜观察及流式细胞仪检测其转染效率,RT-PCR检测PR-39基因表达,并检测PR-39抗菌活性.结果 成功包装出滴度为2×10~9TU/mL的PR-39慢病毒载体,并转染BMSC,转染72 h后可观察到细胞呈现绿色荧光,转染效率为74.09%,同时RT-PCR检测BMSC表达PR-39基因,其上清液对金黄色葡萄球菌有明显的抑制作用,抑菌率为98.43%(P＜0.05).结论 在慢病毒介导下,BMSC成功表达分泌具有较强抗菌活性的抗菌肽PR-39.
目的 構建脯氨痠精氨痠抗菌肽(proline-arginine rich 39-amino acid peptide,PR-39)慢病毒載體,併轉染兔骨髓間充質榦細胞(bone mesenchymal stem cells,BMSC),檢測其在BMSC中的錶達及抗菌活性.方法 PCR擴增目的 剋隆,併連接入慢病毒載體質粒pGC-FU中,與包裝質粒pHelper1.0、pHelper2.0在脂質體2000介導下共轉染293T細胞生產慢病毒顆粒,Real-Time PCR法檢測其滴度;分離培養BMSC,BMSC轉染PR-39慢病毒,熒光顯微鏡觀察及流式細胞儀檢測其轉染效率,RT-PCR檢測PR-39基因錶達,併檢測PR-39抗菌活性.結果 成功包裝齣滴度為2×10~9TU/mL的PR-39慢病毒載體,併轉染BMSC,轉染72 h後可觀察到細胞呈現綠色熒光,轉染效率為74.09%,同時RT-PCR檢測BMSC錶達PR-39基因,其上清液對金黃色葡萄毬菌有明顯的抑製作用,抑菌率為98.43%(P＜0.05).結論 在慢病毒介導下,BMSC成功錶達分泌具有較彊抗菌活性的抗菌肽PR-39.
목적 구건포안산정안산항균태(proline-arginine rich 39-amino acid peptide,PR-39)만병독재체,병전염토골수간충질간세포(bone mesenchymal stem cells,BMSC),검측기재BMSC중적표체급항균활성.방법 PCR확증목적 극륭,병련접입만병독재체질립pGC-FU중,여포장질립pHelper1.0、pHelper2.0재지질체2000개도하공전염293T세포생산만병독과립,Real-Time PCR법검측기적도;분리배양BMSC,BMSC전염PR-39만병독,형광현미경관찰급류식세포의검측기전염효솔,RT-PCR검측PR-39기인표체,병검측PR-39항균활성.결과 성공포장출적도위2×10~9TU/mL적PR-39만병독재체,병전염BMSC,전염72 h후가관찰도세포정현록색형광,전염효솔위74.09%,동시RT-PCR검측BMSC표체PR-39기인,기상청액대금황색포도구균유명현적억제작용,억균솔위98.43%(P＜0.05).결론 재만병독개도하,BMSC성공표체분비구유교강항균활성적항균태PR-39.
Objective To construct the proline-arginine rich 39-amino acid peptide(PR-39)lentiviral vector,and introduce it into bone mesenchymal stem cells(BMSC),the expression and antibacterial activity of PR-39 was detected.Methods The CDS sequence of PR-39 was amplified by PCR from pBluescript Ⅱ SK-PR-39,and then the gene was recombined into the transfer plasmid pGC-FU.The three plasmids of lentiviral vector,transfer plasmid pGC-FU-PR-39,packaging plasmid pHelperl.0、pHelper2.0,which were cotransfected to 293T cells mediated by lipofectamin 2000 to produce lentiviral particles.The tite of viral particles was detected by real-time PCR.The rabbit BMSC were prepared,and then were infected by PR-39 lentiviral particles.The transfection efficiency was observed with fluorescent microscope and flow cytometry,and the expression of PR-39 was detected by RT-PCR,and the antibacterial activity also was investigated.Results The tite of viral particles was 2×10~9TU/mL,and it was showed transfection efficiency was 74.09%.PR-39 gene was expressed successfully in BMSC,and PR-39 inhibited the staphylococcus aureus significantly,and bacteriostatic rate was 98.43%(P＜0.05).Conclusion Antimicrobial peptides PR-39 is expressed successfully in rabbit BMSC mediated by lentiviral vector,and it shows significant antibacterial activity.
