CAJ | 학술논문

目的:构建携带血管形成素-1(ANG-1)基因腺相关病毒(AAV)载体,通过病毒包装和纯化及滴定,获得高纯度高感染性的病毒液,并转染到乳猪骨髓干细胞中,获得有效表达,为进行血管再生的基因治疗研究奠定基础.方法:利用PCR技术,获取目的基因ANG-1,通过重组DNA技术得到ANG-1与pUC119的重组质粒,采用SalⅠ和BglⅡ双酶将pUC119中的目的基因ANG-1基因片断切出,再克隆到质粒pSNAV2.0中.用Lipofectamine将pSNAV ANG-1质粒转染293细胞后,用G418选择培养获得293/AAV2ANG-1细胞株,产生了有传染性的病毒颗粒,纯化并进行病毒DNA颗粒滴度测定.分别将绿色荧光蛋白(GFP)基因重组腺相关病毒(rAAV2GFP)及rAAV2ANG-1感染猪骨髓干细胞,并对转染效率及转染后表达情况进行初步研究.结果:测序证实ANG-1与GenBank提供的原始序列完全一致.重组载体经酶切鉴定与PCR筛选证实重组质粒和预期结果完全一致,在新的重组质粒中,ANG-1有一套CMV启动子和PolyA终止子系统.293细胞包装病毒效果良好,携带ANG-1的感染性AAV滴度为9×1011v.g/ml,用Western杂交法检测显示猪骨髓干细胞中表达ANG-1.1×106v.g/ml rAAV2GFP感染CD34阳性细胞48h后55%左右的细胞有明显的荧光.结论:ANG-1基因AAV构建成功,并能在骨髓干细胞中有效表达,为严重缺血性疾病基因治疗的研究奠定了基础.
목적:구건휴대혈관형성소-1(ANG-1)기인선상관병독(AAV)재체,통과병독포장화순화급적정,획득고순도고감염성적병독액,병전염도유저골수간세포중,획득유효표체,위진행혈관재생적기인치료연구전정기출.방법:이용PCR기술,획취목적기인ANG-1,통과중조DNA기술득도ANG-1여pUC119적중조질립,채용SalⅠ화BglⅡ쌍매장pUC119중적목적기인ANG-1기인편단절출,재극륭도질립pSNAV2.0중.용Lipofectamine장pSNAV ANG-1질립전염293세포후,용G418선택배양획득293/AAV2ANG-1세포주,산생료유전염성적병독과립,순화병진행병독DNA과립적도측정.분별장록색형광단백(GFP)기인중조선상관병독(rAAV2GFP)급rAAV2ANG-1감염저골수간세포,병대전염효솔급전염후표체정황진행초보연구.결과:측서증실ANG-1여GenBank제공적원시서렬완전일치.중조재체경매절감정여PCR사선증실중조질립화예기결과완전일치,재신적중조질립중,ANG-1유일투CMV계동자화PolyA종지자계통.293세포포장병독효과량호,휴대ANG-1적감염성AAV적도위9×1011v.g/ml,용Western잡교법검측현시저골수간세포중표체ANG-1.1×106v.g/ml rAAV2GFP감염CD34양성세포48h후55%좌우적세포유명현적형광.결론:ANG-1기인AAV구건성공,병능재골수간세포중유효표체,위엄중결혈성질병기인치료적연구전정료기출.
Objective: To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1(ANG-1) gene and to express the ANG-1 in targeting cells. Methods: ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1,the virus stocks in high titer were harvested.The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. Results: The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously.Titration of rAAVANG-1 stock was 9×1011v.g/ml.The expression of ANG-1 gene was detected in transfected cells.Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells,55% cells expressed GFP. Conclusion: The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.