CAJ | 학술논문

目的以重组腺病毒(rAd)为载体构建腺病毒-睫状神经营养因子-内部核糖体进入位点-绿色荧光蛋白(Ad-CNTF-IRES-GFP).方法先构建Psp-CNTF-IRES-GFP质粒,再制备PDC316-CNTF-IRES-GFP质粒,然后在脂质体的作用下,用构建好的PDC316-CNTF-IRES-GFP质粒与骨架质粒PBHG在293-LP细胞中构建Ad-CNTF-IRES-GFP腺病毒,并扩增、纯化,鉴定病毒活性.最后,将Ad-CNTF-IRES-GFP转染人源性骨髓间充质细胞(MSCs),观察MSCs的CNTF表达情况.结果成功扩增CNTF基因,扩增后的CNTF基因与基因文库序列完全相符;成功制备PDC316-CNTF-IRES-GFP质粒及Ad-CNTF-IRES.GFP腺病毒,测得Ad-CNTF-IRES-GFP腺病毒的病毒活性单位(pfu)为2.3x1011;构建好的Ad-CNTF-IRES-GFP成功转染MSCs,而凡转染后的MSCs表达CNTF的量为未转染MSCs表达量的20倍.结论本方法能够成功构建Ad-CNTF-IRES-GFP腺病毒载体,而且转染后的MSCs高度表达CNTF.
목적 이중조선병독(rAd)위재체구건선병독-첩상신경영양인자-내부핵당체진입위점-록색형광단백(Ad-CNTF-IRES-GFP).방법 선구건Psp-CNTF-IRES-GFP질립,재제비PDC316-CNTF-IRES-GFP질립,연후재지질체적작용하,용구건호적PDC316-CNTF-IRES-GFP질립여골가질립PBHG재293-LP세포중구건Ad-CNTF-IRES-GFP선병독,병확증、순화,감정병독활성.최후,장Ad-CNTF-IRES-GFP전염인원성골수간충질세포(MSCs),관찰MSCs적CNTF표체정황.결과 성공확증CNTF기인,확증후적CNTF기인여기인문고서렬완전상부;성공제비PDC316-CNTF-IRES-GFP질립급Ad-CNTF-IRES.GFP선병독,측득Ad-CNTF-IRES-GFP선병독적병독활성단위(pfu)위2.3x1011;구건호적Ad-CNTF-IRES-GFP성공전염MSCs,이범전염후적MSCs표체CNTF적량위미전염MSCs표체량적20배.결론 본방법능구성공구건Ad-CNTF-IRES-GFP선병독재체,이차전염후적MSCs고도표체CNTF.
Objective To construct an adenoviral vector carrying the gene encoding ciliary neurotrophic factor (CNTF). Methods The gene fragment encoding CNTF was amplified from pMEG-CNTF plasmid by PCR and the Psp-CNTF-IRES-GFP and PDC316-CNTF-IRES-GFP plasmids were constructed. Using PDC316-CNTF-IRES-GFP and PBHG plasmids, the Ad-CNTF-IRES-GFP vector was constructed, and the constructed vector was amplified, purified and identified in 293-LP cells. Ectopic overexpression of CNTF was induced using the constructed vector in human bone marrow-derived mesenchymal stem cells (MSCs) to investigate the role of CNTF in promoting remyelination. Results The Ad-CNTF-IRES-GFP vector was successfully constructed with a pfu of 2.3x1011. CNTF concentration in the MSCs transfeeted with Ad-CNTF-IRES-GFP vector was 20-fold higher than that in either non-transfected or Ad-EGFP-transfected MSCs. Conclusion The constructed Ad-CNTF-IRES-GFP vector allows CNTF overexpression in human MSCs by 20 folds, which provides a strategy for gene therapy targeting CNTF.