神经损伤与功能重建
神經損傷與功能重建
신경손상여공능중건
NEURAL INJURY AND FUNCTIONAL RECONSTRUCTION
2013年
4期
235-238
,共4页
脑红蛋白%慢病毒表达载体%骨髓间充质干细胞
腦紅蛋白%慢病毒錶達載體%骨髓間充質榦細胞
뇌홍단백%만병독표체재체%골수간충질간세포
Ngb%lentiviral expression vector%BMSCs
目的：克隆大鼠脑红蛋白(Ngb)基因，构建其慢病毒表达载体Lentivirus-Ngb，并转染原代培养的大鼠骨髓间充质干细胞(BMSCs)。方法：人工合成大鼠Ngb cDNA，连接至pLenti6.3-IRES-EGFP慢病毒载体，选择阳性克隆进行酶切鉴定和测序，并将其包装至慢病毒，共转染293T细胞，收获病毒颗粒，检测病毒滴度，PCR检测Ngb的表达。用包装成功的病毒液感染BMSCs，显微镜下观察BMSCs细胞，Western-blot检测Ngb蛋白在BMSCs中的表达。结果：PCR产物双酶切和基因测序确定Ngb连接正确；感染72 h后，荧光显微镜下可以看到GFP阳性的BMSCs细胞，Westen-blot检测表明Ngb在感染慢病毒的BMSCs中表达。结论：成功构建大鼠Ngb基因慢病毒表达载体pLen-ti-Ngb-IRES-EGFP，并成功转染BMSCs。
目的：剋隆大鼠腦紅蛋白(Ngb)基因，構建其慢病毒錶達載體Lentivirus-Ngb，併轉染原代培養的大鼠骨髓間充質榦細胞(BMSCs)。方法：人工閤成大鼠Ngb cDNA，連接至pLenti6.3-IRES-EGFP慢病毒載體，選擇暘性剋隆進行酶切鑒定和測序，併將其包裝至慢病毒，共轉染293T細胞，收穫病毒顆粒，檢測病毒滴度，PCR檢測Ngb的錶達。用包裝成功的病毒液感染BMSCs，顯微鏡下觀察BMSCs細胞，Western-blot檢測Ngb蛋白在BMSCs中的錶達。結果：PCR產物雙酶切和基因測序確定Ngb連接正確；感染72 h後，熒光顯微鏡下可以看到GFP暘性的BMSCs細胞，Westen-blot檢測錶明Ngb在感染慢病毒的BMSCs中錶達。結論：成功構建大鼠Ngb基因慢病毒錶達載體pLen-ti-Ngb-IRES-EGFP，併成功轉染BMSCs。
목적：극륭대서뇌홍단백(Ngb)기인，구건기만병독표체재체Lentivirus-Ngb，병전염원대배양적대서골수간충질간세포(BMSCs)。방법：인공합성대서Ngb cDNA，련접지pLenti6.3-IRES-EGFP만병독재체，선택양성극륭진행매절감정화측서，병장기포장지만병독，공전염293T세포，수획병독과립，검측병독적도，PCR검측Ngb적표체。용포장성공적병독액감염BMSCs，현미경하관찰BMSCs세포，Western-blot검측Ngb단백재BMSCs중적표체。결과：PCR산물쌍매절화기인측서학정Ngb련접정학；감염72 h후，형광현미경하가이간도GFP양성적BMSCs세포，Westen-blot검측표명Ngb재감염만병독적BMSCs중표체。결론：성공구건대서Ngb기인만병독표체재체pLen-ti-Ngb-IRES-EGFP，병성공전염BMSCs。
Objective:To clone neuroglobin (Ngb) gene of rat and construct the lenti-viral expression vec-tors of Ngb, and then transfect the vectors into bone marrow mesenchymal stem cells (BMSCs). Methods:The Ngb gene was connected to the pLenti6.3-IRES-EGFP and the positive clones were selected for re-striction enzyme digestion and sequencing. The plasmids were inserted into lenti-virusus to construct the lenti-viral expression vectors, and then the vectors were identified by enzyme digestion and sequencing analysis. The lentivirus expression in transfected 293T cells was identified by PCR, and the Ngb protein in transfected BMSCs was detected by western-blot. Results:The products were identified by enzyme diges-tion and sequencing analysis. GFP positive BMSCs can be seen under fluorescence microscope 72 hours after transfection. Westen blot analysis showed the expression of Ngb protein in BMSCs. Conclusion:The lentiviral expression vectors of Ngb were constructed and transfected into BMSCs successfully.
