CAJ | 학술논문

目的构建携带人基质细胞衍生因子(SDF)1α基因的反转录病毒真核表达载体pLEGFP-N1-SDF-1α,转染恒河猴骨髓基质细胞(BMSC),观察外源性SDF-1α和增强型绿色荧光蛋白(EGFP)基因在BMSC中的表达情况.方法应用基因重组技术,从pBudce4.1-SDF-1α获得SDF-1α基因片段,重组到pLEGFP-N1真核表达载体上.pLEGFP-N1-SDF-1α经病毒包装,转染至恒河猴BMSC,用Western免疫印迹和免疫细胞化学检测表达情况.结果酶切、PCR和DNA序列鉴定均证实插入基因片段的正确性.BMSC转染pLEGFP-N1-SDF-1α后,在荧光显微镜下发出绿色荧光.western免疫印迹和免疫细胞化学证实SDF-1α在细胞内有效表达.结论成功构建本研究pLEGFP-N1-SDF-1α,经病毒包装转染至恒河猴BMSC,SDF-1α和EGFP基因在BMSC内有效表达.为BMSC-SDF-1α-EGFP工程细胞自体移植治疗相关疾病提供了依据.
목적 구건휴대인기질세포연생인자(SDF)1α기인적반전록병독진핵표체재체pLEGFP-N1-SDF-1α,전염항하후골수기질세포(BMSC),관찰외원성SDF-1α화증강형록색형광단백(EGFP)기인재BMSC중적표체정황.방법 응용기인중조기술,종pBudce4.1-SDF-1α획득SDF-1α기인편단,중조도pLEGFP-N1진핵표체재체상.pLEGFP-N1-SDF-1α경병독포장,전염지항하후BMSC,용Western면역인적화면역세포화학검측표체정황.결과 매절、PCR화DNA서렬감정균증실삽입기인편단적정학성.BMSC전염pLEGFP-N1-SDF-1α후,재형광현미경하발출록색형광.western면역인적화면역세포화학증실SDF-1α재세포내유효표체.결론 성공구건본연구pLEGFP-N1-SDF-1α,경병독포장전염지항하후BMSC,SDF-1α화EGFP기인재BMSC내유효표체.위BMSC-SDF-1α-EGFP공정세포자체이식치료상관질병제공료의거.
Objective To construct a retrovirus eukaryotic expression vector pLEGFP-N1-stromal cell-derived factor-1α (SDF)- 1α that contains human SDF-1α and transfects bone marrow stromal cells (BMSCs) of rhesus, and to examine the expression of exogenous SDF- 1α and EGFP genes in BMSCs .Methods SDF-1α gene obtained from pBudce4.1-SDF-1α was recombined into pLEGFP-N1 vector to generate pLEGFP-N1-SDF-1α by use of genetic recombination techniques. pLEGFP-N1-SDF-1α packaged in virus was transfected into BMSCs of rhesus. Western blotting and immunocytochemistry were used for detection of its expression. Results The inserted gene was verified by enzyme restriction analysis, PCR and DNA sequencing. After transfected with pLEGFP-N1-SDF-1α, the BMSCs emitted green fluorescence, and expressed SDF- 1α as confirmed by Western blotting and immunocytochemistry. Conclusion After transfected to BMSCs of rhesus in virus, pLEGFP-N 1-SDF-1α may effectively express SDF-1α and EGFP,which provides evidences for auto-grafting of BMSC-SDF-1α-EGFP engineered cells in treatment of certain diseases.