外源性绿色荧光蛋白和蛋白酪氨酸激酶受体B基因在大鼠骨髓源性神经干细胞内稳定表达的检测特征

Detection characteristics of the stable expression of recombinant plasmid enhanced green fluorescent protein-C2-protein tyrosine kinase B gene in the neural stem cells derived from the bone marrow stromal cells of rats

目的:利用本实验室构建的携带蛋白酪氨酸激酶受体B基因的荧光真核表达质粒-pEGFP-C2-蛋白酪氨酸激酶受体B,转染大鼠骨髓基质细胞源性的神经干细胞,观察其在神经干细胞中的表达情况。方法:实验于2005-06/2006-06在南方医科大学珠江医院神经医学研究所完成。实验选用SD大鼠进行骨髓的采集及骨髓基质细胞的获取,培养1周后得到较纯化的骨髓基质细胞,进行细胞传代培养,传代培养的细胞加入10%胎牛血清及维甲酸以诱导骨髓基质细胞向神经干细胞分化。细胞培养2周后,pEGFP-C2-蛋白酪氨酸激酶受体B经LIPOEECTAMINE.2000脂质体转染培养的大鼠骨髓基质细胞源性的神经干细胞,24h后观察绿色荧光蛋白的瞬时表达情况,以酶切和测序鉴定质粒的正确性,凝胶电泳鉴定PCR产物大小,应用免疫组化定量分析法检测转染该质粒的细胞中蛋白酪氨酸激酶受体B的含量,并与正常细胞作比较。结果:①细胞转染24h后,荧光显微镜下可观察到绿色荧光蛋白的表达,表达率约18.1%,随着培养时间的延长,表达绿色荧光蛋白的细胞数量逐渐增多,于1周左右达到高峰,表达率约42.8%,继续观察1个月未发现荧光表达减弱。②酶切、PCR和DNA序列鉴定均证实插入片段的正确性,扩增的PCR产物凝胶电泳结果与设计引物间的片段大小1461bp完全一致,转染细胞电泳未见目的条带。③免疫组化结果表明,所有细胞均含有蛋白酪氨酸激酶受体B抗原成分,转染pEGFP-C2-蛋白酪氨酸激酶受体B的细胞该抗原成分较未转染细胞明显增加(19.76±3.98,32.17±6.29,P<0.05)。结论:LIPOEECTAMINE.2000是一种简单而高效的转染大鼠骨髓基质细胞源性神经干细胞的方法,转染的pEGFP-C2-蛋白酪氨酸激酶受体B基因在细胞内可以稳定表达,增强型绿色荧光蛋白可以作为该基因表达的标记。

AIM： To observe the expression of the protein tyrosine kinase B gene （PtkB） in the neural stem cells derived from the bone marrow stromal cells （NSCs-D-BMSC, s） by observing the expression of enhanced green fluorescent protein （EGFP） after transfecting the pEGFP-C2-PtkB plasmid to NSCs-D-BMSCs of the rats. METHODS： The experiment was done in Neuromedical Institute, Zhujiang Hospital of Southern Medical University from June 2005 to June 2006. Bone marrow was collected and BMSCs were obtained from SD rats. After 1-week culture, purified BMSCs were obtained for cell passage culture. 10% fetal bovine serum and retinoic acid were added in cultured cells to induce the differentiation of BMSCs into NSCs. Two weeks after cell culture, the pEGFP-C2-TrkB plasmid was transfected into NSCs-D-BMSC, s with LIPOEECTAMINE.2000. After 24 hours, the transient expression of EGFP was observed. The plasmid＇s correctness was evaluated by the restriction enzyme analysis and sequence. Size of PCR produce was assessed by gel electrophoresis. TrkB content of the cells, was assessed by immunohistochemical quantitative assay, and compare it with the normal cells. RESULTS.①EGFP were successfully expressed 24 hours after nucleofection, and the expressive rate of positive EGFP expression was 18.1%. The positive EGFP expression was enhanced gradually alone with the prolonged culture time, and showed the strongest one week after marked, with about 42.8%. The expression intensity of EGFP did not attenuate even one month after marked. ②Correct construction of pEGFP-C2-TrkB was identifed by restriction enzyme analysis, PCR amplification and nucleotide sequence determinaUon. PCR amplification result was the same as 1 461 bp designed primers, and there was no target strap in electrophoresis. ③The immunohistochemisty results were indicated that all cells contained TrkB antigenic, but the content of this antigenic in the cells taking along TrkB plasmid was obviously more than the normal cells （19.76 ±3.98032.17 ±6.29, P 〈 0.05）. CONCLUSION： LIPOEECTAMINE.2000 is an efficient gene transfer method for the introduction of the genes into rat NSCs-D-BMSCs, The pEGFP-C2-TrkB gene transferred can stabilize expression. EGFP plays a symbol role in the expression of the pEGFP-C2-TrkB gene.