含核受体真核表达载体的构建及其转染真皮多能干细胞(英文)

Construction of recombinant plasmid pEGFPN1-tailless-like protein and transfection into dermal multipotential stem cells

背景:研究表明含核受体基因在神经系统发育过程中具有重要意义,是维持成体干细胞增殖和成神经分化的关键基因。目的:拟构建含核受体真核重组表达质粒pEGFPN1-TLX,并筛选出能稳定表达含核受体的真皮多能干细胞。设计、时间及地点:细胞基因学实验,于2007-03/12在解放军第三军医大学基础医学部病原生物学教研室完成。材料:成年SD大鼠1只,由解放军第三军医大学实验动物研究所提供;真皮多能干细胞由解放军第三军医大学全军复合伤研究所分离培养;质粒载体pEGFPN1及细菌DH5α由徐文岳教授惠赠。方法:首先以大鼠的大脑组织总RNA为模板,RT-PCR扩增出含核受体的编码cDNA序列,T/A克隆至pMDl8-T载体上,然后用BamHI、HindⅢ双酶切释放出经测序鉴定为阳性的重组质粒pMDl8-T-TLX中的含核受体片段,亚克隆到真核载体pEGFPN1中,从而构建真核重组表达质粒pEGFPN1-TLX,最后采用阳性脂质体将pEGFPN1-TLX转染到真皮多能干细胞中。主要观察指标:转染后24h荧光显微镜下观察绿色荧光,RT-PCR检测含核受体mRNA的表达,免疫组化染色观察向神经细胞的分化情况。结果:PCR、酶切和测序结果证明,成功地将含核受体全长cDNA克隆到pEGFPN1质粒中,并成功构建pEGFPN1-TLX重组质粒。与未转染真皮多能干细胞相比,10d后可见pEGFPN1-TLX转染的真皮多能干细胞继续生长,并于15d时形成抗性克隆,荧光显微镜下有绿色荧光蛋白表达;而未转染的真皮多能干细胞10d时已全部死亡。RT-PCR结果显示,pEGFPN1-TLX转染真皮多能干细胞表达TLXmRNA。诱导后3d与单纯真皮多能干细胞比较,pEGFPN1-TLX转染的真皮多能干细胞诱导成NF200阳性细胞数明显增加,诱导成GFAP阳性细胞数明显减少。结论:实验成功构建含核受体真核表达载体,并成功转染了真皮多能干细胞。转染后能明显提高真皮多能干细胞向神经元分化的效率,而向胶质细胞分化受到抑制。

BACKGROUND：It is reported that tailless-like protein（TLX） plays critical roles in the regulation of early developmental processes in vertebrates,and it plays a key role in stem cells proliferation and differentiation into neurons. OBJECTIVE：To construct recombinant plasmid pEGFPN1-TLX and study the transfection into dermal multipotential stem cells. DESIGN,TIME AND SETTING：Cytogene experiment was performed at the Department of Pathogen Biology,School of Basic Medical Science,the Third Military Medical University of Chinese PLA from March to December 2007. MATERIALS：An adult SD was obtained from the Experimental Animal Center of the Third Military Medical University of Chinese PLA;dermal multipotential stem cells（DMSCs） were cultured by the Institute of Combined Injury of the Third Military Medical University of Chinese PLA;pEGFPN1 and DH5α was gifted by professor Xu. METHODS：Total RNA was extracted from rat brain tissue to amplify TLX-coded cDNA sequence using RT-PCR. T/A was cloned on pMD18-T vector and determined using BamHI and HindIII. The products were positive recombinant plasmid pMD18-T-TLX segments,which were sub-cloned in pEGFPN1 to construct recombinant plasmid pEGFPN1-TLX. Finally,pEGFPN1-TLX was transfected into DMSCs. MAIN OUTCOME MEASURES：The fluorescence protein expression was observed under fluorescence microscope at 24 hours after transfection;TLX mRNA expression was detected using RT-PCR;neuronal differentiation was observed using immunohistochemical staining. RESULTS：TLX full length cDNA was successfully cloned into pEGFPN1,and pEGFPN1-TLX was successfully constructed by means of sequence analysis and enzyme cutting identification. As compared with non-transfected DMSCs,pEGFPN1-TLX transfected DMSCs were observed after 10 days,formed resistant clones after 15 days,and shown a green fluorescent protein expression. However,non-transfected DMSCs died at day 10. RT-PCR indicated that pEGFPN1-TLX transfected DMSCs could express TLX mRNA. At day 3 after induction,NF200 positive cells were increased,but glial fibrillary acidic protein positive cells were decreased after induction of pEGFPN1-TLX transfected DMSCs. CONCLUSION：TLX was successfully constructed and transfected into DMSCs. After transfection,neuronal differentiation of DMSCs was enhanced,and the differentiation to gliocytes was inhibited.