摘要
目的:Persephin是近年来发现的对多巴胺神经元的发育与分化起调控作用重要基因,单纯神经干细胞系C17.2移植治疗帕金森病在体内分化为多巴胺能神经元比例不高。实验构建了人Persephin基因重组腺病毒载体,并观察其在C17.2神经干细胞的表达。方法:实验于2005-06/12在中山大学附属第二医院林百欣实验中心完成(广东省重点实验室)。①实验材料:流产胚胎由中山大学附属第二医院妇产科提供,产妇及其家属均签署知情同意书。病毒骨架质粒载体pAdEasy-1、穿梭载体PAdTrack-CMV(含绿色荧光蛋白基因)、大肠杆菌菌株BJ5183和DH5α、人胚肾293细胞均为本室保存。C17.2神经干细胞由美国哈佛大学医学院Snyder教授惠赠。②实验方法:采用反转录聚合酶链式反应从人胚胎脑获得PersephincDNA。将重组质粒pAdTrack-CMV-Persephin与骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,获得重组腺病毒质粒。将重组腺病毒质粒转染293细胞包装获得重组腺病毒AdCMVPersephin,转化体外培养的C17.2神经干细胞。③实验评估:用原位杂交、免疫组化、Westernblot法检测Persephin在C17.2神经干细胞中的表达。结果:①人Persephin基因的克隆与测序分析:琼脂糖凝胶电泳检测PCR扩增产物,其Persephin基因片段长约310bp,与预期分子量相符。Persephin基因被克隆于载体pMD18-T,以SaiⅠ和XhoⅠ双酶切可回收长约310bp的克隆片段,测序结果证实所克隆的为人PersephincDNA。②大肠杆菌同源重组获得克隆化重组腺病毒基因组及鉴定:Persephin重组腺病毒质粒pAdTrack-CMV-Persephin用PacⅠ酶切可切出4.1kb的目标片断,PCR鉴定可从重组腺病毒质粒中可扩增出PersephincDNA片断(310bp)。病毒滴度在脂质体转染后为1.9×107pfu/L。用收集的上清4次重复感染293细胞扩增重组腺病毒后,病毒滴度经绿色荧光蛋白检测达3.0×1011pfu/L。③Persephin基因在C17.2神经干细胞中的表达:免疫组化与原位杂交显示转染pAdPersephin的C17.2神经干细胞胞浆中有Persephin蛋白和mRNA表达。提取感染病毒的C17.2神经干细胞的总蛋白,经Westernblot检测可见一特异性蛋白条带存在。结论:采用RT-PCR法成功克隆人PersephincDNA并构建其腺病毒载体,获得了稳定表达Persephin的神经干细胞克隆,为进一步分析转基因神经干细胞治疗神经退行性疾病提供可行的操作方法。
Recent studies suggest that Persephin plays an important role in regulating the development and differentiation of dopaminergic neurons, and it is difficult for the transplanted C17.2 neural stem cells for treating Parkinson disease to differentiate into dopaminergic neurons. In this study, human Persephin gene recombinant adenovirus vector is constructed, so as to observe its expression in C17.2 neural stem cells. METHODS： The experiment was conducted in the Lin Bai-xin Experimental Center of Second Hospital of Sun Yat-sen University from June to December 2005. ①The abortion embryo was provided by Department of Genaecology and Obstetrics, Second Hospital of Sun Yat-sen University with the informed consent from the parturient and her family. Adenoviral backbone plasmid pAdEasy-1, shuttle plasmid PAdTrack-CMV containing green fluorescent protein gene, E. Coli BJ5183, DH5α, and human embryo kidney 293 cells were preserved in the laboratory. C17.2 neural stem cells were gifted from Professor Snyder, Harvard Medical School.②Human Persephin cDNA was amplified by RT-PCR from human embryo brain. Recombinant shuttle plasmid PAdTrack-CMV-Persephin was co-transformed into E.Coli BJ5183 with adenoviral backbone plasmid pAdEasy-1 to obtain the recombinant adenoviral plasmid, which was transfected into 293 cell lines, and the recombinant adenovirus AdCMVPersephin was packaged and transfected into C17.2 neural stem cells. ③The expression of Persephin in C17.2 neural stem cells was observed by hybridization in situ, immunohistochemical staining and Western blot analysis. RESULTS： ①Agarose gel electrophoresis detection showed that the Persephin gene segment was about 310 bp, which was accorded with the expectation. Cloned sequence about 310 bp was obtained by Sai Ⅰ and Xho Ⅰ digestion after Persephin gene was cloned into pMD18-T, and the sequencing results indicated that it was human Persephin cDNA. ②Recombinant plasmid PAdTrack-CMV-Persephin was Pac Ⅰ digested and 4.1 kb target sequence was obtained. Persephin cDNA （310 bp） was amplified by PCR with virus titer of 1.9x10^7 pfu/L after transfected by liposome, and 3.0x 10^11 pfu/L after transfected with supernatant for 4 times. ③lmmunohistochemical staining and hybridization in situ showed that Persephin recombinant adenovirus could infect 293 cell lines and replicate in the cells. Neural stem cell stably overexpressed the transgene. Western blot detected an specific protein band in the total protein extracted from C17.2 neural stem cells. CONCLUSION： Human Persephin gene is successfully cloned and its recombinant adenovirus vector has been constructed by RT-PCR. Clone stably overexpressed Persephin is obtained. This provides the approach for the further study on neural stem cell therapy for neural degenerative diseases.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第33期6548-6551,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
教育部科研基金资助(20020558074)~~
