摘要
目的以低氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)基因重组腺病毒体外转染胚胎大鼠神经干细胞,建立能稳定表达HIF-1α的基因工程化神经干细胞,为进一步的实验研究提供理论和物质基础。方法①分离SD大鼠胎鼠的间脑,加入神经生长因子EGF、bFGF和B27的神经干细胞条件培养基中克隆培养。通过检测神经干细胞的特异性抗原神经巢蛋白(nestin)、自我增殖能力和分化能力来鉴定神经干细胞;②用含有HIF-1α基因的腺病毒感染培养第2代的神经干细胞,通过绿色荧光蛋白的表达证明转染基因的表达,Western-blot检测转染后HIF-1α蛋白表达的变化,免疫原位杂交检测转染后HIF-1αmRNA阳性细胞。结果①分离培养出了大量具有不断增殖能力的神经干细胞;②70%～80%的神经干细胞能感染HIF-1α基因重组腺病毒,Western-blot和免疫原位杂交实验显示HIF-1α蛋白大量表达。结论成功建立了神经干细胞的分离培养方法及能长期稳定表达HIF-1α的基因工程化神经干细胞。
【Objective】To establish the neural stem cell line that can express HIF-1α stably via the transfection of recombinant virus containing HIF-1α gene in vitro for futher research.【Methods】Tissues of diencephalons were isolated from a SD rat embryo, then the neural stem cells were cultivated in special culture medium containing bFGF, EGF and B27 .The cells were identified by detecting the differential NSC antigen-nestin, the ability to proliferate continuously and differentiate into neurons and glial cells by inducement. The neural stem cells of 2nd generation were infected with recombinant adenovirus containing HIF-1α gene segment. The protein expressed by external gene can be detected by detecting the expression of GFP. The cells were identified with Western blotting and to In situ hybridization technique.【Results】The isolated and cultured neural stem cells had the ability to proliferate continuously. seventy to eighty percent neural stem cells＇ were transfected by recombinant adenovims containing HIF1α gene segment successfully. In situ hybridization and Western-blot showed that a great deal of HIF-1α were expressed in these stem cells.【Conclusions】The isolated and cultured methods of the neural stem cells from rat embryo and the stem cell line which can stably express HIF-1α were successfully established.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2010年第5期679-683,688,共6页
China Journal of Modern Medicine
