腺病毒介导转录因子X盒结合蛋白1转染对神经干细胞增殖及在缺氧环境下凋亡的影响

Effects of adenovirus mediated XBP1 transfection on NSC proliferation and apoptosis under hypoxic conditions

目的:以重组腺病毒为载体,将X盒结合蛋白1基因转染胚鼠海马神经干细胞,观察其是否可以促进干细胞增殖以及在缺氧环境下的抗凋亡能力。方法:取孕16dSD大鼠胚鼠的海马组织进行神经干细胞的分离、克隆、nestin免疫荧光检测,以及传代和扩增;将重组腺病毒Ad-XBP1-EGFP质粒转染胚鼠海马神经干细胞,得到基因修饰后的胚鼠海马神经干细胞,选取普通神经干细胞标记为对照组,转染后的神经干细胞标记为转染组。通过细胞计数和MTT比色法检测对照组和转染组的增殖情况,连续检测7d,绘制生长曲线;取两组神经干细胞用CoCl2诱导缺氧,流式细胞术检测对照组和转染组的凋亡情况。结果:转染组的胚鼠海马神经干细胞增殖能力明显增强(P<0.05);在缺氧条件下,转染组神经干细胞凋亡程度较缺氧对照组减轻(P<0.05)。结论:利用重组腺病毒作为载体成功可以将X盒结合蛋白1基因导入胚鼠海马神经干细胞中;转染后的神经干细胞增殖能力和在缺血、缺氧条件下抗凋亡能力较普通神经干细胞明显增加。

Objective To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector.Method Hippocampi from embryonic,Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning,followed by immunofluorescence for Nestin and sub-culturing.The recombinant adenovirus Ad-XBP1-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs,and then CoCl2 was applied to induce hypoxia.Cell quantification and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBP1-transfected NSCs for 7 consecutive days.Flow cytometry was used to measure apoptosis.Results NSC proliferation was significantly enhanced following XBP1 gene transfection（P0.05）.Under hypoxic conditions,NSC apoptosis decreased following transfection（P0.05）.Conclusion The XBP1 gene can be successfully transfected into rat embryonic hippocampal NSCs by using a recombinant adenovirus vector.NSC proliferation following transfection,as well as anti-apoptotic effects under hypoxia,is significantly increased.