人脐静脉间充质干细胞体外诱导分化为神经元的可行性

Induction differentiation of human umbilical vein-derived mesenchymal stem cells into neuron-like cells in vitro

目的：探讨来源于人脐静脉内皮及内皮下层间充质干细胞体外诱导分化为神经元样细胞的可行性，分析体外分离纯化、原代及传代培养的最佳条件，为神经组织修复选择理想的种子细胞来源。方法：实验于2006—04／12在辽宁医学院解剖学实验室完成。①对象：取正常健康产妇顺产或剖宫产的新生儿脐带20条，由辽宁医学院附属第一医院提供，产妇及其家属均签署知情同意书，实验经医院医学伦理委员会批准。②实验方法：无菌条件下用1g/L Ⅰ型胶原酶消化脐静脉内皮及内皮下层，收集的细胞按5× 10^7 L^-1，1× 10^8 L^-1，5× 10^8 L^-1，1× 10^9 L^-1，3× 10^9 L^-1密度接种进行原代培养，待细胞80％融合时胰酶消化传代。取第2代细胞，诱导组经含有3μmo／L β-巯基乙醇、20％胎牛血清的DMEM预诱导液处理后，换成含10g/L二甲基亚砜、100mmol／L丁化羟基茴香醚的无血清DMEM诱导液进行诱导。未诱导组将诱导液更换为无血清DMEM。③实验评估：记录原代培养过程中不同接种密度细胞贴壁所需时间。免疫细胞化学检测诱导前细胞表面抗原血管性血友病因子、CD166的表达及诱导后细胞巢蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白的表达，倒置相差显微镜下计数阳性细胞分化率。结果：①不同接种密度细胞贴壁所需的原代培养时间比较：5× 10^7 L^-1组仅有1孔细胞贴壁呈克隆样生长，但培养至28d时仍不能传代，其余培养孔均无细胞贴壁生长。与1× 10^8 L^-1组比较，5× 10^8 L^-1，1× 10^9 L^-1，3× 10^9L^-1组细胞贴壁生长的原代培养时间均明显缩短（P〈0．05），且后3组比较差异无显著性意义（P〉0．05）。②贴壁细胞表面抗原检测：CD166呈阳性，血管性血友病因子呈阴性。③诱导分化：诱导组脐静脉间充质干细胞巢蛋白、神经元特异性烯醇化酶呈阳性表达，不表达胶质纤维酸性蛋白。巢蛋白阳性细胞率为（9．5±1．23％，神经元特异性烯醇化酶阳性细胞率为（15．6±2．6）％。未诱导组以上3种标志均不表达。结论：①在体外可以成功分离培养获得人脐静脉内皮及内皮下层的间充质干细胞，其原代培养细胞的种植密度以5× 10^8 L^-1～1× 10^9 L^-1为佳。②细胞传2代后性质相对均一，基本无造血细胞和内皮细胞污染，达到纯化目的，具有间充质干细胞特点。③在化学诱导剂β-巯基乙醇、二甲基亚砜、丁化羟基茴香醚联合作用下，其可以向神经元样细胞分化。

AIM： To explore the feasibility of using mesenchymal stem cells derived from endothelial and subendothelial cells of human umbilical vein to regenerate neuron-like cells in vitro, to analyze the optimal condition of in vitro isolation, purification, primary and passaged culture, and to select seed cells for nerve tissue repair. METHODS： Experiments were conducted in the Department of Anatomy, Liaoning Medical University between April and December 2006. ①Twenty infant umbilical cords were obtained from healthy lying-in women with natural deliveries or caesarean birth after each mother and her family numbers signed an informed consent according to a protocol approved by The First Affiliated Hospital, Liaoning Medical University. The experimental procedures were also authorized by Ethics Committee of the hospital. ②By 1 g/L collagenase 1 digestion, endothelial and subendothelial cells were isolated from human umbilical vein. The isolated human umbilical vein cells were divided into groups according to different implantation density： 5 × 10^7 L^-1, 1 × 10^8 L^-1, 5 × 10^8 L^-1, 1 × 10^9 L^-1, 3 × 10^9 L^-1. Cells of 80-90% confluence were digested with trypsin. Passage 2 cells in the induced group were firstly dealt with DMEM containing 20%fetal bovine serum, 3 μmo/L β -mercaptoethanol, and then induced by serum-free DMEM containing 10 g/L dimathyl sulfoxide, 100 mmol/L dihydroxyacetone. Cells in the non-induced group were dealt with serum-free DMEM. ③The primary culture time were recorded in different groups. Immunocytochemistry analysis for CD166 and von Willebrand factor was performed before induction, as well as for nestin, neuron-specific enolase and glial fibriliary acidic protein after induction. Differentiation rate of positive cells was determined under an inverted phase contrast microscope. RESULTS： ①Comparing the primary time in different groups： The cells in the group of 5 × 10^7 L^-1 were clone-likely in only one well, but could not passage until 28 days. There were no adhered cells in other wells. Compared to the group of 1× 10^8 L^-1, the times of the groups of 5 × 10^8 L^-1, 1 × 10^9 L^-1, 3 × 10^9 L^-1 were all shorted obviously （P 〈 0.05 ） , and there was no significant difference in last 3 groups （P 〉 0.05 ） . ②Immunocytochemistry staining： The adherent cells were negative for von Willebrand factor, but positive for CD 166. ③All of the induced mesenchymal stem cells expressed nestin, neuron-specific enolase, but glial fibriliary acidic protein was not found. The positive cells proportion of nestin were （9.5±1.2） % and neuron-specific enolase were （15.6±2.6 ） %. Above mentioned 3 markers were not expressed in the non-induction group. CONCLUSION： ①Mesenchymal stem cells from the endothelial and subendothelial layers of umbilical cord vein can be cultured and expanded. It was reasonable that mesenchymal stem cells were plated at 5 × 10^8 L^-1- 1 × 10^9 L^-1. ②After passage 2, the cells have the same characteristics of mesenchymal stem cells and have been purified. ③The cells have the potential of differentiating into neuron-like cells under specific culture conditions （ β -mercaptoethanol, dimathyl sulfoxide, dihydroxyacetone） in vitro.