摘要
目的：血管重建手术在多数情况下以自体血管作为替代品,但来源有限。胚胎干细胞具有发育全能性,目前其定向诱导机制尚不明确。通过选择适当的诱导剂,探讨胚胎干细胞体外向血管平滑肌细胞分化的趋势。方法：实验于2006-08/2007-02在南昌大学第二附属医院血液病研究所完成。①动物及细胞系：清洁级孕12.5d昆明小白鼠1只,由南昌大学医学院动物中心提供,实验过程中对动物的处置符合动物伦理学标准。小鼠胚胎干细胞系129X/SvJ编号为SCRC-1018,由American Type Culture Collection提供。②实验方法：取孕12.5d鼠胚胎,去除头部、内脏及四肢,将组织块剪碎,胰酶消化,分离培养胚胎成纤维细胞,传至3～5代时更换为含体积分数为0.15胎牛血清的DMEM高糖培养基,24h后收集培养液,加入2.0mmol/LL-谷氨酰胺,1×非必需氨基酸,0.1mmol/Lβ-巯基乙醇,1000U/mL白血病抑制因子,此即为条件培养基。常规复苏小鼠胚胎干细胞系129X/SvJ,以1×10^6密度接种,传代时用差速贴壁法分离去除已分化的胚胎干细胞,加入条件培养基5mL,经过悬滴-悬浮培养,构建拟胚体分化模型。设立3组,各组均置于明胶包被的T25培养瓶中,每瓶加入50个拟胚体,使其均匀分布于培养瓶底。诱导组7～10d加入10^-9mol/L全反式维甲酸和3μg/L转化生长因子β1,10～21d加入20μg/L血小板源性生长因子。血清对照组仅加入去生长因子胎牛血清,全反式维甲酸组仅加入全反式维甲酸。诱导21d的拟胚体,用胰蛋白酶和胶原酶Ⅱ联合消化为单个细胞,再加入20μg/L血小板源性生长因子继续诱导7d。③实验评估：应用RT-PCR法检测诱导细胞血管平滑肌肌动蛋白、血管平滑肌肌球蛋白重链基因的表达。通过免疫细胞化学技术检测血管平滑肌肌动蛋白的表达来鉴定细胞性质。结果：①胚胎干细胞生长及拟胚体诱导分化：胚胎干细胞在体外能自发形成拟胚�
AIM： Blood vessels from the host are mostly used in the reconstructive vascular operation, but the resource is limited. Embryonic stem cell （ESC） has the totipotency, but the induction mechanisms are not identified at present. This study explored the differentiation of ESCs to vascular smooth muscle cells （VSMC） by suitable inductors. METHODS： This experiment was finished in the Blood Disease Institute, the Second Affiliated Hospital of Nanchang University from August 2006 to February 2007.（1）Animal and cell line： One depuratory grade Kunming Mus musculus albus pregnant for 12.5 days, was provided by the Nanchang University Medical Animal Center. The disposition of the animal accorded with the ethical standard. ESC line 129X/SvJ （No. SCRC-1018） was provided by the American Type Culture Collection.（2）Empirical method： Mouse embryo fibroblast was segregated from the 12.5-day fetus mouse following cutting and trypsinization after removing head, viscera and limbs. As the fibroblasts were serially passaged to 3-5 generations, the medium was changed to the high DMEM with 15% fetal bovine serum, then the high DMEM was collected 24 hours later, and added with 2.0 mmol/L L-glutamine, 1× non-essential amino acid, 0.1 mmol/L β -mercaptoethanol, and 1 000 U/mL leukaemia inhibitory factor. ESC line 129X/SvJ after routine resuscitation was inoculated at the density of 1×10^6 for suspended drops and culture, after the differentiated ESCs were separated using differential attachment. All the cultured cells were placed in the T25 culture flask treated by gelatin with the condition medium （5 mL）, and formed into embryoid body differentiation model. The experiment was set up to 3 groups, in the experimental group 10^-9 mol/L all-trans-retinoic acid （ATRA） and 3 μ g/L transforming growth factor- β 1 were added from the 7^th day to the 10^th day, and 20 μ g/L platelet-derived growth factor was used in this experiment from the 10^th day to the 28^th day. In the serum control group, only fetal
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2272-2276,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30360102)~~
