丙戊酸诱导鼠脂肪源性干细胞向许旺细胞分化的实验研究  

The study for induction and differentiation of rat＇s adipose-derived stem cells by valproic acid in vitro

作　　者：吴飞[1] 贾婷 杨越[1] 孙志博 刘丰[1] 张向阳[1] Wu Fei;Jia Ting;Yang Yue;Sun Zhibo;Liu Feng;Zhang Xiangyang(Department of Endocrinology,Wuhan Central Hospital,Huazhong University of Science and Technology,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院骨外科,武汉430060 [2]华中科技大学同济医学院附属武汉中心医院内分泌科

出　　处：《中华显微外科杂志》2018年第4期356-360,共5页Chinese Journal of Microsurgery

基　　金：国家自然科学基金（81301052,81501894）

摘　　要：目的探讨丙戊酸（VPA）对脂肪源性干细胞（ADSCs）的诱导分化作用。方法2016年11月至2017年10月，取3周龄SD大鼠2只，体外分离、培养ADSCs并传至第3代；经含10ng／ml bFGF的完全培养液培养24h后，分4组。空白对照组（A组）仅加入D-Hank液；实验组加入不同浓度VPA对其进行诱导：B、C、D组分别每孔加入O.5、1.0和10.0mmo/L丙戊酸200μl。每天观察细胞形态变化。培养7d后采用实时荧光定量PCR检测各组细胞神经组织蛋白（S-100）、神经元特异性烯醇化酶（NSE）及巢蛋白（Nestin）基因表达.免疫荧光染色检测S-100蛋白表达。t检验进行组间比较，P〈0.05为差异有统计学意义。结果培养4d后B、C、D组细胞经VPA诱导后逐渐出现许旺细胞样或神经细胞样形态改变.以C组变化最为明显：A组细胞形态未见明显改变，仍以梭形或圆形为主。S-100免疫荧光染色示，B、C、D组染色阳性率较高，以C组阳性率最高；A、D组细胞阳性率较少，其中A组最少。实时荧光定量PCR检测示,S-100基因表达量随VPA浓度升高呈上升趋势，在丙戊酸浓度为1.0mmol／L时达到峰值，之后开始缓慢下降；C组表达量显著高于A、B、D组（P〈0.05），B、D组间差异无统计学意义（P〉0.05）。NSE基因表达与S-100呈相同趋势，且C组表达量显著高于A、B、D组（P〈0.05），B、D组间差异亦有统计学意义（P〈0.05）。Nestin基因表达量各组间差异无统计学意义（P〉0.05）。结论合适浓度的VPA在体外能够诱导ADSCs向许旺细胞样细胞分化.1.0mmol／L可能为其最佳诱导浓度.Objective To study the inducting differentiation effects of the vaproic acid （VPA） on rats adipose derived stem cells （ADSCs） in vitro. Methods From November, 2016 to October, 2017, the ADSCs were isolated from 2 healthy 3-weeks-old Sprague-Dawley （SD） rats and cultured to passage 3, which were treated with 10 ng/ml bFGF for 24 hours before induction. Then the induction media contained the VPA with different concentrations： group B （0.5 mmol/L, 200 μl） VPA; group C （1.0 mmol/L, 200 μl） VPA; group D （10 mmol/L, 200 μl） VPA, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed every day. At 7 days of induction, the gene expressions of neuron-specific enolase （NSE）, nestin （NES）, and S-1O0 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunofluorescence staining. Significance of difference was determined using independent t test. Probabilities lower than 5% （P〈0.05） were considered statistically significant. Results At 4 days after induction, some ADSCs of groups B, C, and D showed the morphology of Schwann-fike cells or neuron-like cells, the change of group C was more obvious; and the AD-SCs of group A had no obvious change, which still present spindle. The S-100 immunofluorescence staining showed higher ratio of positive expression in groups B, C, and D （more obvious in group C） and lower ratio of positive expression in groups A and D （more obvious in group A）. The gene expression of S-100 showed dose-dependent increases in groups C, which was significantly higher than that of groups A, B, and D （P〈0.05）, but no significant differ ence was found between groups B and D （P〉0.05）. The gene expression of NSE showed the same tendency as S-1O0,which reached the peak in group C; the gene expression of NSE in group C was significantly higher than that of groups A, B, and D （P〈0.05）, and groups B and D showed significant difference （P〈0.05）.

关 键 词：脂肪源性干细胞 许旺细胞 丙戊酸 诱导分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]