大鼠骨髓间质干细胞经三七总皂甙体外诱导分化为神经元样细胞:表型特征、凋亡蛋白的表达及存活

Effect of total Panax notoginseng saponins on inducing rat bone mesenchymal stem cells differenting into neuron-like cells in vitro:Expression of phenotype and apoptosis protein and survival of neuron-like cells

目的:研究证实三七总皂甙能促进骨髓间质干细胞向神经细胞的横向分化,但分化细胞能否表达神经递质?存活时间是否延长?采用三七总皂甙对大鼠骨髓间质干细胞进行体外诱导,并检测分化的神经元样细胞表型特征、凋亡蛋白表达及存活情况。方法:实验于2006-01/2007-01在安徽医科大学实验中心完成。①实验方法:脱颈椎法快速处死大鼠,取股骨,剪断骨两端,IMDM冲洗骨髓腔,分离培养骨髓间质干细胞。取第5代细胞,用含体积分数为0.2胎牛血清、1mmol/L2巯基乙醇的完全培养液预诱导24h,然后更换含不同诱导剂的无血清培养液诱导分化,设立3组:三七总皂甙组的诱导剂含2.0g/L三七总皂甙,2巯基乙醇+丁羟茴醚组的诱导剂含200μmol/L丁羟茴醚+10mmol/L2巯基乙醇,空白对照组不添加任何诱导剂。②实验评估:观察诱导期间细胞形态变化及分化细胞的存活时间。免疫细胞化学检测分化的神经元样细胞表型特征及凋亡相关蛋白bcl-2、bax的表达情况,并采用TUNEL法检测细胞凋亡指数。结果:①加入三七总皂甙诱导后约2h可见细胞胞质收缩,逐渐有突起形成。约5h后多数细胞呈明显的神经元样细胞形态,胞体呈圆形,突起较长,双极或多极形,在突起末端出现分支,部分相邻细胞的突起连接成网。②2巯基乙醇+丁羟茴醚诱导8h后即有部分细胞贴壁,但不牢靠,1d后约半数细胞死亡,3d后仅少数细胞存活。三七总皂甙诱导后细胞死亡现象缓慢发生,1周内少数神经元样细胞出现漂浮、崩解,三四周仍有部分细胞存活。③分化细胞能够表达神经干细胞标志巢蛋白、神经元标志神经元特异性烯醇化酶、神经递质标志乙酰胆碱酯酶及γ-氨基丁酸,不表达酪氨酸羟化酶及胶质纤维酸性蛋白。④诱导后12,24h与2巯基乙醇+丁羟茴醚组比较,三七总皂甙组分化细胞bcl-2蛋白阳性表达率明显升高(P<0.01),bax蛋白阳性表达率显著降低(P<0.01),细胞凋亡指数明显降低(P<0.01)。结论:三七总皂甙在体外能够有效诱导大鼠骨髓间质干细胞分化为神经元样细胞,表达神经递质表型特征;与传统的诱导因子比较,可延长细胞存活时间,且具有抗细胞凋亡作用。

AIM： Many studies demonstrate that, mesenchymal stem cells （MSCs） can be differentiated into neuron-like cells （NLCs） by total Panax notoginseng saponins （tPNS）, but the differentiated cells can express neurotransmitters, whether to extend the survival time？ This paper is designed to investigate the differentiation of rat MSCs into NLCs with tPNS in vitro, and detect the phenotypic characteristics, apoptosis protein expression and survival of differentiated NLCs. METHODS： The experiment was completed from January 2006 to January 2007 in Anhui Medical University Experiment Center. （1）Experimental methods： Rats were killed by cervical dislocation, then the MSCs were separated from femur marrow, flushed out with IMDM, and cultured for 5 passages. Culture medium containing 0.2 volume fraction of fetal bovine serum and 1 mmol/L 2-mercapto-ethanol （2-ME） were used for 24-hour pre-induction, then were replaced with different inducers of serum-free culture medium. The study was assigned into three groups： tPNS group containing 2.0 g/L tPNS, 2-ME ＋ butylated hydroxyanisole （BHA） group containing 200 μmol/L BHA ＋ 10 mmol/L 2-ME, and blank control group without the addition of any induction agent. （2）Experimental assessment： Morphological changes and survival time of induced MSCs were observed during cell differentiation and survival. Immunocytochemical detection was used for the phenotypic characteristics and expression of apoptosis-related protein bcl-2, bax of NLCs, and TUNEL technique was used to detect apoptosis index. RESULTS： （1）Cytoplasmic contraction of differentiating MSCs was seen and gradually formed in a protruding 2 hours after tPNS induction. Most of cells were induced to differentiate into NLCs, the cell bodies were round, longer protruding became bipolar or multipolar shape, protruding branches in the end and some of the adjacent cell connected to form a network by tPNS after about 5 hours. （2）Some of adherent cells that were induced to differentiate into NLCs by 2-ME/BHA became precarious 8 hours, about half of the differentiated cells died 1 day later, and only a small number of cells survived 3 days later. However, cell death occurred slowly in the cells that were induced to differentiate into NLCs by tPNS, and NLCs appeared floating and collapse within 1 week, there were still some of NLCs survived after three weeks. （3）The differentiated MSCs expressed neural stem cell markers nestin, neuronal specific markers such as neuron-specific enolase, neurotransmitter acetylcholinesterase signs and γ-amino butyric acid, but it was failed to detect the expression of tyrosine hydroxylase and astrocyte marker glial fibrillary acidic protein under this induction condition.（4） Compared with 2-ME/BHA group, bcl-2 protein expression of differentiated NLCs by tPNS was significantly increased at hours 12 and 24 （P 〈 0.01）, while bax protein expression of differentiated NLCs by tPNS and apoptosis index were significantly reduced （P 〈 0.01）. CONCLUSION： Rat MSCs can be induced to differentiate into NLCs effectively with tPNS in vitro, express neurotransmitters phenotypic characteristics, extend cell survival time, and resist apoptosis.