【摘要】 目的:验证β-巯基乙醇、神经生长因子对大鼠骨髓基质细胞体外定向诱导分化为神经细胞的可行性。方法:实验于2005-05/2006-02在锦州医学院外科实验室完成。①选取清洁级1月龄SD大鼠30只,全骨髓培养法从大鼠骨髓中分离培养骨髓基质细胞。②取第3代骨髓基质细胞,以含胎牛血清的DMEM培养液培养消化后,计数细胞量,描绘细胞增殖曲线。③取第3～5代骨髓基质细胞,接近70%～80%融合后,分为3组:β-巯基乙醇诱导组:加入含1mmol/Lβ-巯基乙醇的无血清培养基预诱导24h后,去除预诱导液,再加入含5mmol/Lβ-巯基乙醇的无血清培养基诱导5h;神经生长因子诱导组:加入碱性成纤维细胞生长因子20μg/L进行预诱导24h后,去除预诱导剂,磷酸盐缓冲液洗涤3次,再加入含神经生长因子50μg/L无血清培养基诱导24h;阴性对照组:仅加等量无血清培养基。观察各组诱导后细胞的形态学改变。④免疫组化法检测诱导后各组细胞的神经元特异性烯醇化酶、微管相关蛋白和胶质纤维酸性蛋白的表达和分化率。结果:①骨髓基质细胞的生长曲线:培养第1～4天为潜伏期,此后细胞增殖加速并进入对数生长期,第9天达到高峰,以后细胞增值速度减慢,进人平台期。骨髓基质细胞倍增时间约为32h。②骨髓基质细胞诱导分化后的形态学观察结果:β-巯基乙醇诱导组经β-巯基乙醇诱导1h,细胞形态即发生改变,胞体收缩成球形或锥形,折光性增强,部分细胞周围有光晕,有较多长突起。5h后,神经元样细胞明显增多,可形成神经网络状结构,但是死亡细胞逐渐增多;神经生长因子诱导组形态学变化出现较迟,在12h才有明显的形态学改变,表现为胞体变大,有类似轴突和树突的细长突起。24h表现更为明显,部分细胞间也可见网络状排列,细胞死亡现象不明显;阴性对照组无明显的形态学改变,仍为长梭形细胞。③神经元样细胞免疫组化测定结果:β-巯基乙醇诱导组、神经生长因子诱导组神经细胞特异性烯醇化酶、微管相关蛋白表达均为阳性,表现为胞体和突起被染成棕黄色,而胶质纤维酸性蛋白呈阴性;阴性对照组无阳性结果出现。β-巯基乙醇诱导组神经细胞特异性烯醇化酶、微管相关蛋白分化率分别为(71.2±3.8)%和(64.1±2.7)%,神经生长因子诱导组分别为(46.2±2.5)%和(42.9±1.9)%,两组间差异有显著性意义(P<0.05,t=-5.205)。结论:β-巯基乙醇、神经生长因子均可成功诱导大鼠骨髓基质细胞分化为神经元样细胞。更多还原

【Abstract】 AIM: To verify the feasibility of β-mercaptoethanol and nerve growth factor (NGF) in the induction of in vitro oriented differentiation of rat marrow stromal cells (MSCs) into nerve cells. METHODS: The study was conducted in the Surgery Laboratory of Jinzhou Medical College from May 2005 to February 2006. ①MSCs were obtained from 30 SD rats aged 1 month in clean grade. ②After 3 passages were obtained,and then digested by DMEM medium containing fetal calf serum,cell amount was counted and the proliferation curve of MSCs was depicted. ③After the 3-5 passages of MSCs were induced,when it was near to 70%-80% fusion,MSCs were assigned into 3 groups: β-mercaptoethanol group: MSCs were maintained in the serum-free medium consisting of 1 mmol/L β-mercaptoethanol for 24 hours,and then the preinduction medium was removed,and the cells were transferred to the neuronal induction medium composed of 5 mmol/L β-mercaptoethanol for 5 hours; NGF group: MSCs were maintained in the preinduction medium consisting of 20 μg/L basic fibroblast growth factor (bFGF) for 24 hours,the preinduction medium was removed,MSCs were washed with phosphate buffer saline (PBS) for 3 times,and then the cells were transferred to the neuronal induction medium composed of 50 μg/L NGF for 24 hours; negative control group: they were cultured in the serum free medium. The changes of morphology were observed after induction. ④After induction,expression and differentiation rate of neuronspecific enolase (NSE),microtubule-associated protein (MAP) and glial fibrillary acidic protein (GFAP) were detected with immunocytochemical method. RESULTS: ①The proliferation curve of MSCs: the first 1-4 days after cultivation was latent period,and then MSCs proliferated rapidly and reached the exponential phase of growth. The number of MSCs reached the peak time at the 9th of culture and since then the proliferation decreased and entered flatform phase. The double time of MSCs was about 32 hours. ②The morphologies of MSCs were observed after induction: The β-mercaptoethanol induction scheme: 1 hour after adding inductive agent,the form of MSCs changed,cell body contracted,the spherical or taper cells were found with strong refraction. Partial cells had halation with many long processes. The neuron-like cells obviously increased at hour 5,and the MSCs became net structure,but death cells were increasing. The NGF induction scheme: The morphologies were found at hour 12,showing large cell body and similar thin long processes of axon and dendrite,and obviously at hour 24,partial cells were with net-shaped arrangement,but death cells were few. The negative control induction scheme: no markedly morphological changes,still were long spindle cells. ③The immunohistochemical assay result of neuron-like cells: The differentiated neuron-like cells in the β-mercaptoethanol group and NGF group were NSE and MAP staining positive,showing buffy cell body and process,but negative in GFAP. No positive result appeared in the negative control group. The differentiation rate of NSE and MAP positive cells in the β-mercaptoethanol group were(71.2±3.8)% and(64.1±2.7)%,respectively. The rate of NSE and MAP positive cells in the NGF group were (46.2±2.5)% and (42.9±1.9),respectively. The difference among two groups were significant (P < 0.05,t =-5.205).CONCLUSION: β-mercaptoethanol and NGF can successfully induce the differentiation of MSCs into neuron-like cells.更多还原