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目的观察肌萎缩侧索硬化患者脑脊液(cerebrospinal fluid,CSF)对大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)增殖分化的影响,探讨MSCs经CSF内移植治疗肌萎缩侧索硬化(amyotro-phic lateral sclerosis,ALS)的启动机制。方法通过密度梯度离心联合贴壁培养法分离培养大鼠MSCs,取生长状态良好的第3代细胞,按0.5×105cells/孔的浓度接种于24孔培养板中,分别加入ALS患者CSF、病例对照组CSF以及和等量条件培养液(空白对照)共培养7 d。MTT法分别检测共培养1 d、4 d、7 d后的MSCs增殖能力,免疫荧光法检测神经细胞特异性表面标志物巢蛋白(Nestin)和神经元特异性烯醇化酶(neuron-specific enolase,NSE)的表达水平。结果 (1)MSCs共培养1 d后,各组细胞形态学无明显变化;4 d后,ALS患者组部分细胞开始收缩并出现细胞突起等形态学变化,病例对照组可见少量细胞出现细胞突起等形态学变化,空白对照组细胞仍无明显形态学变化;7 d后ALS患者组及病例对照组突起细胞数较前明显增多,空白对照组仅可见少量突起细胞;(2)ALS患者组及病例对照组1、4、7 d的细胞平均吸光度值(A)明显高于空白对照组(P<0.01),但ALS患者组及病例对照组无明显差异(P>0.05);(3)MSCs共培养7 d后,ALS患者组和病例对照组Nestin、NSE染色均有阳性细胞表达,而空白对照组仅有极少量表达;ALS患者组Nestin及NSE染色阳性率均显著高于病例对照组(P<0.05)。结论 MSCs分化需要一定的微环境,ALS患者CSF能诱导其分化成为神经元样细胞,可能在MSCs定向分化和神经组织修复机制的启动环节中起作用。
Objective To observe the effect of cerebrospinal fluid (CSF) on the proliferation and differentiation of rat mesenchymal stem cells (MSCs) after amyotrophic lateral sclerosis (ALS) and to investigate the effect of amyotro- phic lateral sclerosis, ALS). Methods Rat MSCs were isolated and cultured by density gradient centrifugation and adherent culture. The third generation cells with good growth status were seeded into 24-well plates at the concentration of 0.5 × 105cells / well. The CSF of ALS patients and the control Group CSF and the same amount of conditioned medium (blank control) co-cultured for 7 days. The proliferation of MSCs was detected by MTT assay at 1 d, 4 d and 7 d after co-culture. Nestin and neuron-specific enolase were detected by immunofluorescence. NSE) expression levels. RESULTS: (1) After 1 day of MSCs co-culture, there was no significant change in morphology of cells in each group. After 4 days, some cells in ALS group began to shrink and showed morphological changes such as cell protrusion. In the control group, a few cells showed cell protrusion Morphological changes, the blank control group cells still no significant morphological changes; 7 days after ALS patients and cases control group significantly increased the number of prominent cells, blank control group only a small amount of protruding cells; (2) ALS patients and cases The average cell absorbance value (A) of the control group at 1, 4 and 7 d was significantly higher than that of the blank control group (P <0.01), but there was no significant difference between the ALS group and the case control group (P> 0.05). (3) After cultured for 7 days, the expression of Nestin and NSE in ALS patients and NSCs was positive in NSE staining, but only in small amount in control group. The positive rates of Nestin and NSE in ALS patients were significantly higher than those in control group (P <0.05). Conclusion MSCs differentiation requires a certain microenvironment. CSF of ALS patients can induce their differentiation into neuron-like cells, which may play a role in the initiation of MSCs differentiation and neural repair mechanism.