肌萎缩侧索硬化患者脑脊液对间充质干细胞增殖分化的影响

The Effect of the cerebrospinal fluid of amyotrophic lateral sclerosis patients on the proliferation and differentiation of bone marrow mesenchymal stem cells

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 study the effect of the cerebrospinal fluid （CSF） from amyotrophic lateral sclerosis （ALS） patients on the proliferation and differentiation of bone marrow mesenchymal stem cells （MSCs） in rat, and explore the activated mechanism of MSCs transplantation through CSF for treating ALS. Methods Rat MSCs were isolated by density gradient centrifugation joint adherent culture. The marrow cells of third generation were collected, inoculated in 24-well culture plates with concentration of 0.5×10^5 cells / hole, added the CSF of patients with ALS and case-control, and equal amount of conditioned medium （blank control）, and co-cultured for 7 days. The proliferation of MSCs was detected by MTT assay after 1, 4 and 7 days of co-culture, respectively. The expression levels of neural cell-specific surface markers nestin （Nestin） and neuron-specific enolase （NSE） were detected by immunofluorescence. Results （1）No significant change of cell morphology was found in all three groups after one day of co-culture with MSCs. 4 days later, some cells in ALS patient group began to shrink and morphological changes such as cell protrusion occurred, a small amount of cell protrusions were observed in the case-control group, whereas there was still no morphological change in the blank control group. 7 days later, the number of cell protrusion increased significantly in the groups of ALS patient and case-control group, whereas only a small amount of protruding cells was found in the blank control group. （2）The mean absorbance value （A） of cells by MTT assay in the groups of AIrS pa- tient and case-control was higher significantly than that in the blank control group after 1, 4 and 7 days of co- culture with MSCs （P〈0. 01）. （3）There were positive expressed cells by Nestin and NSE staining both in the groups of ALS patient and ease-control, whereas there was only a very small amount of expression in the blank control group after 7 days of co-culture with MSCs; and the rate of positive staining by Nestin and NSE in the groups of ALS patient and case-control was higher significantly than that in the blank control group （P〈O. 05）. Conclusions MSCs differentiation requires a certain micro-environment, and the CSF of ALS patients was able to induce their differentiation into neuron-like cells, which may play a role in the initial steps of the directed differentiation of MSCs and nerve tissue repair mechanisms.