Co-culture with dental stem cells ameliorates neuronal apoptosis induced by 1-methyl-4-phenyl pyridinium

doi:10.3969/j.issn.2095-4344.0524

Abstract

Abstract:

BACKGROUND: How to ameliorate neural injury by dental pulp stem cells is of clinical benefit for neurodegenerative diseases.
OBJECTIVE: To investigate the effect of co-culture with dental pulp stem cells (DPSCs) on 1-methyl-4-phenyl pyridinium (MPP⁺)-induced apoptosis of neurons.
METHODS: Human DPSCs were isolated and identified by morphological observation, adipogenic and osteogenic differentiation. Rat midbrain neurons were isolated and co-cultured with DPSCs by Transwell. After treatment with MPP⁺ for 48 hours, the neurons were observed in morphology, and the effect of DPSCs on neuronal apoptosis induced by MPP⁺ was detected by western blot and TUNEL staining.
RESULTS AND CONCLUSION: DPSCs were spindle-like cells capable of differentiating into adipogenesis and osteogenesis. The apoptosis of neurons induced by MPP⁺ were alleviated via co-culture with DPSCs. Further detection by western blot showed that the neurons treated with MPP⁺ reduced the expression of Bcl-2 and promoted the expression of Bax and cleaved caspase3; however, the changes in the expression of these proteins were reduced under the co-culture system of DPSCs. All the above results reveal that DPSCs may repair or prevent neuronal injury induced by MPP⁺ through paracrine actions.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Dental Pulp, Stem Cells, Neurons, Coculture Techniques, 1-Methyl-4-phenylpyridinium, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Zhu Hong-qian. Co-culture with dental stem cells ameliorates neuronal apoptosis induced by 1-methyl-4-phenyl pyridinium[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2727-2732.

Figures/Tables 1

2.1 牙髓干细胞的分离和鉴定结果倒置显微镜下观察牙髓干细胞为成纤维细胞样生长(图1A)。为了进一步确定分离细胞即为牙髓干细胞，进行了成脂和成骨诱导以验证其多能性，经成骨诱导培养液培养后茜素红染色呈阳性(图1B)；经成脂诱导培养液培养后油红O染色呈阳性(图1C)。 2.2 牙髓干细胞共培养可减轻MPP+对神经元的损伤为观察牙髓干细胞对MPP+所致神经元的氧化应激损伤是否具有保护作用，采用Transwell小室将牙髓干细胞与中脑神经元进行共培养，随后使用MPP+损伤中脑神经元以建立帕金森病细胞模型。未经处理的中脑神经元(对照组)形态饱满，细胞突起连续并不断延伸，生长状态良好(图2A)；MPP+组细胞胞体大量萎缩、碎裂，细胞突起断裂消失，大量细胞死亡，状态极差(图2B)；MPP++牙髓干细胞组细胞的胞体饱满度、形态和突起、存活情况较MPP+组明显改善(图2C)。进一步的MTT结果显示，原代中脑神经元经MPP+处理后其细胞存活数较对照组显著降低，差异有显著性意义(P < 0.05，图2D)；MPP++牙髓干细胞组细胞存活数较单纯MPP+组显著提高，差异有显著性意义(P < 0.05，图2D)。以上结果一致提示，牙髓干细胞共培养可有效减轻MPP+对中脑神经元的损伤而起到保护神经元的作用。 2.3 牙髓干细胞共培养对MPP+诱导的神经元凋亡的影响为进一步明确牙髓干细胞对MPP+诱导的神经元损伤的影响，进一步检测和观察了牙髓干细胞对MPP+诱导的神经元凋亡的影响。正常培养的对照组神经元仅有极少量细胞出现凋亡情况(图3A-C)；经MPP+处理之后，MPP+组神经元的凋亡较对照组显著增加(图3D-F)；相反，与牙髓干细胞预先共培养的MPP++牙髓干细胞组细胞凋亡则较MPP+组显著减少(图3G-I)。统计结果(图3J)显示，MPP+组细胞凋亡比例较对照组显著升高(P < 0.05)；而MPP++牙髓干细胞组细胞凋亡比例则较MPP+组显著降低(P < 0.05)；以上结果提示牙髓干细胞可显著减轻MPP+诱导的神经元凋亡。 2.4 牙髓干细胞共培养对神经元凋亡相关蛋白表达的影响为深入探索牙髓干细胞减轻MPP+诱导的神经元凋亡的分子机制，进一步检测了各组细胞中Bax、Bcl-2、Cleaved caspase3和Casepase3等凋亡相关蛋白的表达变化。经MPP+处理之后，MPP+组的促凋亡蛋白Bax表达较对照组明显升高，而保护性蛋白Bcl-2的表达则显著下调，二者间差异有显著性意义(P < 0.05，图4A，B)；与牙髓干细胞预先共培养后，MPP++牙髓干细胞组的促凋亡蛋白Bax表达较MPP+组明显降低，而保护性蛋白Bcl-2的表达则显著上调，两组间差异有显著性意义(P < 0.05，图4 A，B)；Cleaved caspase3被认为是凋亡的标志性蛋白，经MPP+处理之后，MPP+组的促凋亡蛋白Cleaved caspase3表达较对照组明显升高(P < 0.05，图4C，D)，与牙髓干细胞预先共培养后，MPP++牙髓干细胞组细胞Cleaved caspase3表达较MPP+组明显降低(P < 0.05，图4C，D)。以上结果提示牙髓干细胞很可能是通过凋亡相关蛋白来减轻MPP+诱导的神经元凋亡。"

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