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目的研究雪旺细胞(Schwann cells,SCs)对BMSCs来源内皮细胞分泌一氧化氮(nitric oxide,NO)功能的影响,为进一步探究组织工程骨成骨过程中神经因素对血管因素的作用奠定实验基础。方法取1日龄SD乳鼠坐骨神经及臂丛神经,体外分离获取SCs,采用S-100免疫组织化学染色鉴定;取2周龄SD大鼠胫骨及股骨骨髓,体外分离获取BMSCs,诱导分化成为内皮细胞,采用血管性血友病因子与CD31免疫荧光染色鉴定。采用Transwell共培养板对SCs与BMSCs来源内皮细胞进行非接触共培养作为实验组,单纯培养BMSCs来源内皮细胞作为对照组,分别于1、3、5、7 d检测培养液中NO浓度,1、3、7、10 d采用实时荧光定量PCR(real-time fl uorescence quantitative PCR,RT-qPCR)检测一氧化氮合酶2(nitric oxide synthetase 2,NOS2)、NOS3 mRNA表达。结果分离及诱导分化的细胞通过形态学及免疫组织化学和免疫荧光染色鉴定为SCs和内皮细胞。实验组及对照组内各时间点间释放NO浓度比较,差异均有统计学意义(P<0.05);除3 d外,其余各时间点实验组释放NO浓度均显著高于对照组(P<0.05)。RT-qPCR检测结果示,培养各时间点实验组NOS2 mRNA相对表达量均显著高于对照组(P<0.05);两组内各时间点间比较差异均有统计学意义(P<0.05)。除10 d外,其余各时间点实验组NOS3 mRNA相对表达量均显著高于对照组(P<0.05);实验组内各时间点间NOS3 mRNA相对表达量比较差异均无统计学意义(F=6.673,P=0.062),对照组内各时间点间比较差异均有统计学意义(F=36.581,P=0.000)。结论 SCs可促进BMSCs来源内皮细胞释放NO,可能与SCs促进NOS活性有关。
Objective To study the effect of Schwann cells (SCs) on the secretion of nitric oxide (NO) from endothelial cells derived from BMSCs, and lay a foundation for further exploration of the role of neurological factors in the process of osteogenesis of tissue engineered bone . Methods The sciatic nerve and brachial plexus of SD suckling rats were collected at 1 day old. SCs were isolated and identified by S-100 immunohistochemical staining. BMSCs were obtained from the tibia and femur bone marrow of 2-week-old SD rats and were induced to differentiate into endothelial Cells were identified by von Willebrand factor and CD31 immunofluorescence staining. Transwell co-culture plates were used for non-contact co-culture of SCs and BMSCs derived endothelial cells as experimental group. BMSCs-derived endothelial cells were cultured as control group. The concentrations of NO, The expression of nitric oxide synthetase 2 (NOS2) and NOS3 mRNA were detected by real-time fl uorescence quantitative PCR (RT-qPCR) at 3, 7 and 10 days. Results Isolated and differentiated cells were identified as SCs and endothelial cells by morphological and immunohistochemical staining and immunofluorescence staining. The concentrations of NO released in experimental group and control group at different time points were significantly different (P <0.05). Except for 3 days, NO concentration in experimental group was significantly higher than that in control group (P < 0.05). The results of RT-qPCR showed that the relative expression of NOS2 mRNA in experimental group was significantly higher than that in control group at each time point (P <0.05). There was significant difference between the two groups in time points (P <0.05). Except for 10 days, the relative expression of NOS3 mRNA in experimental group was significantly higher than that in control group at each time point (P <0.05). There was no significant difference in the relative expression of NOS3 mRNA among experimental groups at each time point (F = 6.673, P = 0.062). There were significant differences among the control groups at various time points (F = 36.581, P = 0.000). Conclusion SCs can promote the release of NO from BMSCs-derived endothelial cells, which may be related to the promotion of NOS activity by SCs.