人脐带间充质干细胞中神经营养因子的表达

Expression of neurotrophic factors in human umbilical cord-derived mesenchymal stromal cells

目的通过检测人脐带间充质干细胞（hUE．MSCs）的神经营养因子谱，以进一步认识hUC—MSCs通过分泌神经营养因子治疗神经损伤的作用。方法组织块培养法分离培养hUC-MSCs．流式细胞仪鉴定干细胞表面标记物，Raybio人细胞因子抗体芯片检测hUC-MSCs神经营养因子表达情况，RT—PCR检测hUC．MSCs神经营养因子的基因表达，ELISA法定量检测hUC．MSCs上清液中分泌的脑源性神经营养因子（BDNF）、胶质细胞源性神经营养因子（GDNF）、肝细胞生长因子（HGF）、神经营养因子3（NT．3）含量。结果流式细胞分析结果显示hUC—MSCs表面标记物CD29、CD44、CD90阳性（94．05％、90．75％、98．12％），CD34、CD45阴性（3．09％、0．80％）。Raybio人细胞因子抗体芯片结果显示，与神经营养相关的因子BDNF、表皮生长因子（EGF）、GDNF、HGF、NT-3、血管内皮细胞生长因子（VEGF）、胰岛素样生长因子．I（IGF一1）、神经营养因子4（NT．4）在hUC．MSCs中均有表达。RT．PCR检验也证实，GnJ、VEGF、神经生长因子（NGF）、HGF、GDNF、NT-4、BDⅣF和Ⅳ卜3基因在hUC—MSCs表达。ELISA检测细胞上清液中BDNF、GDNF、HGF、NT．3的含量分别为（475．20±32．22）pg／mL、（82．33±3．39）pg／mL、（704．50±12．86）pg／mL、（230．41±16．66）pg／mL，分别与空白对照组相比，差异均有统计学意义（P〈0．05）。结论hUC．MSCs富含神经营养因子，是一种理想的用于神经损伤治疗的种子细胞。

Objective To observe the profile of neurotrophic factors in human umbilical cord-derived mesenchymal stromal cells （hUC-MSCs） with Raybio antibody chip system, and provide a new cell seed source for spine cord injury or peripheral nerve regeneration. Methods The hUC-MSCs were cultured by tissue culture. Characteristics of cell phenotype were confirmed by flow cytometry. Raybio human cytokine antibody array and RT-PCR were employed to detect the neurotropic factors in hUC-MSCs. ELISA was used to determine the contents of growth factors （brain-derived neurotrophic factor [BDNF], glial cell line-derived neurotrophic factor [GDNF], hepatocyte growth factor [HGF], neurotrophin-3 [NT-3]） secreted by hUC-MSCs. Results The hUC-MSCs showed positive results for CD29 （94.05%）, CD44 （90.75%） and CD90 （98.12%）, and negative results for CD34 （3.09%） and CD45 （0.80%）. Array results showed that hUC-MSCs expressed BDNF, epidermal growth factor （EGF）, GDNF, HGF and NT-3, vascular endothelial growth factor （VEGF）, insulin-like growth factor 1 （IGF-1） and NT-4. The gene expressions of IGF-1, VEGF, nerve growth factor （NGF）, HGF, GDNF, NT-4, BDNF and NT-3 were further verified by RT-PCR. ELISA results showed that the contents of BDNF, GDNF, HGF and NT-3 in the conditioned medium were （475.20±32.22） pg/mL, （82.33±3.39） pg/mL, （704.50±12.86） pg/mL, and （230.41 ±16.66） pg/mL, respectively. These were obviously higher than that in the controlgroup. Conclusion The hUC-MSCs, containing plenty of growth factors, especially neurotrophic factors, may be promising seed cells for spine cord injury and nerve regeneration.