Lu Meng, Dong Jun, Lu Teng, Lv Hongjun, Yang Pinglin, Cheng Zhijian, Li Jin, Liang Baobao, Xu Junkui, Li Haopeng, He Xijing
Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, 157 West Five Road, Xincheng District, Xi'an 710004, Shaanxi, China.
Department of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Yanta West Road, No. 277, Xi'an 710061, China.
Int J Mol Sci. 2014 Dec 26;16(1):420-38. doi: 10.3390/ijms16010420.
Transplantation of olfactory ensheathing cells (OEC) is a promising therapy in spinal cord injury (SCI) treatment. However, the therapeutic efficacy of this method is unstable due to unknown reasons. Considering the alterations in the culture environment that occur during OEC preparation for transplantation, we hypothesize that these changes may cause variations in the curative effects of this method. In this study, we compared OEC cultured in medium containing different types and concentrations of serum. After purification and passage, the OEC were cultured for 7 days in different media containing 5%, 10%, 15% or 20% fetal bovine serum (FBS) or rat serum (RS), or the cells were cultured in FBS-containing medium first, followed by medium containing RS. In another group, the OEC were first cultured in 10% FBS for 3 days and then cultured with rat spinal cord explants with 10% RS for another 4 days. An MTT assay and P75 neurotrophin receptor immunofluorescence staining were used to examine cell viability and OEC numbers, respectively. The concentration of neurotrophin-3 (NT-3), which is secreted by OEC into the culture supernatant, was detected using the enzyme-linked immunosorbent assay (ELISA). RT-PCR was applied to investigate the NT-3 gene expression in OEC according to different groups. Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01). OEC cultured with spinal cord explants secreted less NT-3 than OEC cultured alone (F = 9.611, df = 5.139, p < 0.01). Meanwhile, the order of application of different sera was not influential. There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01). In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.
嗅鞘细胞（OEC）移植是脊髓损伤（SCI）治疗中一种很有前景的疗法。然而，由于未知原因，该方法的治疗效果不稳定。考虑到在制备移植用OEC过程中培养环境的变化，我们推测这些变化可能导致该方法疗效的差异。在本研究中，我们比较了在含有不同类型和浓度血清的培养基中培养的OEC。纯化传代后，将OEC在含有5%、10%、15%或20%胎牛血清（FBS）或大鼠血清（RS）的不同培养基中培养7天，或者先将细胞在含FBS的培养基中培养，然后在含RS的培养基中培养。在另一组中，OEC先在10% FBS中培养3天，然后再与含10% RS的大鼠脊髓外植体一起培养4天。分别采用MTT法和P75神经营养因子受体免疫荧光染色检测细胞活力和OEC数量。使用酶联免疫吸附测定（ELISA）检测OEC分泌到培养上清液中的神经营养因子-3（NT-3）浓度。应用RT-PCR研究不同组OEC中NT-3基因的表达。与FBS相比，RS降低了OEC增殖相关的OEC计数（χ2 = 166.279，自由度 = 1，p < 0.01）、MTT法中的光密度（OD）值（χ2 = 34.730，自由度 = 1，p < 0.01）以及上清液中NT-3浓度（χ2 = 242.997，自由度 = 1，p < 0.01）。与单独培养的OEC相比，与脊髓外植体一起培养的OEC分泌的NT-3较少（F = 9.611，自由度 = 5.139，p < 0.01）。同时，不同血清的应用顺序没有影响。当血清浓度为15%时，不同组间NT-3基因表达存在统计学显著差异（χ2 = 64.347，自由度 = 1，p < 0.01）。总之，不同的血清条件可能导致OEC增殖和功能的差异。
