| 李征亚,邹秀兰,彭亮红,张 楚,周文杰,陈轩阁,邹玉平.体外共培养模式下神经干细胞对视网膜小胶质细胞生物学功能的影响[J].,2020,(10):1829-1834 |
| 体外共培养模式下神经干细胞对视网膜小胶质细胞生物学功能的影响 |
| Effects of Neural Stem Cells on the Biological Behavior of Retinal Microglia after Co-culture in Vitro |
| 投稿时间：2019-10-29 修订日期：2019-11-25 |
| DOI：10.13241/j.cnki.pmb.2020.10.007 |
| 中文关键词: 视网膜小胶质细胞 神经干细胞 体外共培养 免疫调控作用 |
| 英文关键词: Retinal microglia Neural stem cells Co-culture in vitro Immunomodulation |
| 基金项目:广东省医学科学技术研究基金项目(A2018372) |
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| 中文摘要: |
| 摘要 目的：通过构建体外共培养体系，探讨神经干细胞(NSCs)对脂多糖(LPS)活化后的视网膜小胶质细胞(RMG)生物学功能的影响及TIMP1/MMP9途径在其中的可能作用。方法：采用振荡分离法获取C57/BL6小鼠原代RMG，通过免疫荧光技术检测细胞Iba1的表达对其进行鉴定。采用含有LPS的培养基(终浓度为1 μg?mL-1)刺激RMG 24h后，将其分为LPS对照组、NSCs组、TB-NSCs组，其中NSCs组将RMG与NSCs共培养24 h，TB-NSCs组将RMG与用中和性抗体封闭TIMP1的NSCs共培养24h；同时，未予以LPS刺激的RMG作为空白对照组。采用免疫荧光技术检测各组RMG的Ki67表达情况，观察其增殖能力；TUNEL技术检测各组RMG凋亡情况；ELISA方法检测各组RMG上清液中TNF-α、IL-1β的蛋白质量浓度。结果：采用振荡分离法获取的原代RMG经免疫荧光染色鉴定Iba1呈阳性。NSCs组Ki67阳性率较LPS对照组降低(P＜0.05)，而TB-NSCs组Ki67阳性率较NSCs组升高(P＜0.05)。NSCs组TUNEL阳性率较LPS对照组明显升高(P＜0.05)，而TB-NSCs组TUNEL阳性率与NSCs组间差异无统计学意义(P＞0.05)。空白对照组、LPS对照组、NSCs组、TB-NSCs组RMG上清液中TNF-α蛋白质量浓度分别为(2.10±0.65)、(25.69±2.01)、(20.01±1.63)、(23.76±1.45)ng?mL-1，总体比较差异显著(F TNF-α =302.65，P TNF-α＜0.05)；IL-1β蛋白质量浓度分别为(1.77±0.74)、(15.38±1.18)、(10.88±0.95)、(13.45±1.41)ng?mL-1，总体比较差异非常显著(F IL-1β =179.84，P IL-1β＜0.05)；其中，NSCs组TNF-α及IL-1β蛋白质量浓度均较LPS对照组显著降低(P＜0.05)，TB-NSCs组TNF-α及IL-1β蛋白质量浓度较NSCs组明显升高(P＜0.05)。结论：体外共培养模式下，NSCs可抑制RMG增殖能力，提高其凋亡水平，并抑制其分泌促炎因子TNF-α及IL-1β，该效应可能与调控TIMP1/MMP9相关。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the biological effects of NSCs on lipopolysaccharide (LPS)-activated RMG after co-culture in vitro, and the signal role played by TIMP1/MMP9 on this interaction. Methods: Primary RMG were isolated from C57/BL6 mice and purified by shaking methods. Immunofluorescence technique was applied to detect the expression of Iba1 to identify microglia. RMG from three groups including LPS control group, NSCs group (co-cultured with NSCs for 24 hours) and TB-NSCs group (co-cultured with TIMP1-blocking-NSCs for 24 hours) were pre-stimulated by LPS (1 μg?mL-1) medium for 24 hours. The cells without LPS stimulation were blank control group. The proliferation status of RMG (Ki67 positive rate) was investigated by immunofluorescence technique and the apoptosis statue was investigated by TUNEL. The release contents including tumor necrosis factor-α(TNF-α) and interleukin-1β (IL-1β) of RMG were detected by ELISA. Results: Primary RMG, which were successfully purified by shaking method, were mostly Iba1 positive. The percentage of Ki67 positive cells was significantly different among the four groups (F=211.26, P＜0.05). That of NSCs group was lower than that of LPS control group (P＜0.05), while that of TB-NSCs group was higher than that of NSCs group (P＜0.05). The percentage of TUNEL positive cells was also significantly different among the four groups (F=98.65, P＜0.05). That of NSCs group was higher than that of LPS control group (P＜0.05), while there was no statistical difference between TB-NSCs group and NSCs group (P＞0.05). The contents of TNF-α in RMG supernatant from blank group, LPS control group, NSCs group, and TB-NSCs group were respectively (2.10±0.65) ng?mL-1, (25.69±2.01) ng?mL-1, (20.01±1.63) ng?mL-1 and (23.76±1.45) ng?mL-1, which were statistically different (F TNF-α =302.65, P TNF-α＜0.05). The contents of IL-1β in RMG supernatant from blank group, LPS control group, NSCs group, and TB-NSCs group were respectively (1.77±0.74) ng?mL-1, (15.38±1.18) ng?mL-1, (10.88±0.95) ng?mL-1 and (13.45±1.41)ng?mL-1, which were also statistically different (F IL-1β =179.84，P IL-1β＜0.05). The contents of TNF-α and IL-1β in NSCs group were both lower than that in LPS control group (P＜0.05), while those in TB-NSCs group were both higher than those in NSCs group (P＜0.05). Conclusion: NSCs might suppress proliferation ability, promote apoptosis level and inhibit proinflammatory cytokines releasing of LPS-activated RMG through TIMP1/MMP9 signal. |
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