体外骨髓间充质干细胞对脂多糖活化的视网膜小胶质细胞生物学功能的影响

背景 视网膜小胶质细胞(RMG)的活化在视网膜变性疾病的发病过程中发挥重要作用,趋化因子CX3C模体配体1(CX3CL1)参与小胶质细胞稳态的调节.骨髓间充质干细胞(BMSCs)可通过旁分泌的方式释放可溶性因子,保护中枢神经系统组织细胞的生物功能,但其对治疗视网膜变性疾病的途径和靶细胞是否为RMG尚不清楚. 目的 观察BMSCs对脂多糖(LPS)活化的RMG生物学功能的影响,探讨CX3CL1/CX3CR1信号通路对二者相互作用的影响.方法 采用视网膜胶质细胞混合培养和振荡分离的方法分离培养SD大鼠RMG,采用免疫荧光染色法观察细胞中CD11b、Iba1和谷氨酰胺合成酶(GS)的表达以鉴定培养的RMG.在细胞培养液中添加1 mg/ml的LPS液2μl以刺激RMG 24 h,然后将细胞分为LPS对照组、BMSCs组和CB-BMSCs组,其中BMSCs组将RMG与BMSCs共培养24 h,CB-BMSCs组将RMG与中和性抗体封闭CX3CL1的BMSCs共培养24 h,未予LPS刺激的RMG作为空白对照组.采用ELISA法检测共培养体系中RMG分泌肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的变化;采用EdU法观察各组RMG的增生能力;采用流式细胞术检测RMG吞噬荧光微球后的平均荧光强度(MFI);利用Transwell小室试验检测RMG的迁移细胞数.结果 用视网膜胶质细胞混合培养和振荡分离的方法成功分离和培养出RMG,细胞中CD11b和Iba1阳性表达呈绿色荧光,GS呈阴性表达.空白对照组、LPS对照组、BMSCs组和CB-BMSCs组细胞上清液中TNF-α的分泌量分别为(2.55±0.97)、(24.91±3.07)、(20.38±2.97)和(24.90±1.88) ng/ml,总体比较差异有统计学意义(F=119.90,P＜0.05);IL-1β的分泌量分别为(1.12±0.36)、(10.40±2.76)、(7.00±1.75)和(9.55±1.11)ng/ml,总体比较差异有统计学意义(F=34.96,P＜0.05);其中BMSCs组TNF-α和IL-1 β分泌量均低于LPS对照组(均P＜0.05),而CB-BMSCs组与LPS对照组间TNF-α和IL-1β分泌量的差异均无统计学意义(均P＞0.05).各组间RMG增生率的总体比较差异有统计学意义(F=42.94,P＜0.05),其中BMSCs组RMG增生率明显低于LPS对照组,差异有统计学意义(P＜0.05),而BMSCs组与CB-BMSCs组间差异无统计学意义(P＞0.05).各组间RMG的MFI值和迁移细胞数量的总体比较差异均有统计学意义(F=70.55、15.49,均P＜0.05),其中BMSCs组MFI值和迁移细胞数量均明显高于LPS对照组,差异均有统计学意义(均P＜0.05),而CB-BMSCs组与LPS对照组间MFI值和迁移细胞数量的比较差异无统计学意义(均P＞0.05).结论 BMSCs能够抑制LPS活化的RMG的增生能力,可能通过CX3CL1/CX3CR1信号通路来抑制活化的RMG分泌促炎性因子,并增强RMG的吞噬和迁移能力.

In vitro effects of bone marrow-derived mesenchymal stem cells on the biological behavior of lipopolysaccharide-activated retinal microglia

Background Retinal microglia (RMG) plays an important role in the pathogenesis of retinal degenerative diseases,while chemokine CX3CL1 participates in the regulation of steady-state of microglia.It has been determined that bone marrow-derived mesenchymal stem cells (BMSCs) have a remarkable role to modulate the immune response and protect the central nervous system through the release of soluble factors in a paracrine fashion and further affect the functional behavior of cells.However,whether BMSCs are able to interact with RMG and activate related signaling pathway for the maintaining of homeostasis in the retina is still unclear.Objective The aim of this study was to investigate the interaction between BMSCs and lipopolysaccharide (LPS)-activated RMG in vitro,and dissect the effects of CX3CL1/CX3CR1 signaling pathway on the biological behavior of BMSCs and RMG.Methods RMG was isolated from SD rats,cultured with mixed culture of retinal glial cells and purified by shaking.The cells were identified by detecting the expression of CD111b,Iba1 and glutamamine synthetase (GS) with indirect immunofluorescence assay.LPS (1 mg/ml,2 μl) was added in the medium for 24 hours to stimulate RMG,and then the cells were divided into LPS control group,BMSCs group (cocultured with BMSCs for 24 hours) and CB-BMSCs group (cocultured with CX3CL1-blocking-BMSCs for 24 hours).The cells without LPS stimulation served as the blank control group.The functions of RMG,including the release content of tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β),the proliferation,phagocytosis,and migration of RMG were examined.Results RMG was successfully isolated and harvested from SD rats by using mixed culture of retinal glial cells and purified by shaking.CD11b and Iba1 showed the positive expression with the green fluorescence in the cells and GS was absent.The contents of TNF-αt in the cell supernatant were (2.55 ±0.97) ng/ml,(24.91 ±3.07) ng/ml,(20.38 ±2.97) ng/ml and (24.90 ± 1.88) ng/ml in the blank control group,LPS control group,BMSCs group and CB-BMSCs group,respectively,showing a significant difference among the groups (F=119.90,P＜0.05).The contents of IL-1 β in the cell supernatant were (1.12±0.36) ng/ml,(10.40±2.76) ng/ml,(7.00± 1.75) ng/ml and (9.55 ± 1.11) ng/ml in the blank control group,LPS control group,BMSCs group and CB-BMSCs group,respectively,showing a significant difference among the groups(F =34.96,P＜0.05).The secretory volume of TNF-α and IL-1 β were evidently lower in the BMSCs group than those in the LPS control group (both at P＜0.05),and no significant differences were found in the secretory volume of TNF-α and IL-1β between CB-BMSCs group and LPS control group (both at P＞0.05).The proliferative rate of RMG was lower in the BMSCs group than that in the LPS control group (P＜0.05),while there was no statistical difference between BMSCs group and CB-BMSCs group (P＞0.05).The mean fluorescence intensity (MFI) and the number of migrated RMG were considerably different among the four groups (F=70.55,15.49,both at P＜0.05),and those in the BMSCs group were significantly increased in comparison with the LPS control group (both at P＜0.05),while there was no significant difference between CB-BMSCs group and LPS control group (both at P＞0.05).Conclusions BMSCs could suppress the proliferation of LPS-activated RMG.Moreover,BMSCs might inhibit proinflammatory cytokines releasing,enhance phagocytosis and migration capabilities of RMG via CX3CL1/CX3CR1 signaling pathway.