中文摘要：

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1mg／ml的LPS液2μl以刺激RMG24h，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24h，CB—BMSCs组将RMG与中和性抗体封闭CX3CL1的BMSCs共培养24h，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-1β分泌量均低

英文摘要：

Background Retinal microglia （RMG） plays an important role in the pathogenesis of retinal degenerative diseases,while ehemokine 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 CD11b,Ibal 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-1β （ 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. CDllb and Ibal showed the positive expression with the green fluorescence in the ceils and GS was absent. The contents of TNF-α in the cell supernatant were （2. 55 ±0. 97 ） ng/ml, （24.91 ±3.07 ） ng/ml, （20.38 ±2.97 ）