中国修复重建外科杂志

中国修复重建外科杂志

BMSCs 条件培养液诱导小胶质细胞分泌精氨酸酶 1 的初步研究

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目的 初步探讨 BMSCs 条件培养液对小胶质细胞(microglia,MGs)激活及其分泌精氨酸酶 1(arginase 1,Arg1)的影响。 方法 取 4 周龄 SD 大鼠股骨和胫骨骨髓,采用差速贴壁法分离培养原代 BMSCs,行 Vimentin 免疫荧光染色鉴定。取新生 3 日内 SD 大鼠全脑,采用胰蛋白酶消化法分离培养原代 MGs,行 Iba1 免疫荧光染色鉴定。收集对数生长期 BMSCs 培养液制备 BMSCs 条件培养液,取原代 MGs,分别使用含 BMSCs 条件培养液的 DMEM/F12 培养基(实验组)和普通 DMEM/F12 培养基(对照组)培养。培养 48 h 后采用倒置相差显微镜观察 MGs 的形态改变;Iba1 免疫荧光染色观察 MGs 激活状态;Iba1 和 Arg1 免疫荧光双标染色及 Western blot 检测 MGs 中 Arg1 的表达。 结果 倒置相差显微镜观察示 BMSCs 约 14 d 进入对数生长期,Vimentin 免疫荧光染色结果示 98% 以上细胞呈阳性反应;MGs 约 21 d 进入对数生长期,Iba1免疫荧光染色结果示约 80% 细胞呈阳性反应。倒置相差显微镜观察示实验组 MGs 被激活,激活后的 MGs 胞体增大、突起变短、呈阿米巴样变化。免疫荧光染色示实验组 Iba1 阳性细胞显著多于对照组,比较差异有统计学意义(t=0.007,P=0.000);免疫荧光双标染色示实验组 Iba1 和 Arg1 双阳性细胞显著多于对照组,比较差异有统计学意义(t=0.007,P=0.000);Western blot 示实验组 Arg1 蛋白相对表达量显著高于对照组,比较差异有统计学意义(t=0.001,P=0.000)。 结论 BMSCs 条件培养液可激活 MGs,并促进 MGs 表达 Arg1。

Objective To observe the effect of bone marrow mesenchymal stem cells (BMSCs) conditioned medium on microglia (MGs) and its secretion of arginase 1 (Arg1). Methods The BMSCs separated through differential adhesion method from the femur and tibia marrow of 4-week-old Sprague Dawley (SD) rats were cultured and identified by Vimentin immunofluorescence staining; whereas MGs separated through trypsin digestion method from the brain of 3-day-old SD rats were cultured and identified by Iba1 immunofluorescence staining. The primary MGs were cultured with DMEM/F12 medium containing BMSCs conditioned medium (experimental group) and with single DMEM/F12 medium (control group), respectively. After 48 hours of culture, the morphology of MGs was observed by inverted phase contrast microscope, the activated state of MGs was detected by using Iba1 immunofluorescence staining, and Arg1 expression of MGs was assessed by Iba1-Arg1 double-labelling immunofluorescence staining and Western blot method. Results Inverted phase contrast microscope observation showed that BMSCs entered logarithmic growth phase at 14 days after culture, and more than 98% cells were positive to Vimentin immunofluorescence staining; whereas MGs entered logarithmic growth phase at 21 days after culture, and around 80% cells were positive to Iba1 immunofluorescence staining. Inverted phase contrast microscope observation displayed that in the experimental group, MGs were activated with increased size of soma, shortened process, and amoeba change. Immunofluorescence staining displayed that the Iba1 positive cells number in the experimental group was significantly higher than that in the control group (t=0.007, P=0.000); double-labelling immunofluorescence staining revealed that the Iba1-Arg1 positive cells number in the experimental group was significantly higher than that in the control group (t=0.007, P=0.000); and Western blot results elucidated that the relative expression of Arg1 protein in the experimental group was significantly higher than that in the control group (t=0.001, P=0.000). Conclusion BMSCs conditioned medium can activate MGs and induce MGs to express Arg1.

关键词: BMSCs; 小胶质细胞; 精氨酸酶 1; 大鼠

Key words: Bone marrow mesenchymal stem cells; microglia; arginase 1; rat

引用本文: 陈云慧, 余曙光, 吴巧凤, 唐勇, 姜岑, 庄志奇, 赵娜, 黄彪, 谢璐霜. BMSCs 条件培养液诱导小胶质细胞分泌精氨酸酶 1 的初步研究. 中国修复重建外科杂志, 2017, 31(12): 1500-1505. doi: 10.7507/1002-1892.201710038 复制

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