骨髓间充质干细胞外泌体促进大鼠脑微血管内皮细胞的增殖和迁移

Bone Marrow Mesenchymal Stem Cell Exosomes Promote Brain Microvascular Endothelial Cell Proliferation and Migration in Rats

目的探讨骨髓间充质干细胞(bone marrow mesenchyml stem cells, BMSCs)外泌体正常状态下对大鼠脑微血管内皮细胞的增殖和迁移的影响。方法提取大鼠BMSCs并进行鉴定,将其与脑微血管内皮细胞(bEnd.3细胞)通过Transwell小室共培养24 h(BMSCs共培养组);提取BMSCs外泌体进行鉴定,荧光显微镜下定性观察细胞吞噬外泌体的行为,CCK8法检测细胞活力选定最佳BMSCs外泌体工作浓度,将其与bEnd.3细胞共培养24 h(BMSCs外泌体共培养组)。另设bEnd.3细胞单独培养组。培养24 h后通过EDU和细胞划痕实验,分别检测以上3组bEnd.3细胞的增殖和迁移能力。结果第3代BMSCs表面CD90、CD29为阳性,CD45显示为阴性,可成骨、成脂,表明所提取的BMSCs纯度较高。透射电镜下BMSCs外泌体形态为类圆形,直径在100 nm左右;NTA分析发现BMSCs外泌体的直径分布范围为50~600 nm,其中粒径峰值为150 nm;免疫荧光显示内皮细胞能够吞噬BMSCs外泌体;CCK8显示20μg/mL外泌体加入对细胞增殖影响达到最佳。EDU检测显示,相较于对照组,BMSCs共培养组、BMSCs外泌体共培养组均可促进bEnd.3细胞的增殖(P<0.05),且促进增殖的能力相当(P>0.05)。细胞划痕实验示,BMSCs外泌体共培养组细胞的迁移率高于对照组(P<0.05),但与BMSCs共培养组差异不明显(P>0.05)。结论 BMSCs外泌体可代替BMSCs,有效促进脑微血管内皮细胞的增殖和迁移,并为卒中后的血管新生提供新的潜在治疗手段。

Objective To study the effect of bone marrow mesenchyml stem cell(BMSC) exosomes(Exo) on the proliferation and migration of brain microvascular endothelial cells in rats. Methods BMSCs were extracted from rats and identified. The BMSCs were co-cultured with bEnd.3 cells in Transwell chamber for 24 h(BMSCs group).Extracted and identified the BMSCs exosomes(BMSC-Exo). Observed and qualitatively evaluated the cells’ abilities on swallowing the BMSC-Exo under a fluorescence microscope. The optimal work concentration of BMSC-Exo was selected by detecting the cell vitality under different BMSC-Exo concentrations by CCK8 method. bEnd.3 cells were co-cultured with BMSC-Exo for 24 h(BMSC-Exo group). bEnd.3 cells cultured alone was set as control group. The proliferation and migration of bEnd.3 cells in the three groups were respectively detected by EDU and cell scratching experiment after 24 h of culture. Results Flow cytometry showed that P3 BMSCs were CD90 and CD29 positive and CD45 negative, with osteogenic differentiation and adipogenesis differentiation, indicating the extracted BMSCs high purity. The BMSC-Exo under transmission electron microscopy was round-shaped with a diameter of about 100 nm;NTA analysis found the diameter distribution of BMSC-Exo ranged from 50 to 600 nm, with a peak size of 150 nm. Immunofluorescence showed that the endothelial cells could swallow BMSC-Exo. CCK8 showed that supplement of 20 μg/mL BMSC-Exo had the best effect on cell proliferation. EDU results showed that BMSCs group and BMSC-Exo group could promote the proliferation of bEnd.3 cells compared with the control group(P<0.05), and there was no difference between BMSCs group and BMSCExo group(P>0.05). Cell scratch test showed that the cell mobility of the BMSC-Exo group was higher than that of the control group(P<0.05), but there was no significant difference between the BMSC-Exo group and the BMSCs group(P>0.05). Conclusion BMSC-Exo can replace BMSCs in effectively promoting the proliferation and migration of cerebral microvascular endothelial cells, which provide a new potential treatment for angiogenesis after stroke.