骨髓间充质干细胞来源外泌体通过miR-181c对脊髓损伤的修复作用研究

Effect of exosomes derived from bone marrow mesenchymal stem cells on spinal cord injury via miR-181c

目的探索骨髓间充质干细胞(BMSC)来源的miR-181c对脊髓损伤(SCI)的修复作用。方法用全骨髓贴壁法分离大鼠BMSC,流式细胞术检测CD29、CD90、CD45及CD34表达。根据转染物质的不同分为Control组(未处理)、miR-181c组(转染miR-181c mimc质粒)及miR-NC组(转染miR-NC质粒)。提取外泌体,用透射电子显微镜观察外泌体形态。构建SCI大鼠模型,造模后1 h,SCI+外泌体组大鼠尾静脉注射BMSC来源的外泌体;SCI+miR-181c组大鼠尾静脉注射转染miR-181c mimic的BMSC来源的外泌体;SCI+miR-NC组大鼠尾静脉注射转染miR-NC的BMSC来源的外泌体;Sham组、模型组大鼠尾静脉注射等体积的0.9%氯化钠溶液(生理盐水)。处理后第1天、第3天、第7天、第14天及第28天采用行为学评分(BBB)评估大鼠的运动功能,TUNEL染色检测脊髓组织细胞凋亡,酶联免疫吸附分析(ELISA)检测大鼠血清炎性因子肿瘤坏死因子(TNF-α)、白细胞介素1β(IL-1β)表达水平,实时荧光定量聚合酶链式反应(q RT-PCR)检测脊髓组织、外泌体中miR-181c,脊髓组织中TNF-α、IL-1βmRNA水平,Western blot检测脊髓组织中Cleaved半胱胺酸蛋白酶第三型(Caspase 3)、Caspase 3、Bcl关联X蛋白(Bax)及B细胞淋巴瘤基因-2(Bcl2)蛋白,BMSC、外泌体中CD9、CD63表达水平。结果透射电子显微镜下观察,BMSC来源的外泌体呈类圆形,直径约50~100 nm。Control组与miR-NC组外泌体中miR-181c表达水平差异无统计学意义(t=0.297,P>0.05)。与miR-NC组比较,miR-181c组外泌体miR-181c表达水平升高(1.03±0.21 vs 0.34±0.04)(t=7.813,P<0.05)。与SCI组比较,SCI+外泌体组脊髓组织miR-181c表达水平升高(0.42±0.06 vs 0.23±0.03)(t=9.500,P<0.05)。与SCI+miR-NC组比较,SCI+miR-181c组脊髓组织miR-181c表达水平升高(0.22±0.03 vs 1.32±0.13)(t=18.440,P<0.05)。SCI+miR-181c组大鼠BBB评分高于SCI+miR-NC组(t=18.440,P<0.05)。SCI+外泌体组大鼠血清TNF-α、IL-1β含量和脊髓组织中TNF-α、IL-1β表达水平均低于SCI模型组(P<0.05),SCI+miR-181c组大鼠血清TNF-α、IL-1β含量和脊髓组织中TNF-α、IL-1β表达水平低于SCI+miR-NC组(P<0.05)。与SCI组比较,SCI+外泌体组大鼠脊髓组织中Cleaved Cas pase 3/Caspase 3、Bax表达水平降低,Bcl2表达水平升高,脊髓组织凋亡率降低(P<0.05)。与SCI+miR-NC组比较,SCI+miR-181c组大鼠脊髓组织中Cleaved Caspase 3/Caspase 3、Bax表达水平降低,Bcl2表达水平升高,脊髓组织凋亡率降低(P<0.05)。结论BMSC来源的外泌体中miR-181c可修复脊髓损伤,机制可能与抑制炎症、抗凋亡有关。

Objective To explore the effect of miR-181c derived from bone marrow mesenchymal stem cells(BMSC) on spinal cord injury(SCI). Methods The BMSC were isolated by whole bone marrow adherent method, the expression of CD29, CD90,CD45 and CD34 were detected by flow cytometry. According to different transfected substances, BMSC were divided into control group(untreated), miR-181c group(transfected with miR-181c mimic) and miR-NC group(transfected with miR-NC). The exosomes were extracted and observed by transmission electron microscope. The SCI rat model was established, at 1st hour after modeling, rats in SCI + exosome group were injected with BMSC-derived exosomes via tail vein;BMSC-derived exosomes transfected with miR-181c mimic were injected into tail vein of rats in SCI + miR-181c group;Rats in SCI + miR-NC group were injected with BMSC-derived exosomes transfected with miR-NC via tail vein;rats in control group and model group were injected with equal volume of 0.9 % sodium chloride solution(normal saline) via tail vein. On the 1st, 3rd, 7th, 14th and 28th day after treatment, the behavioral score(BBB) was used to evaluate motor function of rats. TUNEL staining was used to detect apoptosis of spinal cord tissue. The enzyme-linked immunosorbent assay(ELISA) was used to detect the expression levels of serum inflammatory factors tumor necrosis factor(TNF-α) and interleukin-1β(IL-1β), the real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was used to detect levels of miR-181c in spinal cord tissue and exosomes, and levels of TNF-α and IL-1β mRNA in spinal cord tissue. Western blot was used to detect the expression levels of Cleaved cysteine-depen dent aspartate-specific profeases(Caspase 3), Caspase 3, Bcl-associated X protein(Bax) and B-cell lymphoma gene-2(Bcl2)protein in spinal cord tissue, CD9 and CD63 in BMSC and exosomes. Results Under transmission electron microscope, BMSCderived exosomes were round-like with about 50-100 nm in diameter, and there was no significant difference in expression level of exosomal miR-181c between control group and miR-NC group(t = 0.297, P>0.05). Compared with miR-NC group, the ex pression level of exosomal miR-181c in miR-181c group was increased(1.03 ± 0.21 vs 0.34 ± 0.04)(t = 7.813, P<0.05). Com pared with SCI group, the expression level of miR-181c in spinal cord tissue of SCI + exosome group was increased(0.42 ± 0.06vs 0.23 ± 0.03)(t = 9.500, P<0.05). Compared with SCI + miR-NC group, the expression level of miR-181c in spinal cord tissue of SCI + miR-181c group was increased(0.22 ± 0.03 vs 1.32 ± 0.13)(t = 18.440, P<0.05). The BBB score of SCI + miR-181c group was higher than that of SCI + miR-NC group(t = 18.440, P<0.05). The contents of TNF-α and IL-1β in serum and ex pression levels of TNF-α and IL-1β of spinal cord tissue in SCI + exosome group were lower than those in SCI model group(P<0.05), the contents of TNF-α and IL-1β in serum and expression levels of TNF-α and IL-1β of spinal cord tissue in SCI +miR-181c group were lower than those in SCI + miR-NC group(P<0.05). Compared with SCI group, the expression levels of Cleaved Caspase 3/Caspase 3 and Bax of spinal cord tissue in SCI + exosome group decreased, the expression level of Bcl2 increased, and the apoptosis rate of spinal cord tissue decreased(P<0.05). Compared with SCI + miR-NC group, the expression levels of Cleaved Caspase 3/Caspase 3 and Bax of spinal cord tissue in SCI + miR-181c group decreased, the expression level of Bcl2 increased, and apoptosis rate of spinal cord tissue decreased(P<0.05). Conclusion It is demonstrated that miR-181c in exosomes derived from BMSC could repair SCI, and mechanism may be related to the inhibition of inflammation and antiapoptosis.