810nm弱激光对脊髓损伤小鼠神经元轴类再生的促进作用及其相关机制  

Promoting effect of 810 nm low-level laser on axonal regeneration of neurons in mice with spinal cord injury and its related mechanism

作　　者：张家玮 孙嘉锴 郑峤 宋基伟 李鲲 梁卓文 胡学昱 王哲 Zhang Jiawei;Sun Jiakai;Zheng Qiao;Song Jiwei;Li Kun;Hu Xueyu;Wang Zhe(Department of Orthopedics,First Affiliated Hospital of Air Force Medical University,Xi’an 710032,China;Research Institute of Orthopedics of PLA,Xi'an 710032,China)

机构地区：[1]空军军医大学第一附属医院骨科,西安710032 [2]全军骨科研究所,西安710032

出　　处：《中华创伤杂志》2019年第4期359-367,共9页Chinese Journal of Trauma

基　　金：国家自然科学基金面上项目(81070996,81572151);陕西省社会发展攻关项目(2016SF-143).

摘　　要：目的探讨810nm弱激光对小鼠脊髓损伤(SCI)后神经元轴突再生的作用及其相关机制。方法体内实验:选取20只Balb/c小鼠,通过钳夹伤方法,建立标准化小鼠SCI模型,按照随机数字表法分为SCI组及SCI后810nm弱激光照射组(弱激光组),每组10只。弱激光组采用选定参数的弱激光(连续波波长810nm,功率密度2mW/cm2,光斑面积4.5cm2,照射时间50min,获得能量密度6000J/cm2)连续照射损伤区,14d后免疫荧光染色观察SCI组及弱激光组损伤区M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、M2型巨噬细胞标志物精氨酸酶1(Arg-1)及巨噬细胞普遍标志物F4/80的表达情况。体外实验:选取20只Balb/c小鼠,体外获取并培养小鼠原代骨髓源性巨噬细胞,而后使用脂多糖(LPS)和γ干扰素(INF-γ)诱导为M1型巨噬细胞。按照随机数字表法将巨噬细胞培养板分为M1型巨噬细胞组(M1组)和弱激光照射组(M1+弱激光组),每组细胞数目相同。M1组不做处理,M1+弱激光组采用体外标准化810nm弱激光-巨噬细胞照射模型进行照射。照射24h后RT-qPCR和ELISA法检测两组白细胞介素1受体拮抗剂(IL-1RA)、白细胞介素10(IL-10)的表达情况。照射48h后Westernblot分析两组iNOS、Arg-1、分化抗原簇206(CD206)、蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、环磷腺苷效应元件结合蛋白(CREB)和磷酸化环磷腺苷效应元件结合蛋白(p-CREB)的表达。照射48h后,分别使用两组巨噬细胞条件培养基培养背根神经节神经元(DRG),培养48h后测量DRG轴突生长的长度。结果体内实验中,相较于SCI组,弱激光组损伤区F4/80+iNOS+的荧光强度显著下降(1.00±0.08∶0.06±0.04)(P<0.05);F4/80+Arg-1+的荧光强度显著上升(1.00±0.07∶2.15±0.12)(P<0.01)。在体外实验中,与M1组相比,M1+弱激光组中M1型巨噬细胞标志物iNOS的表达降低(1.00±0.11∶0.08±0.01)(P<0.01);M2型巨噬细胞标志物Arg-1(1.00±0.14∶2.44±0.16)、CD206的表达升高(1.00±0Objective To investigate the effect of 810 nm low-level laser on neuronal axonal regeneration of mice with spinal cord injury and its related mechanism. Methods In vivo experiment: 20 Balb/c mice were randomly divided into the spinal cord injury group (SCI group) and the 810 nm low-level laser irradiation group (low-level laser group) after spinal cord injury according to the random number table method, with each group containing ten mice. A mice SCI model was established through clamp injury and the low-level laser group continuously irradiated the damaged area with weak 810 nm low-level laser with selected parameters (continuous wave with wave length 810 nm, power density 2 mW/cm2, spot are 4.5 cm2, irradiation time 50 minutes, energy 6 000 J/cm2). Then immunofluorescence staining was used to observe the M1 macrophage marker-inducible nitric oxide synthase (iNOS), the M2 macrophage marker arginase 1 (Arg-1) and the universal marker F4/80 of macrophages after 14 days. Furthermore, in the in vitro experiment, standardized low-level laser-macrophage irradiation model was established. Another 20 Balb/c mice were used to obtain primary bone marrow-derived macrophages which were induced into M1 macrophages using lipopolysaccharide (LPS) and interferon-gamma (INF-γ). The M1 macrophages were randomly divided into the M1 macrophage group (M1 group) and the low-level laser therapy group (M1+ low-level laser group) equally according to the random number table method. The M1 group was not treated, and the M1+ low-level laser group was treated with low-level laser of selected parameters. RT-qPCR and ELISA were used to detect the expression of interleukin-1 receptor antagonist (IL-1RA) and interleukin-10 (IL-10) in M1 macrophages 24 hours after irradiation. Western blot was used to analyze the expression of iNOS, Arg-1, differentiation antigen cluster 206 (CD206), protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), cyclic adenosine response element binding protein (CREB) and phosphorylated cyclic adenosine resp

关 键 词：脊髓损伤 激光疗法 巨噬细胞 神经再生 

分 类 号：R651.2[医药卫生—外科学]