过氧化氢诱导的大鼠盆腔神经节体外损伤模型的构建

Establishment of hydrogen peroxide-induced rat major pelvic ganglion injury model in vitro

目的探索成年大鼠盆腔神经节(MPG)体外损伤模型的构建方法,为盆腔神经损伤相关疾病的基础研究提供有效模型。方法选取8周成年SD大鼠,处死后获取MPG离体组织,以RPMI-1640培养基进行体外培养,1周后分别加入0、100、200、300、400、500μmol/L不同浓度过氧化氢(H_(2)O_(2)),诱导MPG氧化应激损伤。免疫组织化学法检测神经标志物表达、ELISA法检测神经营养因子水平,并进行外周神经丝生长长度的比较,确定合适的损伤诱导浓度。结果大鼠MPG离体培养3 d后,可见组织贴合,有神经丝样组织生长;神经丝蛋白标志物NF-H染色阳性,证实为神经组织。加入H_(2)O_(2)后,随着浓度增加,MPG组织形态出现不同程度破碎裂解,神经标志物S100β表达下调,神经丝生长长度受到抑制;同时,培养基脑源性神经营养因子及神经生长因子表达量逐渐下降。结论使用RPMI-1640培养基培养,并以200μmol/L H_(2)O_(2)诱导后,可以构建稳定的成年SD大鼠MPG体外损伤模型。

Objective To establish adult rat major pelvic ganglion(MPG)injury model in vitro in order to provide an effective model for basic research of pelvic nerve injury related diseases.Methods Adult SD rats at 8 weeks were selected and sacrificed to obtain MPG tissue in vitro.The MPG tissue was cultured on RPMI-1640 medium.One week later,hydrogen peroxide(H_(2)O_(2))was added with different concentrations of 0,100,200,300,400,500μmol/L to induce MPG oxidative stress injury.The expression of neural marker was detected by immunohistochemistry.In addition,the level of neurotrophic factors was detected by ELISA,and the growth length of peripheral neurofilament was compared to determine the appropriate concentration of injury induction.Results Tissue adhesion and neurofilament growth were observed 3 days after in vitro culture of adult SD rat MPG.The staining of neurofilament protein marker NF-H was positive,which confirmed the regeneration of nerve tissue.With the increase of H_(2)O_(2) concentration,the tissue morphology of MPG revealed different degrees of fragmentation.Meanwhile,the expression of neural marker S100βwas down-regulated,and the growth length of neurofilament was inhibited.Accordingly,the expression levels of brain derived neurotrophic factor and nerve growth factor in the cultured medium decreased gradually.Conclusion Stable in vitro injury model of MPG in adult SD rats can be established after cultured on RPMI-1640 medium and induced with 200μmol/L H_(2)O_(2).