过氧化氢诱导的大鼠盆腔神经节体外损伤模型的构建

doi:10.3877/cma.j.issn.1674-0793.2021.06.001

中华普通外科学文献(电子版) ›› 2021, Vol. 15 ›› Issue (06) : 401 -405. doi: 10.3877/cma.j.issn.1674-0793.2021.06.001

论著

过氧化氢诱导的大鼠盆腔神经节体外损伤模型的构建

施万程¹, 杨文德¹, 刘千惠¹, 黄旭娜², 杨晓锋¹, 杨尚斌¹, 方佳峰¹^,^†(

)

^1. 510630　广州，中山大学附属第三医院胃肠外科
^2. 510630　广州，中山大学附属第三医院中心实验室

收稿日期:2021-08-26 出版日期:2021-12-01
通信作者: 方佳峰
基金资助:
国家自然科学基金青年项目(81901471); 广东省自然科学基金面上项目(2021A1515010577)

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

Wancheng Shi¹, Wende Yang¹, Qianhui Liu¹, Xu'na Huang², Xiaofeng Yang¹, Shangbin Yang¹, Jiafeng Fang¹^,^†()

^1. Department of Gastrointestinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
^2. Central Laboratory, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

Received:2021-08-26 Published:2021-12-01
Corresponding author: Jiafeng Fang

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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、500 μmol/L不同浓度过氧化氢（H₂O₂），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H₂O₂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200 μmol/L H₂O₂诱导后，可以构建稳定的成年SD大鼠MPG体外损伤模型。

关键词: 盆腔神经节, 成年大鼠, 过氧化氢, 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 (H₂O₂) was added with different concentrations of 0, 100, 200, 300, 400, 500 μmol/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 H₂O₂ concentration, the tissue morphology of MPG revealed different degrees of fragmentation. Meanwhile, the expression of neural marker S100β was 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 200 μmol/L H₂O₂.

Key words: Major pelvic ganglia, Adult rats, Hydrogen peroxide, Injury model

图1 成年大鼠盆腔神经节（MPG）体外培养模型的成功构建　前列腺背侧叶表面可见白色半透明斑块的MPG组织（A）；MPG培养3 d后，可见外周神经丝样结构生长（B）；MPG于RPMI-1640培养基联合基底胶（A组）或单纯RPMI-1640培养基（B组）培养后，均可见外周细胞生长（C）；MPG外周生长细胞NF-H免疫荧光染色阳性，呈红色荧光表达（D）

图2 过氧化氢（H₂O₂）诱导成年大鼠盆腔神经节（MPG）损伤及神经蛋白成分丢失S100β染色可见神经组织形态渐出现破碎裂解，在300 μmol/L及以上浓度时，MPG组织已接近失去正常神经节结构（A）；免疫量化分析结果显示，随着H₂O₂浓度增加，S100β表达量逐渐下降（B）；与0 μmol/L组相比，^*P<0.05；^**P<0.01

图3 过氧化氢（H₂O₂）抑制成年大鼠盆腔神经节（MPG）神经细胞生长　随着H₂O₂浓度增加，MPG外周神经丝生长逐渐受到抑制（A）；各组神经丝长度比较（B）；^*两组间比较，P>0.05；^**与0 μmol/L组相比，P<0.01

图4 过氧化氢抑制成年大鼠盆腔神经节（MPG）分泌神经营养因子　A、B分别为各组MPG培养基神经生长因子（NGF）、脑源性神经营养因子（BDNF）分泌水平；^*两组间比较，P>0.05；^**与0 μmol/L组相比，P<0.01

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