面神经离断后Cathepsin S基因敲除小鼠小胶质细胞反应的实验研究

Microlial Responses after Facial Nerve Axotomy in CS Deficient Mice.

目的:研究面神经损伤后CS基因敲除(CS-/-)小鼠小胶质细胞的反应。方法:两种小鼠经脾分别注射CFDA,24h后离断右侧面神经。术后7d,取出含面神经核(FMN)的脑干进行切片,小胶质细胞和面神经元免疫荧光双重染色,作Westernblot分析,并进行CS、CB的RT-PCR定量分析。术后14d、50d,进行小胶质细胞免疫荧光双重染色。分别培养CS-/-和野生型小胶质细胞后进行ATP诱导小胶质细胞位移实验、游走实验、增殖实验。结果:神经离断后,CS和MMP-9在野生型小胶质细胞增生表达,而不出现在CS-/-小胶质细胞。CB仅在野生型面神经元内显著性增加。CFDA和CD3仅在野生型小鼠FMN内出现。CS和MMP-9在野生型小鼠FMN内明显增加,而CB的水平无变化,并且CB在CS-/-小鼠FMN内显著性增加。CS-/-小胶质细胞的位移和游走能力大幅降低,但增殖能力两者无明显差别。结论:面神经离断后,CS的缺失会使单核、淋巴细胞的动员游走能力受到损害,表现出了神经元毒性和吞噬作用。

Objective：To investigate the role of CS in the recruitment and response of microglia after facial nerve axotomy.Methods：CFDA was slowly injected into the spleen of CS-/-and wild-type mice.24 hlater,the right side facial nerves of these animals were axotomized at the stylomastoid foramen after injection.In some animals,The distal free axotomized end of the facial nerve was filled with DiI as a tracing marker of facial motoneuron.For double staining,sections of the brain stem were incubated with anti-CS,CB,chAT,MMP-9antibody,then further treated with Iba1 or F4/80 antibody.After14,50 days of axotomy,sections were incubated with anti-CD3 antibody,then further treated with Iba1 antibody.The specimens obtained from CS-/- and wild-type mice after a 7-day-axotomy were incubated with anti-CS,anti-CB and anti-MMP-9for Westernblot analysis.The specimens were also treated with CS and CB RT-PCR analysis.ATP-induced migration/transmigration and GM-CSF-induced proliferation of primary cultured microglia assay were performed.Results：After axotomy,CS and MMP-9were highly expressed in wild-type but not in CS-/- migroglia.CB was only expressed in wild-type motoneuron.CFDA and CD3 were only detected in wild-type FMN.RT-PCR and Westernblot analysis indicated that CS and MMP-9increased only in wild-type FMN and CB increased only in CS-/- FMN after axotomy.Migration and transmigration assay indicated that CS deficiency impaired the invasive ability of monicitic cells including micrglia.There was no difference between both groups in microglial proliferation assay.Conclusion：CS deficiency impaired the recruitment and infiltration of monocitic cells and T lymphocytes into FMN after axotomy.The CS deficiency caused microglia to become neurotoxic and phagocytic.