Cathepsin S基因敲除小鼠的胶质细胞对面神经损伤后神经再生的影响

Effects of cathepsin S in microglia on regeneration of facial nerve after injury

关键词:  CS基因敲除小鼠  面神经切断  面神经元生存

Key words:  CS deficient mice  facial nerve axotomy  motoneuron survival

摘要:

目的 研究股质细胞内丝蛋白水解酶Cathepsin S(CS)对小鼠面神经损伤后神经再生的影响。方法 将CS基因敲除（CS-/-)小鼠和野生型小鼠的一侧的面神经在茎乳孔处离断，术后2、4、7、14、30、50 d取出含面神经核（FMN)的脑干进行切片，以Iba-l、Tenasin-R(TNR)、chAT抗体分别染色胶质细胞和面神经元并计数。以CS、CystanC(CysC)、Iba-l或者F4/80抗体结合反应进行肢质细胞免疫焚光双重染色。术后7 d,取两组含FMN的脑干，用HBG标记的CS、CysC和TNR抗体作Westemblot分析。术后7 d取两组含FMN的脑干，用Quickprep mRNA套装进行CS、CysC的RT-PCR定量分析。结果 面神经离断后7 d,野生型胶质细胞包绕神经元，但CS-/-胶貭细胞仅仅接触而不包绕神经元。免染结果提示，CS出现在野生型肢质细胞，而极少出现在CS-/-胶质细胞,CysC在两组均增加，同时,CS-/-胶质细胞对TNR的降解能力显著低于野生型胶质细胞。RT-PCR和 Westemblot分析显示，神经离断后,CS在野生型FMN内明显增加，但CysC在两组中均增加。TNR在野生型FMN 内明显减少。对chAT染色的面神经元统计显示，术后50 d，CS缺失造成了神经元存活率降低，与野生型有显著性差异。结论 面神经离断后，CS的缺失会使神经元容易死亡，CS在激活的胶质细胞内的表达对损伤的神经元的保护及恢复起到重要作用。

Abstract:

Objective To investigate the effect of lysosomal cysteine protease cathepsin S (CS) in regeneration of facial nerve after injury. Methods The right facial nerves of CS -/- and wild-type mice were transected approximately 1 mm at the stylomastoid foramen. The brain stem containing facial motor nucleus (FMN) were obtained from CS -/- and wild-type mice at 2, 4，7，14，30，and 50 days after an axotomy，the specimens were immunohistochemically stained with anti-Ibal，anti-choline acetyltransferase (chAT)，and anti-tenascin R (TNR) antibody. For double staining, sections of the brain stem were incubated with anti-CS and anti-CysC antibody, then further treated with Ibal or F4/80 antibody. The specimens obtained from CS -/- and wild-type mice 7 d after axotomy were incubated with anti-CS，anti-TNR and anti-CysC for Western blot analysis ； the expression of CS and CysC mRNA was analyzed by RT-PCR. Results The facial motoneurons were encysted by microglia in wild-type mice at 7 d after axotomy ； however not in CS -/- mice. CS was observed in activated wild-type microglia but not in CS -/- microglia. CysC increased in activated microglia of both groups. There was a marked reduction in the immunoreactivity for TNR in the FMN from wild-type mice after axotomy, compared with the of the FMN from CS -/- mice. Both RT-PCR and Western blot analysis indicated that CS increased only in FMN of wild-type mice and CysC increased in both groups after nerve axotomy. Meanwhile, TNR decreased markedly in wild-type FMN. The counting of chAT-positive cells indicated that the deficiency of CS in CS -/- mice significantly decreased the survival of injured motoneurons 50 d after axotomy. Conclusion CS in activated microglia may play an important role in facial nerve motoneuron survival after nerve axotomy.