体内冷冻的正常及镉处理小鼠睾丸内血清蛋白质的免疫组织化学研究

Immunohistochemical Study of Serum Proteins in Normal and Cadmium-Treated Mouse Testis by in vivo Cryotechnique

目的:了解正常和镉(Cd)处理小鼠睾丸内血清蛋白质渗透性的时相性变化,揭示支持细胞间紧密连接的屏障功能。方法:保持正常动力学的血循环下,运用活体内冷冻技术对曲细精管内白蛋白和IgG进行快速冷冻固定。冷冻固定的睾丸行冷冻置换后作石蜡包埋。5μm厚连续切片分别用于抗鼠血清白蛋白和IgG的免疫组化染色和H.E.染色。结果:正常曲细精管,白蛋白免疫反应产物主要定位于管周肌样细胞周围,以及间质细胞之间和毛细血管内,部分免疫反应产物呈拱型出现在曲细精管上皮基底室内,沿精原细胞表面分布。在依据生精细胞排列而区分的曲细精管上皮周期的不同发育时相,拱型免疫反应产物的数量不同。正常小鼠睾丸内IgG免疫反应的定位型式与白蛋白的免疫定位相似。Cd处理后24h,部分曲细精管上皮内空泡形成和管腔增大,白蛋白和IgG免疫反应产物不仅出现在基底室,而且也见于近腔室的支持细胞和生精细胞之间。结论:运用活体内快速冷冻技术结合血清白蛋白和IgG的免疫组化方法,能清楚地揭示正常和Cd处理小鼠曲细精管内血清蛋白质的不同免疫染色型式,反映体内支持细胞间紧密连接结构的变化。

Objective： To investigate time-dependent changes of serum proteins permeability in the normal and cadmium（Cd）-treated mouse testis, reflecting tight junctional （TJ） barriers of Sertoli cells. Methods： The serum proteins, albumin and immunoglobulin-G（IgG）, in the seminiferous tubules were firstly immobilized by the ＂in vivo cryotechnique＂, in which the dynamic blood circulation was always kept. The cryofixed testicular tissues were then processed for the freeze-substitution method, and embedded in the paraffin wax. Serial sections of 5μm thickness were immunostained by anti-mouse albumin or IgG antibody with peroxidase immunostaining, and also stained with hematoxylin-eosine （HE） for morphological observation. Results： In normal seminiferous tubules, albumin immunoreaction products were localized around peritubular myoid cells and among Leydig cells, as well as in blood vessels. They were also localized as arch-like patterns around some spermatogonia in basal compartments. The number of the immunopositive arch structures was different according to developmental stages of the seminiferous cycle, judging from the arrangement of germ cells by HE-staining. The patterns of localization of IgG immunostaining in normal mouse testis were similar to that of albumin. In 24 h after Cd-treatment, some enlarged spaces and vesicular formation in the seminiferous epithelium were observed on the paraffin sections by HE- staining. The albumin or IgG immunolocalization was seen not only in the basal compartments, but also in the adluminal compartments between Sertoli cells and germ cells. Conclusion： The structural changes of inter-Sertoli TJ barriers in vivo, such as different immunostaining patterns of serum proteins between the normal and Cd-treated mouse seminiferons tubules, could be clearly detected by the ＂in vivo cryotechnique＂ with albumin or IgG immunohistochemistry.