黄鳝成体性腺组织生殖干细胞的体外培养及鉴定

Culture and Characterization of Germ Stem Cell in the Gonad of Monopterus Albus

通过黄鳝成体性腺组织的体外培养，探讨黄鳝成体性腺生殖干细胞体外分离培养与鉴定的方法．分离黄鳝性腺组织进行植块培养，倒置相差显微镜下观察细胞生长状况．利用H．E、油红0染色、碱性磷酸酶（AP）细胞化学染色等方法对培养细胞进行鉴定．黄鳝成体性腺组织原代培养3～5d，可见组织周围有细胞迁出，并形成生长晕．15d左右，生长晕直径可达3～3．2mm，生长晕中的细胞主要包括：成纤维细胞、神经样细胞、类生殖干细胞三种类型．成纤维细胞呈不对称的长梭形，突起短而多，细胞核较浅，核仁明显，有2～3个核仁，是生长晕的主要细胞成分．神经样细胞具有放射丝状细胞突起，油红O染色呈阳性反应．类生殖干细胞呈圆形，单核或多核，核质比大，AP染色呈现出阳性反应．探索了适于黄鳝成体性腺组织体外培养的条件和方法，并对原代培养的细胞进行了初步鉴定，为进一步利用体外培养的黄鳝成体性腺生殖干细胞开展黄鳝性逆转分子机制研究奠定了基础．

Objective： To culture gonad tissues of Monopterus Albus and To explore the methods of the purification, culture and identification of the germ line stem ceils of Monopterus Albus in vitro. Method： The tissue block pasted culture method were used to culture gonad tissues of Monopterus Albus and the germ line stem cells of Monopterus Albus was observed under inverted phase contrast microscope. The cultured cells were identified by H. E, Oil red O staining, alkaline phosphatase （AP） cell staining. Results： The cells migrated out around tissues after 3-5 days culturing and the growth halo emerged. About 15 days, the diameter of the growth halo is up to 3 - 3.2 mm, the cells of the growth halo include three types： fibroblasts, nerve-like cells, and germ stem cell similar cells. Fibroblast was the main components of the growth halo. Its body was fiat. The morphology was unregular and there were many short prominences and the light nuclear of cell had 2-3 obvious nucleoluses, the main components of the growth halo. The morphology of Nerve-like cells. Nerve-like cells with radiation filamentous cell protrusions were positive in oil red O staining. Germ stem cell similar cells are round, single-core or multi-core, and the ratio of the nucleus and cytoplasm is mass, AP staining shows positive. Conclusion： the suitable conditions and methods for the cultivation of gonad tissues of Monopterus Albus in vitro and preliminary identificated the cells of primary cul- ture, Therefore, this study has laid a foundation for the cultivation of the germ line stem cells of Monopterus Albus in vitro as well as for the mechanism of sex reversal of Monopterus Albus on the molecule level.