以鼠尾胶为贴黏剂的小鼠淋巴管内皮细胞培养体系的建立

Establishment of culture system for mouse lymphatic endothelial cells adhered to rat-tail collagen

目的:建立一个简便、廉价和稳定的小鼠淋巴管内皮细胞(LEC)培养体系.方法:应用不完全弗氏佐剂诱导小鼠腹腔淋巴管瘤形成,消化法分离获得LEC,置于自制的鼠尾胶包被的培养瓶(板)中培养.以免疫荧光化学法检测LEC特异性标记物VEGFR-3和LYVE-1的表达,以3H-TdR掺入率测定和细胞倍增时间检测LEC的增殖活力,以淋巴管形成试验判定LEC能否形成淋巴管样结构.结果:不完全弗氏佐剂能诱导小鼠腹腔淋巴管瘤形成,所获LEC活细胞数大于98%.免疫荧光化学检测表明,LEC表达VEGFR-3和LYVE-1.在鼠尾胶包被的培养瓶(板)中,LEC生长状况良好,3H-TdR掺入率明显增加,倍增时间为(42.7±3.2)h;在鼠尾胶凝胶中,LEC能形成淋巴管样结构.结论:鼠尾胶为贴黏剂的体系是一种简便、廉价和稳定的小鼠LEC培养体系.

AIM： To establish a convenient, cheap and stable culture system for mouse lymphatic endothelial cells （LEC）. METHODS： Mouse lymphangiomas in abdominal cavity were induced by incomplete Freund＇s adjuvant, then disrupted and digested to obtain LEC, which were cultured in the flask or plate previously coated with rat-tail collagen. Expressions of LEC specific markers, VEGFR-3 and LYVE-1, were identified by immunofluorescence. The proliferation activity of LEC was evaluated by ^3H-TdR incorporation efficiency and cell doubling time. The capability of LEC to form lymphatic vessel-like structures was assessed by the in vitro lymphatic vessel formation assay. RESULTS： Mouse lymphangiomas in abdominal cavity were induced successfully by incomplete Freund＇s adjuvant, and more than 98% living LEC were harvested. The expressions of VEGFR- 3 and LYVE-1 were positive in LEC. In the rat-tail collagen coated flask or plate, LEC grew well, with 3 H-TdR incorporation efficiency increasing obviously and cell doubling time being （42.7 ± 3.2 ） h. In the gel formed by rat-tail collagen, LEC could form lymphatic vessel-like structures. CONCLUSION： Mouse LEC culture system using rat-tail collagen as adhesive is a convenient, cheap and stable culture system.