大鼠肠系膜淋巴管内皮细胞体外培养方法的建立及鉴定

目的　建立分离大鼠肠系膜淋巴管的技术及其内皮细胞的培养方法并证实体外培养的大鼠肠系膜淋巴管内皮细胞具有合成一氧化氮及第Ⅷ因子相关抗原的能力。方法　利用手术显微镜及显微手术器械分离大鼠肠系膜淋巴管,采用植块培养法进行内皮细胞原代培养并传代培养。用免疫组织化学的方法鉴定第Ⅷ因子相关抗原及一氧化氮合酶在体外培养的大鼠肠系膜淋巴管内皮细胞中的表达。测定一氧化氮代谢产物的含量。结果　体外培养的大鼠肠系膜淋巴管内皮细胞呈梭形或多角形,形成单层后呈典型的鹅卵石样或铺路石样排列。第Ⅷ因子相关抗原的免疫过氧化氢酶染色反应阳性,阳性率 98%。一氧化氮合酶表达阳性率为83%,阳性反应产物在细胞核周围呈棕黄色。一氧化氮代谢产物含量(10 26±2 17)mol·L-1 (n=7 )。结论　本实验采用培养方法可行性强,简单可靠,保持了内皮细胞的结构和功能。并证实了体外培养的大鼠肠系膜淋巴管内皮细胞能表达一氧化氮合酶及第Ⅷ因子相关抗原。

Culture and evaluation of rat''''s mesenteric lymphatic endothelium in vitro

Aim To establish the technology of isolation rat’s mesenteri c lymphatics and the culture method of their endothelium and confirm the ability o f synthesis nitric oxide and Factor Ⅷ related antigens in the endothelium in vitro. Methods With an operating microscope and microsurgical in struments, rat’s mesenteric lymphatics were isolated and the endothelium were p rimarily cultured and subcultured. Exerting immunohistochemical method, Factor Ⅷ related antigens and nitric oxide synthase were evaluationed. The contents of NO metabolite were measured. Results The rat’s mesenteric lympha tics were isolated and the endothelium were Fusiform shape or polygon, the monol ayer cultures displayed typical cobblestone or pavingstone morphology. Factor Ⅷ related antigens were expressed and their positive rate was 98%. The positive rate of nitric oxide synthase was 83%. Positive reaction products were buffy ar ound nuclear. NO metabolite was (10.26±2.17) mol·L -1(n=7). Conclusion The culture method we introduced was very practical, simple and reliable. The endothelium is in normal structure and function characters. It also certificated that the endothelium in vitro could express nitric oxide synthase and Factor Ⅷ related antigens.