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目的：建立体外分离及培养人真皮微血管内皮细胞的方法，为进一步开展创面愈合及相关领域的细胞和分子生物学研究提供物质基础和实验模型。方法：健康成人中厚皮组织经中性蛋白酶消化后去除表皮；然后，采取薄片真皮组织胰蛋白酶低湿慢速灌注法消化微血管内皮细胞；将获取的微血管内皮细胞于不同条件的培养基内连续培养５～１５ ｄ，观察其基本生物学生长特征，并采用免疫细胞化学方法行Ⅷ因子相关抗原染色鉴定。结果：①采用中性蛋白酶及胰蛋白酶分步消化法，所分离获得的细胞体外具有很强的增殖能力；该细胞呈现Ⅷ因子相关抗原阳性，说明为真皮微血管内皮细胞；②细胞单层培养时显示活跃的形成类管腔状结构的生物学特征；③真皮微血管内皮细胞在不同培养基内生长状态不同，含８％ 胎牛血清、２ ％ 人分娩前母体血清及适量ｃＡＭＰ等添加物的ＩＭＤＭ 为其较理想的培养基。结论：经体外分离及培养获得了生长性良好的人真皮微血管内皮细胞。
OBJECTIVE: To establish a method for the isolation and culture of human dermal microvascular endothelial cells in vitro and provide the material basis and experimental model for the further study of wound healing and related cellular and molecular biology. METHODS: The epidermis was removed by neutral protease digestion of healthy adult mesodermal tissues. Then, the endothelial cells were digested by low-speed slow-perfusion with trypsin and low-dampness in the dermis of the dermis. The cultured endothelial cells were cultured continuously in different conditions From 5 to 15 days, the basic biological growth characteristics were observed and identified by immunocytochemical staining for factor Ⅷ related antigen. Results: (1) Neutral protease and trypsin digestion method, the isolated cells have a strong proliferative capacity in vitro; The cells showed Ⅷ factor-related antigen positive, that dermal microvascular endothelial cells; ② cell monolayer culture Showing the biological characteristics of the active formation of lumen-like structure; ③ dermal microvascular endothelial cells in different media in different growth states, containing 8% fetal bovine serum, 2% before delivery of maternal serum and the amount of cAMP and other additives IMDM For its more ideal medium. Conclusion: Human dermal microvascular endothelial cells with good growth were obtained by in vitro isolation and culture.