摘要
目的 微血管内皮细胞 (MEC)增殖和血管发生存在于很多生理和病理过程中 ,为体外重建人增生性瘢痕组织微血管内皮细胞 (HSMEC)生长模型 .方法 正常人包皮和增生性瘢痕经 1.2 5 g· L- 1 dispase和 1.2 5 g· L- 1 胰蛋白酶依次消化 4℃ 2 4h后 ,机械挤压分离组织内 HSMEC和人真皮微血管内皮细胞 (HDMEC) ,经 5 0 0 0 U· L- 1 b FGF,2 0 0m L· L- 1胎牛血清的 DMEM内培养和 0 .1g· L- 1胰蛋白酶 ,0 .15 mmol· L- 1 EDTA的 TE分离、纯化 ,组织学 HE染色和 因子相关抗原免疫组化染色 ,观察 MEC的阳性细胞百分数 .结果 MEC在含 b FGF培养基中增殖旺盛 ,少量混生的成纤维细胞经 TE分离 MEC而被基本遗弃 ,3代 MEC能获得 97.0 %～ 98.0 %的纯度 ,明显高于原代培养 (P<0 .0 1) ,HSMEC形态学与 HDMEC无明显差异 .结论 低浓度b FGF和 TE细胞分离液分别对 MEC的培养和纯化有可靠作用 ,HSMEC的培养成功为烧伤创面愈合和瘢痕增生机制的研究扩展了空间 .
AIM To establish the in vitro growth model of human hypertrophic scar microvascular endothelial cells(HSMEC). METHODS The human normal forskin segments and hypertrophic scar tissues obtained from clinical operations were treated with 1.25 g·L -1 dispase and then 1.25 g·L -1 trypsin solution at 4℃ for 24 hours respectively. After being released from the tissues by mechanical pressing, the human dermal microvascular endothelial cells (HDMEC) and HSMEC were cultivated in Delbeco's modified eagle medium (DMEM) supplemented with 200 mL·L -1 fetal calf serum and bFGF (5000 U·L -1 ). The cultivated MEC were isolated and purified with 0.1 g·L -1 trypsin 0.15 mmol EDTA (TE). Hematoxylin and eosin (HE) staining and immunohistochemistry staining for factor Ⅷ related antigen were performed to identify the MEC. The percentage of MEC was calculated. RESULTS MEC grew and proliferated well in DMEM containing FCS and bFGF and a few of dermal fibroblast contaminating the MEC could be removed almost completely with TE. The purities of both HSMEC and HDMEC in passage 3 were 97.0% to 98.0%, significantly higher than that in primary culture ( P <0.01). CONCLUSION A low concentration of bFGF and the TE isolation exert reliable effects on the MEC growth and purification. The successful culture of HSMEC might widen the range of study on the mechanisms of burn wound healing and scarring.
出处
《第四军医大学学报》
2000年第11期1359-1362,共4页
Journal of the Fourth Military Medical University
