人真皮微淋巴管内皮细胞的分离培养和鉴定

Isolation and characterization of human dermal capillary lymphatic endothelial cells

目的建立人真皮来源淋巴管内皮细胞（LECs）分离和培养的方法。方法采用中性蛋白酶及胶原酶消化方法结合免疫磁珠从真皮组织分选CD34^-／CD31^＋细胞。免疫细胞化学、免疫荧光及逆转录．聚合酶链反应（RT-PCR）方法检测LECs特异标志的表达，噻唑蓝（MTT）比色试验测定多种与内皮细胞增殖相关的细胞因子和低氧条件对其增殖的影响。结果CD34^-／CD31^＋细胞在单层细胞培养时形成内皮细胞典型的铺路石样外观，而在胶原凝胶三维培养时形成管状结构。CD34^-／CD31^＋细胞表达淋巴管内皮细胞特异性标志，包括LYVE-1，VEGFR-3和Prox-1。RT-PCR显示Prox1和VEGFR-3mRNA在CD34^-／CD31^＋细胞中特异性表达。特异作用于淋巴管内皮细胞的VEGF-C对LECs的促增殖作用明显（P〈0．01）。低氧条件和Ang2在体外未显示对LECs有促进增殖的作用。结论应用中性蛋白酶及胶原酶消化方法结合免疫磁珠分选可以成功分离获取人真皮LECs。

Objective To estabhsh a noval approach of isolation and culture of human dermal capillary lymphatic endothelial cells （LECs） in vitro. Methods After dispase digestion to remove epidermis, collagenase was used to digest and harvest all the dermal cells. Immunomagnetic beads were then applied to isolate CD34^-/CD31^＋ cells. These isolated cells were tested by immunocytochemistry, immunofluoresence and RT-PCR with LECs specific markers. Proliferation of isolated cells in response to angiogenic factors and hypoxia was tested by MTF method. Results Cells exhibited their typical cobblestone morphology as monolayer cultures under standard growth conditions and tube formation appeared in threedimensional cell culture system. Cultured cells were positively stained for LYVE-1, Prox-1 and VEGFR-3 specificly. RT-PCR revealed the specific expression of Proxl and VEGFR-3 mRNA in cultured cells, consistent with LECs. LECs responded to VEGF-C significantly （ P 〈 0.01 ）, but angiopoietin-2 （Ang2） and hypoxia were inactive to proliferation of LECs in vitro. Conclusion Using collagenase digestion procedure followed by immunomagnetic beads sorting, human dermal LECs could be harvested successfully.