人真皮淋巴管内皮细胞的分离及冷冻保存

Practice of human dermal lymphatic endothelial cell isolation and cryopreserving technique

目的建立分离培养人真皮淋巴管内皮细胞(LECs)及其冷冻保存技术。方法采用中性蛋白酶及胶原酶消化结合免疫磁珠方法,分选CD34-/CD31+细胞。所获内皮细胞进行体外培养,经免疫荧光染色、RT-PCR鉴定证实为LECs。采用梯度降温法冻存内皮细胞,经复苏后进行形态学观察,以台酚蓝试验、MTT试验、流式细胞术细胞检测等方法鉴定细胞生物学特性。结果中性蛋白酶及胶原酶消化方法结合免疫磁珠分选,可从真皮组织获取高纯度的LECs。与新鲜细胞相比,复苏的内皮细胞活力保持在92%以上。复苏后的内皮细胞形态学、生物学特性及生长曲线与新鲜细胞差异无统计学意义。冻存内皮细胞的凋亡率较新鲜内皮细胞高,为(9.15±0.34)%vs(5.31±0.23)%,但无统计学差异(P>0.05)。结论中性蛋白酶及胶原酶消化结合免疫磁珠分选,可获取足够数量及纯度的LECs;冻存的内皮细胞复苏后无明显形态学改变,并保持较高的活力及体外增殖能力;可作为种子细胞来源,为淋巴管系统发育和新生研究提供方便。

Objective To establish a noval approach of isolation and cryopreserving of human dermal capillary lymphatic endothelial cells （LECs） in vitro. Methods CD34^-/CD31^± cells were isolated by means of immunomagnetic beads after dispase and collagenase digestion to harvest all the dermal cells. The isolated endothelial cells were tested by immunofluorescence and RT-PCR with LECs specific markers. LECs were suspended in cryopreserving solution and cryopreserved in liquid nitrogen. The characteristics of post-thawed cells and non-frozen cells were evaluated by morphological observation, HE staining, trypan blue staining, MTT method and flow cytometry method. Results Extremely high-purified LECs from human dermal tissues could be successfully harvested by collagenase digestion procedure followed by immunomagnetic beads sorting. Compared with non-frozen endothelial cells,post-thawed endothelial cells showed 92% of vitality and character of cultured LECs in vitro. The cell morphology, biological characteristics and growth curves of post-thawed and non-frozen LECs were coincident. The apoptosis rate of post-thawed LECs was higher than non-frozen ones, （9. 15 ± 0.34） % vs （5.31 ± 0.23）% , however, there was no significant difference（P 〉 0.05）. Conclusion Enough high-purified LECs can be isolated by collagenase digestion procedure followed by immunomagnetic beads sorting. Post-thawed endothelial cells are proved to have high vitality and growth potential in vitro without significant morphological changes. Cryopreserved LECs may serve as a cell choice for research of lymphangiogenesis and lymphatic patterning.