细胞因子诱导及贴壁法提取淋巴内皮祖细胞

Extraction of Lymphatic Endothelial Progenitor Cells by Cytokine Induction and Adhesion

目的:使用细胞因子诱导及贴壁法从C57BL/6小鼠骨髓细胞中分离淋巴内皮祖细胞(LEPCs),并对其进行鉴定。方法:通过贴壁技术获取小鼠骨髓24小时后贴壁细胞,待其增殖融合至80%~90%后,使用Tryple Express酶对贴壁细胞消化1分钟,去除消化掉的细胞,剩余细胞继续培养,待其再次融合至80%~90%时,进行1:2传代,将获得的第三代细胞进行流式细胞术检测其表面特异性标记物,并对其进行淋巴管成管分化实验,鉴定细胞。结果:我们所获取的LEPCs,在体外诱导后,可形成典型的微管样结构,并且经过多次传代后可形成“鹅卵石”样的典型细胞形态;其表面特异性标记物为:CD34、CD113双阳性率为:87.17% ±1.77%;CD113、VEGFR-3双阳性率为:85.17% ±1.65%;CD34、VEGFR-3双阳性率为:83.77% ±0.42%,符合相关文献关于LEPCs的报道。结论:利用细胞因子诱导及贴壁法可获取较高纯度的LEPCs。

Objective: To isolate and identify lymphatic endothelial progenitor cells (LEPCs) from bone marrow cells of C57BL/6 mice by cytokine induction and adhesion. Methods: The adherent cells of mouse bone marrow were obtained by adherent technique for 24 hours. After they proliferated and fused to 80%~90%, the adherent cells were digested with Tryple Express enzyme for 1 minute, the di-gested cells were removed, and the remaining cells continued to be cultured. When they were fused to 80%~90% again, they were passaged at 1:2, and the surface specific markers of the third gener-ation cells were detected by flow cytometry. The experiment of lymphatic differentiation was car-ried out and the cells were identified. Results: The LEPCs we obtained can form a typical microtu-bule-like structure after induction in vitro, and can form a typical “pebble”-like cell morphology af-ter many passages, and its surface specific markers were: CD34, CD113 double positive rate: 87.17% ±1.77%, CD113, VEGFR-3 double positive rate: 85.17% ±1.65%. The double positive rate of CD34 and VEGFR-3 was 83.77% ±0.42%, which was consistent with the reports about LEPCs in related literature. Conclusion: High purity LEPCs can be obtained by cytokine induction and adhesion.