脂肪干细胞诱导分化成淋巴管内皮样细胞的初步研究

Preliminary study of induction of adipose-derived stem cells into lymphatic endothelial-like cells

目的探讨利用血管内皮细胞生长因子C（VEGF—C）（156s）等生长因子诱导脂肪干细胞成为淋巴管内皮样细胞的方法。方法取健康成人脂肪组织，胰酶消化法获得脂肪组织来源的间质干细胞（adipose—derivedstemcell），通过流式细胞仪检测其表面标记，并进行成脂肪、成骨诱导等体外分化能力鉴定。取生长状态良好的P3代细胞进行诱导实验：设置含VEGF—C156s、bFGF等生长因子的诱导液组为实验组，并设空白对照组（常规L—DMEM培养基）。观察诱导前后两组细胞的形态变化，并于10d后进行LYVE一1免疫荧光的鉴定。结果成功分离纯化脂肪干细胞，流式细胞结果显示，CDl3、CD29、CD44、CDl05高表达，CD31、CD34、CD45、HLA—DR极低表达，具有间充质干细胞特性；体外成脂和成骨能力鉴定也取得成功；在体外成功利用含VEGF—C156S、bFGF等生长因子的诱导液将脂肪干细胞诱导为淋巴管内皮样细胞，实验组LYVE一1免疫荧光显色呈强阳性，而对照组则未见到阳性染色；荧光强度定量结果差异有统计学意义（P〈0．01）。结论利用含有VEGF—C156S的诱导液可以将脂肪干细胞诱导分化为淋巴管内皮样细胞。

Objective To investigate the feasibility of induction of adipose-derived stem cells into lym- phatic endothelial-like cells by VEGF-C （ 156 s） and other growth factors in vitro. Methods 0. 25% Tt＇ypsin was used to digest adipose tissue obtained from healthy adults. FACS was employed to detect cell surface markers and in vitro differentiation capacity was identified by adipogenic and osteogenic induction. The third generation of cells with good growth state was utilized to perform the following experiment. Set cells induced by induction medium containing VEGF-C156 s, bFGF as the experimental group, and a control group（ L-DMEM MEDIUM）. After 10 days induction of adipose-derived stem ceils, ceils were stained with anti-LYVE-1, a specific marker for lymphatic endothelial cells. Results Adipose-derived stem cells were successfully obtained by trypsin di- gestion and purification. FACS results manifested similar mesenchymal stem cell characteristic of high positive rate of CD13, CD29, CD44, CD105 and low positive rate of CD31, CD34, CD45, HLA-DR. Lymphatic endo- thelial-like cells induction in vitro by the induction medium containing VEGF-C156s, bFGF and other growth factors was achieved with high expression of LYVE-1. Intensity of LYVE-1 immunoflurorescence was valued by NIH ImageJ software. The differences between experimental and control group with different induciton mediums had statistical differences （P 〈 0. 01 ）. Conclusion Adipose-derived stem cell can be induced into lymphatic endothelial-like cell by the induction medium containing VEGF-CI56s, bFGF and other growth factors in vitro.