小鼠卵黄囊内皮细胞的体外培养及其细胞因子表达的检测

Analysis of cytokines derived from murine yolk sac endothelial cells cultured in vitro

目的 :分离纯化小鼠卵黄囊内皮细胞 (YS -EC) ,并检测其细胞因子的表达。方法 :显微分离小鼠卵黄囊 ,经酶消化得到卵黄囊细胞 ,卵黄囊细胞经贴壁培养、传代纯化得到小鼠YS -EC ,酵母碎片吞噬实验检测YS-EC的吞噬功能 ,免疫组化方法检测YS -ECvWF的表达 ,应用AtlascDNAExpressionArray对卵黄囊内皮细胞表达的细胞因子进行检测。结果 :经体外培养纯化的小鼠卵黄囊内皮细胞大小形态均一 ,为圆形或卵圆形贴壁细胞 ,具有克隆形成能力 ,在传代培养过程中可形成血管条索样结构 ,具有吞噬功能 ,表达vWF ,符合内皮细胞的生物学特征。小鼠卵黄囊内皮细胞中有转化生长因子 β2 (TGF - β2 )、干扰素γ(IFN -γ)、胎肝激酶配基 (FL)、骨形态发生蛋白- 4 (BMP - 4 )、巨噬细胞炎性蛋白 - 1β(MIP - 1β)、骨形态发生蛋白 - 2A (BMP - 2A)、fms样酪氨酸激酶 2 (FLT2 )、内皮素 2 (endothelin 2 )、胸腺素β10 (thymosinβ10 )、白细胞介素 6 (IL - 6 )、白细胞介素 9(IL - 9)、白细胞介素 13(IL - 13)、小分子可诱导性化学因子A5 (SCYA5 )和酰基辅酶A结合蛋白 (ACBP)等细胞因子mRNA的表达。结论 :体外培养纯化的小鼠卵黄囊内皮细胞符合内皮细胞的生物学特征 ,有TGF - β2、IFN -γ、FL、BMP - 4、MIP - 1β、BMP - 2A?

AIM: To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS: The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS: Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor,vWF). The mRNA expressions of cytokines, such as TGF-β2, TNF-α, IFN-γ, FL, BMP-4, MIP-1β, BMP-2A, FLT2, endothelin 2, thymosin β10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION: mYS-EC was purified and expanded in vitro . The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.