VEGF-C在替代性活化的巨噬细胞转分化为淋巴管内皮细胞中的作用

Transdifferentiation of alternatively activated macrophages into lymphatic endothelial cells induced by VEGF-C

目的观察替代性活化的巨噬细胞(aaMphi)在血管内皮生长因子-C(VEGF-C)的诱导下能否转分化为淋巴管内皮细胞(LEC),初步探讨aaMphi促进淋巴管生成的可能机制。方法以重组小鼠白细胞介素-4(IL-4)处理小鼠巨噬细胞(RAW264.7)24h,建立aaMphi模型。分别以不同浓度的小鼠重组VEGF-C处理aaMphi,通过测定后者在基质胶中形成簇样物和管样结构的情况,最终确定以浓度为100ng/ml的VEGF-C建立aaMphi转分化系统。在此系统中,分别于第0、7、14和28天以实时定量RT-PCR法检测aaMphi LEC特异性标志物[血管内皮生长因子受体-3(VEGFR-3)、Prox1]和aaMphi特异性标志物(Fizz1)的情况。连续28天在倒置相差显微镜下观察aaMphi在基质胶中形成管样结构的情况。结果成功地建立了以VEGF-C为诱导剂,以EBM-2为培养基,以基质胶作为支持物的aaMphi转分化系统,其VEGFR-3和Prox1 mRNA的表达逐渐增加,而Fizz1 mRNA的表达逐渐下降,至第14天分别达到最高值和最低值;第28天和第14天的情况无明显差别。从第7天到第28天,可见aaMphi在基质胶中逐渐形成明显的管样结构,且随着时间延长数量逐渐增加。结论VEGF-C通过诱导aaMphi中VEGFR-3和Prox1的表达上调,促使aaMphi转分化为LEC:这是aaMphi促进淋巴管生成的可能机制之一。

Objective To explore whether alternatively activated macrophages （aaMphi） can transdifferentiate into lymphatic endotbefial cells （LEC） under the inducement of VEGF C, and to investigate the possible mechanisms involved in aaMphi-induced lymphangio- genesis. Methods An aaMphi model constructed by treating mouse macrophage cells RAW264. 7 with mouse recombinant IL-4 for 24h was treated with different concentrations of recombinant mouse vascular endothelial growth factor-C （VEGF-C）. After the clustering formed and the tube-like structures were detected in the matrigel, the aaMphi transdifferentiation system was finally decided to be constructed with the VEGF-C in the concentration of 100ng/ml. The mRNA expression of LEC specific markers, VEGFR-a and Proxl, and aaMphi specific marker, Fizzl, were detected by real time quantitative RT-PCR on the 0, 7th, 14th, and 28th day, respectively, after VEGF-C stimulation. Formation of tube-like structure in the matrigel was observed with inverted phase contrast microscope in 28 consecutive days. Results The VEGF-C induced transdifferention system, incubated in EBM-2 medium and sustained by the matrigel, was successfully established. In this system, it was found that the mRNA expression of VEGFR-3 and Proxl gradually increased, whilst that of Fizzl decreased. The mRNA expression of VEGFR-3 and Proxl reached the peak value, whilst that of the Fizzl went down to the nadir, on the 14th day. No significant difference in values was found between the 14th day and the 28th day. During the period of the 7th day to the 28th day, distinct tube-like structures were gradually formed in the matrigel and the numbers increased in a time-dependant manner. Conclusion VEGF-C can induce the transdifferentiation of aaMphi into LEC by up-regulating the mRNA expression of VEGFR-3 and Proxl in aaMphi, which is one of the possible mechanisms involved in aaMphi-induced lymphangiogenesis.