目的

建立一个稳定的大鼠胃癌移植瘤新生淋巴管内皮细胞（LEC）培养体系。

方法

SGC7901胃癌细胞被接种到4周龄的裸大鼠足垫，瘤周围注射1%的Evans蓝显示淋巴管，显微解剖淋巴管，应用免疫磁珠法分选纯化LEC后在EGM-2培养液中培养，细胞免疫组化法检测LEC特异性标记物淋巴管内皮透明质酸受体1（LYVE-1）、膜糖蛋白（podoplanin）、血管内皮生长因子受体3（VEGFR-3）的表达，以管腔形成实验判定LEC能否形成淋巴管样结构。

结果

成功构建裸大鼠胃癌移植瘤动物模型，显微解剖获得瘤周淋巴管。免疫磁珠法分选LEC，其纯度为95%，活细胞数大于70%。免疫组织化学检测表明，LEC表达VEGFR-3、podoplanin和LYVE-1。在Matrigel基质胶上，LEC能形成管腔样结构。

结论

淋巴管解剖和免疫磁珠法可成功分选胃癌移植瘤新生淋巴管内皮细胞，从而建立一种简便和稳定的LEC培养体系。

Objective

To establish a convenient and stable culture system for tumor lymphatic endothelial cells (LEC).

Methods

SGC7901 human gastric adenocarcinoma cells were injected into the footpad of the nude rat.Evan's blue dye in PBS was injected around the xenotransplantational tumor in order to display the lymphatic vessels around the tumor.Microanatomy technique was used to dissect lymphatic vessel.Mamnetic activated cell sorting (MACS)technology was used to purify the LEC.Expressions of LEC specific markers,LYVE-1,podoplanin and VEGFR-3,were identified by immunohistochemical staining.The capability of LEC to form lymphatic vessel-like structures was assessed by the in vitro lymphatic vessel formation assay.

Results

Injection of Evans blue around the tumor permitted the rapid display of the lymphatic vessels around the tumor. About 95%LEC were harvested MACS and among them more than 70% were living cells. The expressions of LYVE-1,podoplanin and VEGFR-3 were positive in LEC.LEC could form lymphatic vessel-like structures.

Conclusion

LEC in tumor can be harvested by microanatomy technique and MACS,which is a convenient and stable culture system.