Basic research of lymphangiogenesis for new treatment of lymph edema

为了确定淋巴管内皮细胞和癌细胞之间分子的生物学选择性，在本研究中，我们尝试分离和培养人淋巴管内皮细胞，从乳腺癌患者前哨淋巴结的传入淋巴管中分离，然后表征细胞的表型。此外，我们还利用cDNA微阵列分析和实时荧光定量PCR技术对人淋巴管内皮细胞的组成基因进行了鉴定，并成功地利用胰蛋白酶消化法从乳腺癌患者前哨淋巴结的丰富的插管淋巴管中分离和建立了人淋巴管内皮细胞。细胞培养于EGM-2培养基中，添加10%FBS，在5%O_2、5%CO_2、90%N_2、37℃条件下培养。培养的细胞呈鹅卵石状单核细胞，并具有明显的Dil-Ac-LDL吞噬作用。这些细胞还结合免疫组织化学 ...更多信息 RT-CR定量分析也显示Podoplania-、VEGF R3-和Prox-1-mRNA在培养的细胞中更有选择性地表达。细胞对ecNOS、iNOS、考克斯-1和考克斯-2呈强免疫反应。利用寡核苷酸芯片技术分析了细胞组成型表达的细胞类型特异性基因。与人脐静脉内皮细胞相比，培养的细胞选择性表达血管生成素样4、氧自由基相关酶、粘附分子及其相关蛋白多糖等88种boa基因。结论：采用分离、插管、酶消化的方法培养人淋巴管内皮细胞，为进一步研究淋巴管内皮细胞的分子生物学和基因组学特性提供了一种简便易行的方法。少

In order to identify biological selective profiles of molecules between the lymphatic endothelial cells and carcinoma cells, in the present study, we have attempted to isolate and culture human lymphatic endothelial cells isolated from afferent lymph vessels of the sentinel lymph nodes in patients with breast cancers and then characterize phenotypically the cells. Furthermore we have identified the characterization of constitutive genes of the human lymphatic endothelial cells using cDNA microarray analysis and quantitative real-time Pat technique.We succeeded to isolate and establish human lymphatic endothelial cells from cannulated affluent lymph vessels of the sentinel lymph nodes in patients with breast cancers by using trypsin digestion. The celbwere cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37℃. The cultured cells exhibited a monokner with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. The cells also bind immunohist … More ochemically lymphatic vessel markers such as Podoplanin, LYVE-1, VEGF receptor 3, and Pocx-1 The quantitative RT-CR analysis also showed that the Podoplania-, VEGF R3-, and Prox-1-mRNA expressed more selectively in the cultured cells. The cells had strong immunoreactivities to anthem of ecNOS, iNOS, COX-1, and COX-2. The constitutively expressed call-type specific genes of the cells were also analyzed by using the oligonucleotide microarray method. Compared with human umbihcal vein endothelial cells, the cultured cells expressed selectively the 88 boa genes such as angiopoietin-like 4, oxygen-radicals-related enzymes, and adhesion molecules and its-related proteoglycans. In addition the findings suggest that the cultured cells seem to be human lymphatic endothelial cells.In conclusion the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be more easily and useful for investigating cellular molecular biological, and genomic properties of the cells. Less