Basic research of lymphangiogenesis for new treatment of lymph edema

为了鉴定淋巴管内皮细胞和癌细胞之间的分子生物学选择性，本研究试图分离和培养乳腺癌患者前哨淋巴结传入淋巴管中的人淋巴管内皮细胞，并对其表型进行鉴定。此外，我们还利用基因芯片技术和实时定量PAT技术对人淋巴管内皮细胞的构成基因进行了鉴定。利用胰酶消化法，成功地从乳腺癌患者前哨淋巴结富含淋巴管的淋巴管中分离和建立了人淋巴管内皮细胞。在含10%胎牛血清的EGM-2培养液中，在37℃、5%O2、5%CO2、90%N_2的条件下培养细胞。培养的细胞呈鹅卵石状的单核细胞，对Dil-ac-LDL有明显的吞噬作用。这些细胞还与免疫组氨酸…结合。更多的淋巴管标记物，如PODOPLAIN、LYVE-1、VEGFR3和POXX-1。定量RT-CR分析也显示PODORANIA-、VEGFR3-和ProX-1-mRNA在培养的细胞中有更高的选择性表达。细胞对ecNOS、iNOS、COX-1、COX-2有较强的免疫反应。利用寡核苷酸微阵列技术分析细胞Call-型特异性基因的组成表达。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蛋白4、氧自由基相关酶、黏附分子及其相关蛋白多糖等88个boA基因。综上所述，我们采用的分离、插管和酶消化法培养人淋巴管内皮细胞可能更容易，更适合于细胞的分子生物学和基因组特性的研究。较少

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