| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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
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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
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