| BackgroundPro-chairman of International Lymphology Academy, Professor JR. Casley Smith indicate even : "Treatment of lymphedema is a notorious difficulty" , We adopt such conservative treatment according to patient's condition as massage acupuncture and so on., or we adopt operative treatment and drug therapy; however, conservative treatment is time-consuming; it only relieve symptom temporary and its effectiveness is not obvious, operation treatment is usually limited in range in treatment.and post-operation occur lymphatic vessel re-blockage .Its short-term effectiveness is satisfactory .long-term effectiveness is not satisfactory。operative treatment。As the aspect of drug treatment, emictory use in especial treatment of lymphedema, which threaten the function of heart, lung. But it did not adopt in common lymphedema. Therefore, there is not ideal treatment method to aim directly at lymphedema in clinic.Since 1996, A critical advance came from the pioneering work by Joukov and colleagues, who identified a ligand of FLT-4 (VEGFR-3), the vascular endothelial growth factor VEGF-C, a member of VEGF/PDGF family and abundant aminothiopropionic acid in C'. After found it, in early stage, most research was developed on its genes, molecular structure, receptor and its structural feature, bionomics, and so on. Mesenchymal Stem Cells (MSCs) developed from mesodermic desmohemoblast, and has the ability differentiate into osteoblasts, chondroblasts, and adipocytes in response to appropriate stimuli, it had been became a kinds of ideal cell in tissue and cell repair for its merits as followed:1. Bone marrow is an alternative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation, which obviates immunologic incompatibilities, is possible.2. Of the various progenitor cells that exist within bone marrow, mesenchymal stem cells (MSC) are particularly attractive for clinical use because they are easily isolated, can be expanded in culture, and can be genetically manipulated using currently available molecular techniques.3. MSCs are adult stem cells, has no ethics questionsSo, it is important for applicated MSCs into degenerative disease, genetic deficiency or others.Objectives1. To separate and culture human bone marrow mesenchymal stem cells. Then identify the stem cells and store cells for further study.2. Induce the differentiation of human bone marrow mesenchymal stem cells by VEGF-C156s and decide the best concentration and time of VEGF-C156s to induce differentiation.3. To study the function of lymphatic endothelial cells induced from MSCs in three dimensional substances.Methods1. Get the human bone marrow mesenchymal stem cells by lymphocyte separation medium (1.073g/ml) and adherent culture. Store cells for further study by passage and freeze cells. Identified the stem cells by flow cytometry to detect the surface antibody as CD14,CD34,CD44,CD105 and CD166. 2. Observe the effect of VEGF-C156s on differentiation of human bone marrow mesenchymal stem cells into lymphatic endothelial cells with different concentration and at different time. Count the ratio of positive stain cells after stained by VI factor and LYVE-1.3. Prepares collagen gel on the ice box and then put into 37℃incubator. Inoculate the cells with good growth condition on the surface of the collagen gel. Then observe the growth of cultured cells by inverted phase contrast microscope. Vertical section was also observed.Results1. Human bone marrow mesenchymal stem cells were get by lymphocyte separation medium and adherent culture. The surface antibody identified by flow cytometry show results with positive CD44,CD105 and CD166 surface antibody. Negative results were get by CD14,CD34 surface antibody analysis. The results are coincidence with references, and proved the isolated cells are mesenchymal stem cells.2. With concentration of 50ng/ml, VEGF-C156s can induce the differentiation of human bone marrow mesenchymal stem cells from the five day. The differentiated cells show positive stain of VI factor and LYVE-1.3. Lymphatic endothelial cells induced from MSCs can grow with tubiform structure.Conclusions1. Isolated and cultured human bone marrow mesenchymal stem cells from bone marrow with high purity and get ready for further study.2. Human bone marrow mesenchymal stem cells can be induced by VEGF-C156s and differentiated into lymphatic endothelial cells.3. Lymphatic endothelial cells induced from MSCs have the function to form vessels and formed lymphatic vessels in vitro.
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