The Relative Study Of Integrinα9in Lymphendothelial Differentiation From Adipose-derived Stem Cells(ADSCs)

ObjectiveTo isolate and culture adipose-derived stem cells(ADSCs) fromhuman adipose tissue. And then explore the changement of integrinα9inlymphendothelial differentiation of ADSCs. So as to find a molecular targetand then making contribution to the molecular treatment of lymphedemawith ADSCs.Methods①ADSCs were isolated and cultured from human adipose tissuewhich derived from the liposuction surgery. Morphological characteristicsof primary and passage cell was observed with inverted phase contrastmicroscope.②In order to indentify the cell, the expression of CD90,CD29,CD34and CD45in the cell membrane were assayed by using flow cytometry(FCM). The growth curve was drawn with MTT assay to difine thegrowing of the passage cell.③The lymphendothelial differentiation of ADSCs was induced withhuman vascular endothelial growth factor C (VEGF-C)、human vascular endothelial growth factor A (VEGF-A) and platelet-derived growth factorBB(PBGF BB) in vitro. Took human lymphatic endothelial cells(LECs) tobe the positive control group.④We detected the expression of the lymphatic endothelialhyaluronan receptor (LYVE-1), integrinα9and CD34with Western Blot.Through analyzing the expression of LYVE-1and CD34, we defined thelymphendothelial differentiation. Finally explored the changement ofintegrinα9during the differentiation.⑤The cell cycles of the three groups were determined with flowcytometry(FCM). So as to analyse the changement of cell cycle duringthe differentiation. Then the data was analysed with SPSS17.0.Results①ADSCs appeared spindle shape and adherent growth. And showedmuch dependence on the cell density. In our experiment we found that thepassage cells grew much faster than the primary cells. The growth curvesof1th,3th and5th passage of ADSCs all presented “S” shape. There was nodifference in cell activity of different passage.②The results of the antigens expression detected by FCM: CD29and CD90were detected, and CD34and CD45was not detected. Theresult was quit the same with MSCs.③From the result of western blot we discovered that thecharacteristic antigen of LECs LYVE-1was detected in the positive control group, but the characteristic antigen of vascular endothelial cells CD34was not detected. and at the same time integrinα9was detected too. Theresult related with the experiment group that LYVE-1was detected andCD34was not proved the formation of LECs, at the same time bothLYVE-1and integrinα9were a little lower than the positive control group.All the three antigens were not detected in the negative control group.④To analyse the proportion of cells which stayed at the G0/G1phase,we found that the data of the experiment group（65.462.73）%and thepositive control group（62.350.50）%were smaller than the negativecontrol group （88.791.53）%. The difference among the three groupswas significant statistically (F=186.77， P﹤0.01). Compared theexperiment group with the experiment group, the difference wassignificant statistically (q=22.06, P﹤0.01). And the same to the positivecontrol group and negative control group (q=25.01,P﹤0.01). But whichbetween experiment group and positive control group was not significantstatistically (q=2.94, P=0.083﹥0.05).Conclusion①ADSCs appear spindle shape and adherent growth. In the primaryculture, ADSCs show colony growth trend and are quit dependent on thecell density. The passage cells grow much faster than the primary cells.There is no difference in the growing of different passage of the ADSCs.Passage cells with high purity can be used in the experiment of lymphendothelial differentiation of ADSCs.②Human ADSCs can differentiate into LECs but not vascularendothelial cells when culture in vitro with VEGF-C, VEGF-A andPDGF-BB. The positive expression of LYVE-1and negative expression ofCD34can support the formation of LECs. And the cell cycle changesduring this differentiation process. The proportion of cells which staying atthe G0/G1phase will reduce.③The expression of integrinα9in LECs is related with LYVE-1. Tobe an important factor of the Prox1signaling pathway, integrinα9maymake an important role in the formation of LECs. We can also make aninference that integrinα9may obtain the opportunity to become amolecular target for the treatment of lymphedema. But as the mechanismof Prox1-integrinα9signaling pathway is not clear yet we should takefurther study on this.