The Activation Of Wnt/?-catenin Signal In The Bone Marrow Microenvironment Regulates The Self-renewal And Expansion Of Hematopoietic Stem Cells

Hematopoietic stem cells?HSCs?are the origin cells of all blood cell lines.They are capable of self-renewal and continuous proliferation,and responsible for maintaining and reconstructing hematopoietic and other important physiological functions.Hematopoietic stem cell microenvironment?Niche?is located around the blood vessels near the trabecular bone area,by bone marrow mesenchymal stromal cells and endothelial cells of maintaining and regulating hematopoietic stem cell physiological functions of local tissue microenvironment^[1],In the bone marrow microenvironment,Niche cells by adhesion molecules and signaling molecules to membrane surface and HSCs direct or indirect contact and regulation of HSCs self-renewal,proliferation and differentiation,and a series of biological behavior.In order to study the expansion of hematopoietic stem cells in vitro,this study investigated the regulation of Wnt/?-catenin signal activation in bone marrow microenvironment on the self-renewal and amplification of hematopoietic stem cells by activating the Wnt/?-catenin signal pathway in stromal cells?OP9?.The paper is divided into four parts:?1?the establishment of four stromal cell lines with different activation types of?-catenin protein;?2?the effect of activation of?-catenin in stromal cells on proliferation of hematopoietic stem cells in vitro;?3?the effect of activation of?-catenin in stromal cells on the ability of hematopoietic reconstruction in mice;?4?study on the molecular mechanism of the effect of?-catenin activated stromal cells on the proliferation of hematopoietic stem cells.The results showed that:1:In vitro serum co-culture conditions,stromal cells activation in?-catenin signal can be expanded hematopoietic stem/progenitor cells?Lin^-c-Kit⁺Sca-1⁺,LSK?,but it can't promote hematopoietic reconstruction ability in irradiated mice,we are going to different degree of?-catenin protein OP9 cell activation and under the condition of LSK cells in serum co-culture conditions after 9 days,the number of groups of LSK cells with?-catenin signal enhancement in turn.Among them,the strongest activate protein?-catenin of experimental group??-catenin DeltaN90group?,the expansion of LSK cell count is more than double that of group did not activate the?-catenin group.we collect the competitive of all cells transplantation after experiment,found that the activation of?-catenin group??-catenin DeltaN90group?relative to the activation group,the expansion of the overall cell did not enhance hematopoietic reconstruction ability in mice.2:In vitro without serum co-culture conditions,stromal cells with different degrees of activation of?-catenin signal were able to amplify hematopoietic stem/progenitor cells,and the higher the degree of activation of?-catenin protein,the more the number of amplified hematopoietic stem/progenitor cells.The number of amplified hematopoietic stem/progenitor cells in the most activated?-catenin group was twice than that of the non-activated group.After amplification of the most activated?-catenin stromal cells,the clonal formation ability of LSK cells was enhanced,and the clonal formation ability of the red line colony was 2 times higher than that of the non-activated group.We will amplify cells competitive after transplantation to receptors in mice,found that the?-catenin activation group B lymphocytes in the peripheral blood and bone marrow of mice and T lymphocytes and Gr-1⁺CD11b⁺granulocyte group were higher than the proportion of not?-catenin activation,explain?-catenin type stromal cells cause expansion of overall cell activation has better to restore the normal hematopoietic function of mice.3:We use the strongest activation?-catenin stromal cells and LSK cells to co-culture in Transwell device and find out:When Transwell isolated OP9 and LSK cell,the number of amplificated HSCs was less than 2000,significantly below contact co-culture system?about 4000?,the number of amplification of?-catenin activation stromal cells secrete factors are likely to be the membrane type factors,This need to contact with the LSK cells to better promote the expansion of HSCs.This study provides a certain culture method and idea for the effective amplification of HSCs in vitro,and reveals the influence of strongly activated?-catenin expression in stromal cells on the self-renewal and amplification of HSCs.At a later stage,the molecular mechanism of activated?-catenin stromal cells regulating HSCs amplification will be explored to provide information for further revealing the complex network components regulating HSCs function in the bone marrow microenvironment.