Study On Pulmonary Telocytes

Telocytes are a new type of interstitial cells that was initially identified by Romania professor L. M. Popescu in2005and was named formally in2010. It is confirmed and accepted that telocytes exist in almost all organs and tissues in mammals. The morphology and ultrastructure of telocytes are completely different from other interstitial cells such as fibroblast、mesenchymal stem cells, and it owns distinctive miniliform prolongation, which could form3D network structure.Through the methods of histology and immunology, it has been identified that telocytes are positive to CD34、c-kit、vimentin, and they connect with other cells such as fibroblast、immune cells、nerve cells et al, and participate in the intracellular signal transduction, especially they connect closely with stem cells and play roles as "stem cell helper cells" during the repair and regeneration of injured tissues.However, it is still a big challenge to isolate、culture and purify telocytes from tissues, and the study about the function of this new type of interstitial cells is limited. Among the studies about telocytes, most of them focus on the morphology and histology, only a few of them focus on the biological functions with slow progress.We believe that the structure and tissue distribution characteristics of a cell must respond to its function. Based on the present study results and through the methods of histology, cellular biology, genetics et al, this project tries to explore the localization and distribution of pulmonary telocytes, to reveal the differences between pulmonary telocytes and other interstitial cells, to explain the potential functions of telocytes, and to provide theoretical evidence and experiment basis for the further application of telocytes in the treatment of pulmonary diseases. Part I Identification, isolation and culture of pulmonary telocytesObjecives:To identify if telocytes exist in pulmonary tissues, to explore the distribution characteristics of telocytes in the lung, and to study the morphology and dynamic changes of cultured pulmonary telocytes.Methods:The distribution of telocytes in the lung was identified through electronical microscopy and immnuohistochemistry; mice/human pulmonary telocytes were isolated and cultured, and the morphology was observed through primary culture and vital staining; the changes of morphology were recorded through dynamic observation with Cell-IQ living cell workstation; biomarkers on the surface of telocytes were detected with flow cytometry.Results:Under the observation from both scanning and transmission electronic microscope, we found that telocytes located in the interstitial spaces of tracheal cartilage and pulmonary tissues, connected with capillaries, bronchia and alveolar epithelial cells, and surrounded pulmonary stem cells. The prolongation of telocytes, namely telopode, was a lumens structure, and consisted of an alternation of thin and thick segments. Immunohistochemistry showed that telocytes were positive to CD34, c-kit and vimentin. The cells we isolated and cultured owned morphology of typical telocytes. Flow cytometry revealed that human pulmonary telocytes were positive to CD44, CD73, CD90, CD105and negative to CD34、CD11b、 CD19、CD45、HLA-DR, which were corresponding to human bone marrow mesenchymal stem cells. Dynamic observation of primary culture confirmed that pulmonary telocytes owned the characteristics of transmigration and chemotaxis, and they could form network structure, secret particulate matter and phagocytize particulate matter secreted from other telocytes. Conclusion:Telocytes also existed in the trachea and pulmonary tissues with characteristic distributions, and they participated with network structure in small vessels and bronchium, and connected closely with alveolar epithelial cells and stem cells. Human pulmonary stem cells expressed the same biomarkers like human bone marrow mesenchymal stem cells. Telocytes could form network structure and might participate in the cellular signal transduction. Part Ⅱ Analysis of gene variation among pulmonary telocytes、 fibroblast and mesenchymal stem cellsObjectives:To study the gene variation among telocytes and other interstitial cells; to identify the telocytes special genes and confirm if telocytes are a new type of interstitial cells.Methods:To isolate and culture mice pulmonary telocytes, and to analyze the gene expression among telocytes, mice fibroblast and mesenchymal stem cells (MSCs) through genomic techniques, and to identify the different and co-expressed genes among these cells.Results:More than2000and4000genes were over-expressed and down-expressed in mice pulmonary telocytes respectively, as compared with either mice MSCs or fibroblasts. There were156co-expressed and variant genes, of which46genes had certain interactions. Varied genes were mainly involved in the regulation of multicellular organismal process, anatomical structure morphogenesis, or development process. Telocytes specially expressed Rbp1.Conclusion:There are significant variations about gene expressions among telocytes、 fibroblast and MSCs, and telocytes specially expressed Rbpl. Telocytes indeed are a new type of interstitial cells. Part Ⅲ The function of pulmonary telocytes on the repair of injured vascular endothelial cellsObjectives:To explore if telocytes could synthesize and secrete angiogenesis associated cytokines; to study if telocytes could promote the proliferation and the repair of injured vascular endothelial cells.Methods:Mice/human pulmonary telocytes were isolated and cultured for24and48hours respectively, and the level of cytokines in the supernatant, such as VEGF, EGF, TNF-α, IFN-γ, GM-CSF, MCP-1, TIMP-1, TIMP-2, were detected with ELISA; the proliferation of human pulmonary microvascular endothelial cells (HPMECs) were detected after the cells were cultured with DMEM complete media and telocytes conditional medium (TCM) respectively; the tubule generation capacity of HPMECs were detected with Matrigel, after the cells were injured with LPS and cultured with telocytes conditional medium simultaneously.Results:The level of certain angiogenesis associated cytokines increased in the supernatant of cultured telocytes, especially VEGF and EGF. Being compared with control group, the proliferation of HPMECs cultured with TCM was increased; TCM promoted tubule generation capacity of LPS injured HPMECs.Conclusions:Telocytes could secret VEGF and EGF, and human pulmonary telocytes could promote the proliferation of HPMECs, and promote the repair of LPS injured HPMECs.