Abstract：CD9, known as motility- related protein- 1(MRP1), is a tetraspanin transmembrane protein. This kind of protein is related to aggressive behavior of cancer cells. CD9 was initially reported as a tumor metastatic suppressor, and its overexpression suppressed metastasis of cancer cells. However, similar studies reported that CD9 expression was rather intensified in cancer tissue in comparison with normal tissues. The role of CD9 in tumor development is still controversy. Deer antlers are the only mammalian appendages capable of full renewal. Unprecedented growth rate (up to 2.75 cm/d) of antlers provides a rare system where fast cell proliferation is elegantly regulated without becoming cancerous. Previous studies reported that antler stem cells expressed high level of CD9 antigen. CD9 is thought to be involved in cell proliferation, motility, differentiation and signal transduction. To examine the function of CD9 in antler stem cells, Western blot analysis, immunofluorescence and flow cytometry were performed to verify expression of CD9 in antler stem cells (including antlerogenic periosteum cells (APC), pedicle periosteum cells (PPC) and reserve mesenchymal cells (PMC) in antler tips). To estimate the biological function of CD9 in cell motility, Anti-CD9 antibody was used in motility assays. And in cell proliferation, MTT assay with Anti-CD9 antibody was performed. The results showed that all of 3 kinds of antler stem cells expressed high levels of CD9 antigen, and more than 90% of primary cultured antler stem cells were CD9 positive. Neutralization of CD9 in antler stem cells did not significantly affect cell migration. Neutralization of CD9 could significantly promote cell proliferation of pedicle periosteum cells and reserve mesenchymal cells, but not antlerogenic periosteum cells. Most of factors involved in extracellular matrix-integrin and focal adhesion signal path were detected in antler stem cells. The results demonstrated that CD9 could serve as a useful marker for isolation of antler stem cells and might play an important role in antler stem cell proliferation. Revealing the function and regulatory mechanism of CD9 in antler stem cells may contribute to the understanding of the role of CD9 in rapid proliferating cells, especially tumor cells. Overall, deer antlers can be developed into a novel model for research of CD9 function.
