Expression And Regulation Of TGF-β Family And Their Receptors In Sika Deer Antler

Deer antler is the unique mammalian tissue which can fully regenerate after losing. The regeneration of deer antler involves the rapid proliferation of a number of tissues, including: epidermis, dermis, cartilage, bone and nerves. Therefore, deer antlers provide an excellent model to study the developmental regulation of these tissues. Recent evidences suggest that antler regeneration is a stem cell-based process that depends on the activation of stem cells located in the pedicle periosteum which are presumed to be neural crest-derived. It has been demonstrated that several developmental pathways involved in antler regeneration arealso known to play a role in the control of skeletal development and regeneration in other vertebrates. However, in contrast to most other natural examples of regeneration of complete body structures, antler regeneration apparently neither depends on a functional nerve supply nor involves a direct contact between wound epithelium and mesenchymal tissue. Therefore, the regeneration process of deer antlers in bone tissue of mammals is different from the epimorphosis of lower grade vertebrates. Deer antler grows very rapidly, and the proliferation speed of mesenchyme in its growth center is faster than the proliferation speed of tumor cells.In mammals, currently, the TGF-βhas three Isoforms: TGF-β₁, TGF-β₂ and TGF-β₃, whose biological characteristics are similar and share the same receptor. TGF-βis a multifunctional growth factor, which shows different physiological effects due to different types of cells, different microenvironment and different self-state of cells. SB-431542 is a specific TGF-βI receptor inhibitor, which can competitively bind ATP site and specifically inhibit TGF-βI receptor so as to prevent phosphorylation of I-receptor and block the expression of specific target genes of TGF-βsignaling pathway.The aim of this study was to investigate the expression of TGF-βisoforms and its receptor in sika deer using in situ hybridization. The results of in situ hybridization showed: the mRNA expression of TGF-β₁, TGF-β₂, TGF-β₃ in the epidermis of Sika deer antler could only be detected at a low level, and the expression level was increased gradually from outside to inside, and it reached the highest value in the basic level of epidermal, which was mainly expressed in the keratin cells of stratum spinosum. TGF-β₁, TGF-β₂, TGF-β₃ mRNA signal was highly detected in the proliferating cells of the dermis, particularly TGF-β₃ and it was mainly expressed in the dermis fibroblast, whole there was no detectable signal in the perivascular cells and hair bulb and sebacecous glands. It was suggested that TGF-βmight contribute to epidermal cell differentiation and maturation of velvet and velvet skin dermis rapid proliferation of fibroblasts, thereby increase the growth rate of the skin and dried quickly, coordinate the rapid growth of deer antler cartilage, while TGF-β₃ might play a leading role. Deer skin sebaceous glands and hair follicle cells might not be directly regulated by TGF-β. The expression of TGF-β₁, TGF-β₂, TGF-β₃ mRNA signal could be detected in the fibrous region of the perichondrium, and the TGF-β₃ mRNA signal level was the highest. The signal was mainly showed on periosteal fibrous cells, while the signals of cells around the blood vessel were slow. TGF-β₁, TGF-β₂, TGF-β₃ mRNA were lowly expressed in the mesenchyme, but the signal of TGF-β₂, TGF-β₃ was higher than TGF-β₁. The positive cells were the undifferentiated senchymal cells. The express level of TGF-β₂ and TGF-β₃ was higher than TGF-β₁ in deer antler mesenchyme. It was suggested that TGF-β₂, TGF-β₃ played an important role in the induction of antler mesenchymal cells and cartilage cell proliferation. TGF-β₁,TGF-β₂ and TGF-β₃ mRNA were highly expressed in the chondrocytes of the cartilage columns, but not in perivascular cell in cartilage and the highest signal level was TGF-β₃. The positive cells were the maturing chondroblasts cells and the mature cartilage region cells. It suggested that TGF-βplayed an important role in maintaining the rapid proliferation of cartilage cells and cartilage cells maturation, while TGF-β₃ was the most important. The expression location of TGF-βI and type II receptor mRNA was similar with TGF-β, only with part difference. At the same time, we used cell culture method in vitro, adding blocker SB-431542 to block TGF-βsignaling pathway to research the changes of proliferation speed of cells in different tissues of Sika deer antler under the conditions of lack of the regulation of TGF-βsignaling. The results of cell experiment showed that antler cells were cultured in new medium on day 3, and the non-adherent cells were removed. Then we observed the antler cells under inverted microscope, the results indicated that antler cells showed clone-like growth, and the adherent cells proliferated rapidly. The results of Trypan blue staining showed that the cell activity was above 90% or more. The cells of dermal layer tightly arranged with thin fibrous morphology, and cell proliferation had not been significantly affected; the cartilage cells arranged in loose, mostly showing slender polygon, and cell proliferation was inhibited significantly; cells in mesenchymal layer showed fusiform or triangular and polygon, cell proliferation had also been inhibited significantly; most of the former cartilage layer cells were fusiform or spindle, few cells were cobblestone-like, and cell proliferation activity had increased. The cells in cartilage layer showed triangular or polygon, we found few stained cells were fusiform or spindle, and the cell proliferation was inhibited.It was suggested that TGF-βmight have little proliferative effect to dermal cell or the dermal cell could be regulated by other complex regulation of the subnet; TGF-βmight promote the proliferation of cartilage cells, mesenchymal cells and cartilage cells; TGF-βmight promote the differentiation of precartilage cells differentiation and inhibit the growth of precartilage cells.The present study will provide important scientific basis to further understand the function of TGF-βisoforms in antler regeneration and the regulation of rapid growth.