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Expression and Regulation of Angiopoietins and Their Receptor in Sika Deer Antler

Author: ZhangZuo
Tutor: YueZhanPeng
School: Jilin University
Course: Basic Veterinary Science
Keywords: Angiopoietins sika deer antlers regeneration
CLC: S825
Type: Master's thesis
Year: 2012
Downloads: 38
Quote: 1
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Deer antler re-growth is a unique regenerative event in mammals which in general are unableto replace bony appendages. This growth appears to be co-ordinated in a manner analogous to fetallong-bone growth, but occurs in a mature animal. Thus deer antler growth provides a uniquenatural model of organic regeneration of mammals,especially of rapid and complete boneregeneration. Recent evidence suggests that antler regeneration is a stem cell-based process thatdepends on the activation of stem cells located in the pedicle periosteum which are presumed to beneural crest-derived. The fact that despite their enormous growth rate the antlers of intact andcastrated deer appear to be resistant to malignant transformation furthermore offers researchopportunities for cancer biology.Angiopoietins(Ang), a family of major angiogenic growth factors, Four members of theAngiopoietin family have been discovered, Tie-2(tyrosine kinase with immunoglobulin andepidermal growth factor homology domains, Tie-2) which possessed the immune globulin and theepidermal growth factor homologous structure of domain is their common receptors. Angiopoietinfamily can effectively promote angiogenesis and participate in control of other angiogenesisfactors, it has been confirmed that Angiopoietin related with regeneration, mature and stability ofthe blood vessels of the network system. Angiopoietins-1(Ang-1), which is a glycoprotein withmolecular weight of70KD, can promote mature and stability of the new blood vessels andstability of the blood vessels with inflammation. Angiopoietins-2(Ang-2) is a natural antagonist ofAng-1, it also can combine with Tie-2with a good affinity, but without phosphorylation of Tie-2.Ang-2can block Tie-2activation from Ang-1, and increase vascular permeability and decline itsstability. Ang-3(mouse) and Ang-4(people) are interspecific homologous body. ThusAngiopoietins is considered as the preferred molecules for study of coordinating angiogenic inantler growth process.The aim of this study was to localize the expression of Angiopoietin(Ang)-1, Angiopoietin(Ang)-2and their receptor Tie-2within the growing antler tip of one-year-old sikadeer and to explore the cell proliferation and the expression of Ang-1,Ang-2of the cartilage cellsin vitro after treatment with oestrogen and testosterone. The expression of Ang-1, Ang-2and Tie-2was explored through the research on growing tip in sika deer antler using in situ hybridizationand western blot, and then compared the expression difference between the tissue zones of sikadeer antler (i.e., antler epidermis, dermis, mesenchyme, precartilage, transition zones, cartilage).The in situ hybridization results showed that Ang-1mRNA was highly expressed in the fibroblastsof the dermis, the maturing chondroblasts of the precartilage and the chondroblasts, chondrocytes,chondroclasts, osteoclasts, the hypertrophic chondrocytes and the blood vessel-associated cells ofthe cartilage columns. The expression of Ang-1mRNA signal could also be detected in theepidermis and transition zones. The positive cells in the epidermis were basal cells and there wasno detectable signal in hair follicle and sebaceous glands in the epidermis of sika deer antler.Ang-1mRNA was expressed in the chondroblasts and chondrocytes of the transition zones. Ang-1mRNA was lowly expressed in the undifferentiated mesenchcymal cells of the mesenchyme layer.The results suggest that Ang-1may contribute to the deer antler dermis fibroblasts rapidproliferation and maturation of the chondroblasts, thereby promote the development of antlercartilage and increase the growth rate of the deer antler skin to coordinate with the rapid growth ofdeer antler cartilage. There was no obvious difference in the intensity of the signal between Ang-1and Ang-2in the tissue zones of sika deer antler. The results showed that Ang-2mRNA wasmainly expressed in the fibroblasts of the dermis, the maturing chondroblasts of the precartilageand the chondroblasts, chondrocytes, osteoclasts, the hypertrophic chondrocytes and the bloodvessel-associated cells of the cartilage columns, Ang-2mRNA was also detected in the basal cellsof the epidermis and the chondroblasts and chondrocytes of the transition zones. However, therewas no detectable signal in the mesenchyme. Tie-2mRNA was only detected in the maturingchondroblasts of the precartilage and the chondroblasts, chondrocytes, osteoclasts and the bloodvessel-associated cells in the cartilaginous column. However, there was no detectable signal in theother tissue zones. Localization of Ang-1and Ang-2in fibroblasts, the maturing chondroblasts,chondroblasts, chondrocytes, osteoclasts, the hypertrophic chondrocytes and the bloodvessel-associated cells suggest that Ang-1, Ang-2and Tie-2may play an important role in theregulation of cell proliferation and differentiation of developing antler skin and cartilage. Thewestern blot analysis also provided evidence that Ang-1and Ang-2expression were localized differentially in the dermis, mesenchyme, precartilage, transition zones, cartilage of the antler deer,however, there are expression difference between the different tissue zones. Western blot analysisshowed that Ang-1protein expressed strongest in mesenchyme and precartilage. In the dermis andtransition zones, Ang-1protein was also detected. However, the expression in cartilage layer wasvery weak. Ang-2protein expressed strongest in dermis and mesenchyme. In the precartilage,transition zones and cartilage, Ang-2protein was weaker.Isolated and cultured chondrocytes from adult healthy sika deer antler by cell culturetechnique, respectively, added oestrogen and testosterone of different concentration into theculture medium of the second generation chondrocytes, after24h and48h, MTS assay wasperformed to detect the cellular proliferation rate, and half quantitative polymerase chainreaction (PCR) was performed to detect influence of the expression of Ang-1and Ang-2from thetwo hormones. The results showed that the chondrocytes in vitro can completely adherencewithin24h. Then we observed the cells under inverted microscope, the results indicated that thechondrocytes showed clone-like growth and closely arranged, the cells were triangle or polygon.The results of Trypan blue staining showed that the cell activity was above90%. MTS assayresults showed that the proliferation activity of chondrocytes with10-7、10-8、10-9、10-10g/mltestosterone treatment was marked higher than the control group, while there was no significantdifference of the proliferation activity of chondrocytes between10-11g/ml testosterone treatmentgroup and the control group. The proliferation activity of chondrocytes with10-9、10-10、10-11g/mloestrogen treatment was marked lower than the control group, while there was no significantdifference of the proliferation activity of chondrocytes between10-7and10-8g/ml oestrogentreatment group and the control group. This suggests that testosterone may play a role inpromoting the proliferation of the chondrocytes and antler growth,and oestrogen may play a rolein inhibiting the proliferation of the chondrocytes and antler growth during antler growingprocess. Half quantitative PCR results show that Ang-1expression of the chondrocytes in10-9and10-10g/ml testosterone was enhanced, especially in10-10g/ml testosterone treatment. Ang-1expression of the chondrocytes in10-8g/ml testosterone was weakened. Ang-2expression of thechondrocytes in10-8、10-9、10-10g/ml testosterone were enhanced. These results showed that alow concentration level of testosterone can promote Ang-1expression in the chondrocytes, whilea high concentration level of testosterone can inhibit Ang-1expression. The influence of Ang-2expression from testosterone concentration level is not obvious. Ang-1did not expressed in the chondrocytes after10-7g/ml oestrogen treatment, Ang-1expression of the chondrocytes in10-8g/ml oestrogen was weakened, Ang-1expression of the chondrocytes in10-9g/ml oestrogen wasenhanced. Ang-2expression of the chondrocytes in10-7、10-8、10-9g/ml were all inhibited. Theseresults showed that a high concentration level of oestrogen can inhibit Ang-1expression in thechondrocytes, while a low concentration level of oestrogen can promote Ang-1expression. Theinfluence of Ang-2expression from oestrogen concentration level is also not obvious. Theseresults will provide important theoretical basis for further study of molecular regulationmechanisms of regeneration and rapid growth in antler.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Deer
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