Study The Regulation The Differentiation And Proliferation Of Epiphysis Stem Cells And The Mechanism By Notch And PTHrP

Part 1: Regulation the growth of epiphysis stem cells by NotchExperiment 1: Culture of epiphysis stem cells and review of their growth characteristics in vitroObjective: To study the culture of mouse epiphysis stem cells in vitro and the review of growth characteristics for the actual application.Methods: Epiphysis stem cells were harvest by collagenase digestion after microoperation, and pured by Percoll density gradient centrifugation, then the cell growth curve, mitotic index and alkaline phosphatase were observed, the types II and X collagen were detected by immuno- histochemistry and immunofluorescence, and the transmission electron microscope was used to observe the content of rough endoplasmic reticulum.Result: Mouse epiphysis stem cells are fibroblast-like, with little alkaline phosphatase and rough endoplasmic reticulum, and much type II collagen.Conclusion: Epiphysis stem cells cultured in vitro can differentiate into the mature cells, which could provid ideal seeds cells for the cartilage or bone tissue engineering. Experiment 2: Regulation the differentiation and proliferation of epiphysis stem cells by NotchObjective: To study the regulation of differentiation and proliferation of epiphysis stem cells cultured in vitro by activated Notch.Methods: The rhNF-κB , an activator of Notch , and the γ — secretase inhibitor II (MW167) were added into the mediums of epiphysis stem cells respectively, and the control was added with PBS buffer. Then the detections were carried out by RT-PCR, immunohistocbemistry, MTT, flow cytometry(FCM), alkaline phosphatase staining and Western Blot.Result: Notch1 and Jagged1 were expressed in epiphysis stem cells. The expressions of PCNA, Collagen II of rhNF-κB group were more than those of control, and the effect of promoting proliferation was obvious (p=0.027), more cells stayed at S phase(26.54%). The expressions of alkaline phosphatase and Collagen X of MW167 group were increased obviously, but the expressions of Collagen II and stathmin became decreased apparently.Conclusion: When the Notch signaling was activated, the epiphysis stem cells were going on proliferating. On the contrary, the cells were going on differentiating when the Notch signaling was suppressed Experiment 3: Study the apoptosis repression and the mechanism of epiphysis stem cells by activated Notch1Objective: Study the apoptosis repression and the mechanism of epiphysis stem cells cultured in vitro by activated Notch1.Method: The expression of Notch was detected by RT-PCR, then rhNF-kB, an activator of Notch, andγ—secretase inhibitor II (MW167) ,an inhibitor of Notch, and HA 14-1, an inhibitor of Bcl-2, were added into the medium of epiphysis stem cells, and the control was added with PBS buffer. After the induced by Celecoxib, the cell vigour was detected by MTT, the apoptosis was detected by flow cytometry, and the proteins Hes-1 and Bcl-2 were detected by Western Blot.Result: Notch1 and Jagged1 were expressed in epiphysis stem cells. In the group of rhNF-kB, the cell vigour and the expressions of Hes-1 and Bcl-2 increased and the cell apoptosis decreased. But in the groups of MW167 and HA 14-1 and control, the cell apoptosis increased obviously, the differences are significant (p<0.05). And there was no expression of Bcl-2 in the groups of HA 14-1 and HA 14-1 with rhNF-kB.Conclusion: Activated Notch1 could repress the apoptosis of epiphysis stem cells by promoting the expressions of the target gene Hes-1 and anti-apoptosis protein Bcl-2. Experiment 4: The growth of mouse limb bud was regulated by NotchObjective:To study the regulative effects of Notch signal system on the vegetations of organs cultured in vitro.Methods: Limb buds of mice were cultured in vitro for 16 days. The proliferation and differentiation of osteoepiphysis stem cells were detected by meassuring the grouth length in portait direction, HE staining and TB staining.Results: To compare with the control group, the limb buds of mice grew slowly in portait direction in rhNF-κB trial group and quicklier in MW167 trial group with significant difference among those groups (p<0.05).It was showed that osteoepiphysis stem cells in quiescent zone were increased markedly and matured cells in proliferation and hypertrophy zones were few in proportion in rhNF- κB trial group; It was showed that osteoepiphysis stem cells were visibly in differentiated state, and very few in quiescent zone,and matured cells in proliferation and hypertrophy zones were increased significantly in proportion.Conclusion:The actived Notch signal system had regulative effects on the proliferation and differentiation of osteoepiphysis stem cells cultured in vitro,the same to the osteoepiphysis stem cells in vivo. Part 2:Experiment 5: Study the regulation the differentiation and proliferation of epiphysis stem cells and the mechanism by Notch and PTHrPObjective: To study the regulation the differentiation and proliferation of epiphysis stem cells and the mechanism by Notch and PTHrP.Methods: The coexpression of Notch and AP-1 and PTHrP was detected by immunohistochemistry. After the transfection of PTHrP, the proliferation was detected by MTT, the collagen X and PTHrP and AP-1 were detected by Western blot.Result: When the Notch was activated, it promoted the expression of PTHrP and AP-1. After the transfection of PTHrP, the proliferation became strong obviously (p<0.05), and the differentiation was repressed. When Notch was activated, the expression of AP-1 and PTHrP increased, on the contrary , they decreased.Conclusion: Notch promoted the expression of PTHrP by AP-1. Notch and PTHrP regulated the growth of epiphysis stem cells by the way of three-dimensional method.