Experimental Study Of Calcitonin Gene-related Peptide On Proliferation And Osteogenic Differentiation In Precartilaginous Stem Cells

Part Ⅰ Effect of CGRP on proliferation of precartilaginous Stem CellsObjective:Isolated, purified and identified of precartilaginous stem cells to establish a simple and effective purification and culture method for precartilaginous stem cells rat (SD) in vitro, providing of cell source for subsequent experiments.Method:1 Isolated, purified and identified of precartilaginous stem cells:Neonatal SD Rats which were in 24 hours of births, surgical microscope ring cut La Croix Central, cultured cells. MACS sorting isolated and purified PSCs, serially passaged, amplified. Observation of cell morphology and growth under inverted phase contrast microscope.2 FGFR-3 expression of cell surface was detected by horseradish peroxidase-labeled immunocytochemistry.3 Cell proliferation assay, the purified 3rd generation PSCS were seeded in 96-well plates, they were treated with CGRP at concentrations of 0,10-8,10-9 and 10-10mol/L, detected by CCK-8 kit at 0,3,7,10 and 14 days, then drew growth curve and analyzed of different concentrations of CGRP on PSCs proliferation.Result:1 Most of purified PSCs were triangular or polygonal, transparent, adherent growth, refraction good, large nuclear with small cytoplasm, and in uniform of cell morphology and size.2 Cell proliferation assay showed that there was no significant statistical significance at different concentrations of CGRP treated in PSCs at the same observation points.Conclusion:1 MACS sorting could obtain a purified stable PSCs, providing of source of subsequent experiments.2 Different concentrations of CGRP have no effect on the proliferation of PSCs in vitro.Part Ⅱ Effect of CGRP on osteogenic differentiation of precartilaginous Stem CellsObjective:To determine CGRP affect PSCs expression of Osteogenic gene (RUNX2, OPN, BGP), Extracellular matrix Collagen I, ALP and Calcified nodules in vitro.Method:1 PSCs which were classified to experimental group and control group were cultured in Osteogenesis induction medium with or without CGRP at concentration of 10-9mol/L.2 Cultured for 14 days, alizarin red staining. Calcified nodules of experimental group and control group were detected by inverted phase contrast microscope and analyzed by image analysis software.3 Alkaline phosphatase (ALP) activity, quantitation and staining assays were performed at day 3,7,10 and 14 after control group and experimental group cultured with 0 or 10-9mol/L CGRP respectively.4 PSCs were collected and total RNA was isolated after induced culture for 7 and 14 days, Real-time PCR assay for RUNX2, OPN, BGP and Collagen I of control group and experimental group respectively.Result:1 After 14 days of CGRP stimulation, experimental group was observed more and lager calcified nodules. Quantitative detection showed Mineralized nodule area between the two groups had significant difference.2 ALP activity showed between the two groups were no significant statistical significance at day3 The experimental group than the control group ALP content was significantly increased trend in 7-14 days.4 RT-PCR showed RUNX2, OPN, BGP and Collagen I of experimental group seemed more significantly increase than control group.Conclusion:CGRP could up-regulate expression of RUNX2, OPN, BGP and Collagen I, ALP and Calcified nodules of PSCs in vitro.Part Ⅲ The role of the canonical Wnt signaling pathway in osteogenic differentiation of precartilaginous Stem Cells induced by CGRPObjective:Discussion of the Wnt pathway involved in CGRP induced PSCs osteogenic differentiation.Method:1 Purified 3rd generation PSCS were seeded in 6-well plates and classified into 4 groups with osteogenic induction culture medium:control, CGRP 10-9mol/L, DKK-1 10-8mol/L and CGRP 10-9mol/L+DKK-1 10-8mol/L. Total protein was isolated after osteogenic induction culture for 7 days and protein concentration was determined by BCA method.2 Expression of β-catenin protein was revealed by Western blot and analyzed by graphics software.3 Real-time PCR assay for RUNX2, OPN, BGP and Collagen I of PSCs of 4 groups respectively.Result:After CRhe eGP stimulate PSCs significantly promote the expression of β-catenin protein. Treat with β-catenin inhibitor DKK-1, β-catenin expression was significantly reduced, while down-regulate expression of RUNX2, OPN, and BGP. However, no remarkable change of Collagen I was detected. CGRP can significantly increase protein β-catenin expression suppressed by DKK-1.Conclusion:CGRP could partly activate canonical Wnt/β-catenin pathway to promote osteogenic of PSCs differentiation.