| Part1Isolated、Purified and Identified of Bone marrow Mesenchymal Stem CellsObjective:To establish a simple and effective purification and culture method for rat (SD) bone marrow derived mesenchymal stem cells (MSCs) in vitro, providing of cell source for subsequent experiments.Methods:Whole bone marrow of two weeks inbred male SD rats were used for mesenchymal stem cells isolation, followed by adherent purification and serial passage amplification. During this period, observe cell morphology changes with Inverted phase contrast microscope, identify mesenchymal stem cell surface membrane antigen by flow cytometer. Further determination was performed after induced differentiation to osteocyte, chondrocytes, and adipocyte.Results:Cells adhered scattered in6h, firmly adhered after3day with increased cell volume and more particles, showing colony growth. After7days cell morphology became narrow, spiral or in spindle-shaped arrangement, and stay uniform after passage. Flow cytometer shows positive rate was respectively, Alizarin Red staining showing a lot of orange calcium nodules after osteogenic differentiation, fat oil red O staining showing a large number of bright red, highly refractive lipid droplets in the cytoplasm, dense texture group formed after cartilage cells induction, and alcian blue staining showing lot of blue intracellular matrix particles. Conclusion:Purified mesenchymal stem cells can be achieved through the whole bone marrow adherent method coupled with serially passaged method, which is a simple and effective method for sorting culture, providing us mesenchymal stem cells in good condition, stable traits, and strong differentiation potential. Our method not only provides a stable source of cells for subsequent experiments, but also is an ideal seed cells for tissue engineering. Part2Effect of the Feedback control between Wnt pathway and Sox9on BMSCsObjective:To control MSCs differentiation precisely and enrich wound healing, regenerative medicine theoretical basis, through investigating the Wnt pathway and Sox9expression and association on the course of chondrogenesis, focusing on the effect and mechanism of Wnt pathway regulates MSCs induction into chondrocytes via Sox9expression, and feedback regulation of Sox9overexpression on Wnt pathway activity.Methods:Stimulate primary bone marrow mesenchymal stem cells in vitro with LiCl, then observe mesenchymal stem cell proliferation by CCK8and Western blot analysis of PCNA. During bone marrow mesenchymal stem cells differentiation into cartilage, Western blot, realtime-PCR were used to detect cartilage marker Sox9, Collagen2a, Aggrecan and Wnt pathway key molecule β-catenin, especially in early and late stage, with or without LiCl and Dkk-1stimulation._Meanwhile, with or without stimulation of LiCl and Dkk-1in differentiation late stage, mature cartilage marker Collagen2a and hypertrophy index Collagen10, early osteogenic differentiation index RUNX2were tested by realtime-PCR and Sox9, P-catenin protein expression were measured by Western blot. Furthermore, after transfected with Sox9lenti-virus,bone marrow mesenchymal stem cells was detected by realtime-PCR of Collagen2a、Aggrecan, the quantitative determination of secreted GAG content, western blot analysis of Sox9and β-catenin; in the21st day of differentiation into cartilage after tranfection, immunofluorescence of Collagen2a expression and Western blot detection of Wnt pathway associated protein GSK-3β, β-catenin expression, p-β-catenin protein were conducted.Results:LiCl enhanced mesenchymal stem cell proliferation, and increased expression of PCNA protein. In chondrogenic differentiation period, Sox9, Collagen2a, Aggrecan mRNA and protein expression increased gradually with time, secreted GAG was gradually increased over time, and a significant increase in the14th day, and β-catenin protein expression appears to increase after the first reduction. In the early induction of differentiation into cartilage, LiCl promoted β-catenin protein expression, while promoting proliferation-associated protein PCNA and Cyclin D protein, and Dkk-1surpressed β-catenin protein expression and reduced protein expression of PCNA and Cyclin D; in LiCl-treated group Sox9, Collagen2a, Aggrecan cartilage related protein expressed, while Dkk-1group had the opposite result. In the late induction of differentiation into cartilage, β-catenin protein expression was increased in LiCl group and was suppressed in Dkk-1group, while Alcian blue staining was significantly enhanced in LiCl group and was decreased in Dkk-1group; secreted GAG show similar results; cartilage index Sox9, Collagen2a, Aggrecan mRNA and protein expression was also significantly higher in LiCl group, while in the Dkk-1group was significantly reduced. In the late induction of differentiation into cartilage, Collagen2a expression gradually decreased with time, Collagen10and RUNX2mRNA expression gradually increased, while P-catenin protein expression gradually increased, and Sox9expression reduced, under LiCl and Dkk-1intervention, we found LiCl upregulated β-catenin protein expression, down-regulated Sox9and Collagen2a expression and increased the expression of Collagen10a and RUNX2,which can be reversed by DKK-1. During chondrogenic differentiation period, transfection with Sox9-lenti-virus significantly increase Collagen2a and Aggrecan mRNA, and secreted GAG while decreased β-catenin protein expression; In the21th day of differentiation, Immunofluorescence showed significantly higher Collagen2a expression in transfected group, in which Western blot analysis showed higher GSK-3β and β-catenin protein phosphorylation levels.Conclusion:LiCl can activate the Wnt pathway to promote bone marrow mesenchymal stem cells proliferation. LiCl, activating Wnt pathway key protein β-catenin, promoted bone marrow mesenchymal stem cells proliferate rapidly and regulate Sox9to induce chondrogenic differentiation in the early induction of differentiation into cartilage, then in the late stage mainly regulate Sox9to promote chondrogenic differentiation; after chondrogenic differentiation Sox9was reduced, coupled with weaker expression of mature cartilage, stronger cartilage hypertrophy and early osteogenesis. Dkk-1, inhibiting the Wnt pathway, had the opposite effect. The feedback of Sox9overexpression inhibited the β-catenin expression and promoted β-catenin protein degradation, and ultimately maintained induced differentiation into cartilage. Part3Effect of the coactions between Wnt pathway and NF-κB pathway on BMSCs in inflammationObjective:To investigate the role of the Wnt pathway in bone marrow mesenchymal stem cells differentiation into cartilage in inflammation environmental, analyze the interaction between Wnt pathway and NF-κB pathway, providing a feasible solution for promoting differentiation into cartilage in inflammation and clinical treatment of articular cartilage injury.Methods:Culture primary bone marrow mesenchymal stem cells, create an inflammatory microenvironment with IL-1, then induce differentiation into chondrocytes, conduct Alcian blue staining to test chondroitin sulfate in cartilage matrix, quantify secreted GAG content. Furthermore Wnt agonists LiCl and inhibitors GSK-3β were administered; in the21thday conduct immunofluorescence for Collagen 2a, quantification of secreted GAG content, realtime-PCR for cartilage marker Collagen2a,Sox9, Aggrecan, Western blot for NF-κB pathway related protein Ikkβ, IκB, NF-κB and their phosphorylation levels, NF-κB expression within the nuclear,as well as Wnt pathway associated protein,β-catenin, GSK-3βand their phosphorylation levels, and nuclearβ-catenin expression.Results:in IL-1treated Group,Alcian blue staining was significantly weakened, secreted GAG content and cartilage index Sox9, Collagen2a, Aggrecan were also reduced;But LiCl reversed this effect while GSK-3β group manifested the opposite result. Furthermore, Western blot suggested LiCl group had higher expression of NF-κB pathway related protein and lower corresponding phosphorylation levels, lower GSK-3βphosphorylation level, lower P-catenin phosphorylation level, less NF-κB within nucleus,moreβ-catenin translocation to nucleus,which behaved in the opposite way in GSK-3βgroup.Conclusion:IL-1induced inflammatory microenvironment was unfavorable for bone marrow mesenchymal stem cells differentiation into chondrocytes. Wnt pathway agonists LiCl significantly inhibited GSK phosphorylation, thus inhibiting β-catenin phosphorylation, increasing its expression, and translocation into nucleus,at the same time,competitively inhibited NF-κB nuclear receptors,regulate NF-κB pathway in turn, thus contributing to mesenchymal stem cells differentiation into cartilage induction in inflammatory environment. |
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