| For a long time, extracorporeal shock wave therapy used to treat urinary system stones, and achieved good clinical efficacy. In recent years found ESWT can activate osteoblasts, and promote fracture healing. Osteoblasts activation is a very complicated biological regulation process, which concerns several aspects, such as circulating hormones, local cytokines, expression of activated proto-oncogenes and signal transduction, furthermore there is a closer relationship between osteoblast activation and systemic calcium metabolism. ESWT studies show that it can prompt hMSCs to differentiate bone cell, induce cell proliferation, increase alkaline phosphatase activity, type I collagen protein expression and osteocalcin synthesis, and ultimately induce bone formation. ESWT as a new non-invasive orthopedic treatment, compared to traditional surgical treatment have many advantages, this treatment has been widely accepted and recognized.The mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in this process. MAPK pathway can be activated by the source of stimulation signal, including mechanical stimulation, cytokines, growth factors, neurotransmitters, hormones, cell adhesion and cell stress,and so on. The pathway was activated and phosphorylate nuclear factor and other protein kinase, phospholipase, cytoskeleton-binding protein, and other substrates, regulate gene transcription and then participate in physiological processes, such as cell growth, development, split and the synchronous functions of cells. Activated protein -1 (AP-1) is a intersection of cell signaling pathways in the nucleus. As the one of substrates of MAPK signaling pathway, it Composite by Jun and Fos family members. AP-1 have a clear role in bone formation and osteoblast differentiation: activation of bone formation, expressed high levels of c-fos and c-jun of the mRNA. However, which MAPK pathway involved in osteoblast differentiation, ERK, JNK or p38MAPK pathway? And the role of ERK, JNK or p38MAPK pathway is not yet very clear.Msenchymal Stem cells (MSCs) are multipotent stem cells derived from mesoderm, possessing potent proliferative capacity and self- renewal and pluripotent. MSCs can differentiate into cells derived from the same germ-layer, for example, osteocytes, adipocytes and muscle cells under the control of different environ- ment and cytokines. The new works show that they can also transdiff- erentiate into liver cells, neuron-like cells and neuroglial cells derived from other germ-layer. MSCs have some advantages, including drawing the materials easily and slightly injury, multiple amplify- cation easy in vitro, no ethical matters and immune rejection and differentiation easy. For this reason, MSCs have been the optimum and main source of seed cells of islet cell transplantion. There are more and more research find that MSCs can differentiate into various cells though specific stimulation, such as osteoblasts and chondroblasts both in vitro and in vivo. As primitive stem cells with multidiferen- tiation potential, MSCs have been widely used as vector cells in tissue engineering and cell engineering. Therefore the applications of hMSCs in these areas have drawn great attention and have been widely explored in clinical and preclinical studies. The intervention research of ESW induce hMSCs are less and the emphasis are varies. In this respect, this experiment focused on human mesenchymal stem cells (hMSCs), hMSCs derived from adult bone were isolated, culture-expanded efficiently in vitro and its biological features are detected. BM cells were obtained from iliac crest aspirates from healthy donors after informed consent. BM was layered over 1.073g/ml Percoll solution and centrifuged at 900g for 30 min. Mononuclear cells collecting at the interface were recovered, resuspended in Dulbecco's Modified Eagles Medium- Low Glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 2mM L-glutamine, and incubated at 37℃in 5%CO2 in air. Then in vitro observe the morphology under microscope, count the amount of cells and draw cell growth curve,detect cell cycle through FACS and osteoblast-differentiation potentiality were detected.,Objective: By density gradient centrifugation isolation and culture of hMSCs. Application 8.5 Kv, 120 frequency shock wave induced hMSCs,observe the role of ERK, JNK and p38MAPK pathway in the shock wave-induced hMSCs to osteoblast differentiation and the expression of c-fos and c-jun mRNA.Methods: Detect alkaline phosphatase activity and calcium deposition to reflect differentiation from hMSCs to osteoblast. Used Western-blot to reflect the MAPK pathway,RT-PCR to investgate the expression of c-fos and c-jun mRNA. Applications ERK, JNK and p38MAPK kinase inhibitor (PD98059, SP600125, SB203580) to inhibit corresponding access, detect the shock wave whether through ERK,JNK or p38MAPK pathway to increase c-fos and c-jun expression and phos- phorylation.Results: hMSC have been successfully isolated and culture -expanded. The isolated and culture-expanded hMSC get the well homogenicity , multi-potential adult stem cells character.Compared with the control group, in the shock wave induced, ERK,JNK and p38MAPK pathway were activated early. ERK pathway inhibitor (PD98059) significantly inhibited the proliferation of hMSCs and the p38 MAPK pathway inhibitor (SB203580) significantly reduced ALP activity and calcium deposition, inhibition the expression of c-fos and c-jun mRNA and hMSCs differentiation to osteoblasts. JNK pathway inhibitor (SP600125) reduced ALP activity and calcium deposition, inhibition of c-fos and c-jun expression, but p> 0.05, no statistics significance.Conclusion: In process of shock wave induced hMSCs to osteoblasts differentiation, through the activation of p38 MAPK pathway, increased the expression c-fos and c-jun mRNA, increase in the number and activity of MAPK substrate AP1, ultimate prompting hMSCs to osteoblasts differentiation. ERK pathway promote cell proliferation.JNK pathway May be involved in the differentiation, but the role needs to be further explored. |
