An Experimental Study Of The Bergapten Effect On Differentiation Ability Of The Bone Marrow Mesenchymal Stem Cells Into Osteoblasts

Background:Bone marrow mesenchymal stem cells have been the subject of scientific attention since it was discovered in the middle of twentieth Century. The source of BMMSCS from the embryonic mesoderm, is a class with self-renewal and multilineage differentiation potential of non-hematopoietic stem cells, mainly exists in systemic solutions interstitial connective tissue and organ,content in the bone marrow is the most abundant.In recent years, with all of the bone marrow mesenchymal stem cell research and awareness of the deepening and cell biological engineering technology unceasing development, the bone marrow mesenchymal stem cells in clinical medicine and regenerative medicine is applied more and more widely, is a hotspot in the field of the tissue repair.BMMSCS isolated in vitro culture method simple, amplified faster, and in certain conditions can be induced to differentiate into osteoblasts, adipocyte, chondrocyte, most clinical applications of BMMSCS is based on the potential of multi-directional differentiation, but BMMSCS in vitro induced guide differentiation conditions are not ripe, the induced efficiency is not stable, pending further study.Osteoporosis (osteoporosis, OP) is characterized by low bone mass and microstructure of bone tissue, which leads to the increase of bone fragility and a systemic metabolic disease which is easy to cause fracture. Especially in postmenopausal women with common and postmenopausal estrogen secretion was significantly reduced, bone resorption and bone formation are speeding up, a high turnover bone metabolism, bone resorption compared to the process of bone short, resulting in bone loss.Osteoblasts, osteoclasts and bone marrow mesenchymal stem cells play an important role in the pathogenesis of osteoporosis. In recent years, the research has the anti osteoporosis effect of active components in traditional Chinese medicine is to promote bone cell proliferation or has been broken bone cell formation and function, and the role of pharmaceutical ingredients of bone marrow mesenchymal stem cells are often ignored. At present, there are few reports about the role of traditional Chinese medicine components in bone marrow mesenchymal stem cells, which can promote the ability to prevent and cure osteoporosis.Bergapten,as Furanocoumarin natural products, a variety of plant secondary metabolites, widely exist in nature, is one of effective components of bergamot, angelica, citron, and notopterygium root and other many Chinese herbal medicines. Bergapten in foreign countries used in the clinical treatment of vitiligo and other skin diseases have decades of history, and achieved good effect.In recent years the role of bergapten in tumor has become a hot topic, the study found, the gastric cancer, liver cancer, breast cancer and nasopharyngeal carcinoma cell with in vitro inhibition, and study of related mechanisms, has made good progress.At the same time, a widely studied of bergapten other effects, a study found that bergapten can by nhibiting the formation of osteoclasts and their precursors to prevent LPS induced in bone loss and absorption. Bergapten can increase BMP-2 (bone morphogenetic protein 2) expression of strengthening bone formation. Thus, bergapten indeed can promote bone formation and inhibit bone resorption function. But bergapten in bone marrow mesenchymal stem cells into the bone effect and the prevention and treatment of osteoporosis animal experiments have not been reported.Wnt signaling pathway includes 3 intracellular signal transduction pathways, namely, Wnt/beta-catenin signal transduction pathway, Wnt/Ca2+ signal transduction pathway and Wnt/planar cell polarity (PCP) signal transduction pathway. Wnt/beta catenin signaling pathway in regulation of embryo of normal development and cell proliferation, migration and differentiation of important physiological processes and plays an important role in bone mass and bone growth regulation is confirmed, is the focus of the skeletal system related disease mechanism and Study on bone metabolism.Bone marrow mesenchymal stem cell differentiation is influenced by many factors, the regulation of multiple signaling pathways, beside the Wnt/beta catenin signaling pathway regression secretion or autocrine manner affect cell differentiation and proliferation.Under normal conditions, the majority of cytoplasmic beta -catenin is bound to extend from the cell membrane to intracellular E-cadherin, while the remaining part and colorectal adenoma like protein and peptide axis multimeric protein complexes binding, is conducive to the cytoplasmic GSK-3 beta (glycogen synthase kinase) and phosphorylation of the complex the beta -catenin is phosphorylated beta -catenin susceptible to degradation of the ubiquitin proteasome system, so as to keep the beta-catenin concentration of cytoplasm free at a relatively low state, the activation of Wnt signaling pathway, through the phosphorylation and degradation of beta antagonist -catenin, the intracellular accumulation of -catenin and the content of P to the nucleus, the nucleus is connected with the TCF/LEF family of transcription factors, regulation of transcription and expression of distal bone health so as to control the downstream target genes. Therefore, to a certain extent, bergapten through the regulation of the Wnt signaling pathway to participate in the regulation of bone marrow mesenchymal stem cells into osteoblasts. Beta catenin as a downstream protein of the Wnt signaling pathway, mTORC1 exercise physiological function are important signal molecules. In practice, the research process, people often by detecting beta catenin expression quantity to measure the activity of Wnt signaling pathway. Therefore, it is not difficult to see beta catenin is in the Wnt signaling pathway is an important protein factorsObjective:Set up different concentration gradient, CCK-8 was used to detect different concentrations of bergapten on the effects of bone marrow mesenchymal stem cell proliferation activity to choose the appropriate drug concentration. By choosing a bone cell differentiation specific signs of alkaline phosphatase (ALP) immunocytochemical staining, also used Western blotting to observe runt related transcription factor 2 (Runx2) and Osteocalcin (OCN) expression and verification of bergapten in vitro on bone marrow mesenchymal stem cells into osteogenic differentiation. Establish classic ovarian resection model of osteoporosis, micro CT, and to observe the HE staining, and immunohistochemical staining technique bergapten in animals on the ability of bone. And on this basis, from the signal pathway revealed bergapten promoting mechanism of bone marrow mesenchymal stem cells differentiating into osteoblasts.Methods:1.Effect of bone marrow mesenchymal stem cells were isolated and cultured and bergapten concentration on its proliferation4 week old female C57BL/6 mice were selected, broken neck were killed after disinfection of lower limbs surgical site, in order to cut the skin and muscle cut with sterile Department of Ophthalmology, asepsis bilateral tibia and femur, and in the PBS cleaning. The two ends of the cut tibial and femoral exposed bone marrow cavity, with a syringe repeatedly absorbing medium tibia and femur bone marrow rinse, repeated pipetting after being uniformly dispersed by centrifugal tube 800r/ min centrifugal 4min, cell precipitate by adding 10% fetal calf serum and 1% double anti (penicillin and streptomycin) DMEN/F12 complete culture medium, inoculation in 25 cm2 culture flasks, density is 1×106/ml, into the incubated with the culture box. After 48 hours out of non adherent cells, the medium was changed to new. Every three days in a culture medium, were digested and passaged cells fused to more than 90%. Select the fourth generation BMMSCS, kind into 96 well plates to complete culture medium added with Bergapten (dissolved in DMSO), the concentration of 0.1,1,10,100uM/L, the control group added the same volume of dimethyl sulfoxide (DMSO) each set of 10 wells and cultured for 14 days, each well to join the 10 uLCCK8 reagent and the culture plate into the culture box to cultured for 2 hours, enzyme labeled instrument 450nm wavelength plate read, optical density value (OD), the understanding of different concentrations of Bergapten on the effects of bone marrow mesenchymal stem cell proliferation.2. Effect of Bergapten on the differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitroTake the 4th generation of BMMSCS into 6-well plate. Every pore and cell number 1X105, add 2ml into bone induction medium (complete culture medium beta glycerophosphate lOmMol/L, ascorbic acid 50 uM/L and dexamethasone 0.1 uM/L), every three days for a liquid. We added different concentrations of Bergapten, namely 0.1,1,10 uM/L, the control group with the same volume of DMSO,11 holes in each group. In the cell culture box after 14 days of culture, so as to measure intracellular ALP activity by measuring the absorbance of reagent cell lysis. Part of the cells were fixed with 4% paraformaldehyde, using BCIP/NBT ALP staining reagent. By Western blot (WB) to detect the expression of RUNX2 and OCN in cells in a variety of ways, a number of indicators of the effect of Bergapten on bone marrow mesenchymal stem cells differentiating into osteoblasts. Study on mechanism, we chose the Western blot of beta-catenin and GSK-3 beta to verify the BMMSCS Wnt pathway in the state, from the signal pathway of BP promotes BMMSCS to reveal the mechanism of bone.3. The study of Bergapten promoting osteogenesis in vivoThirty C57BL/6 mice were divided into three groups:sham operation group (sham group), operation group (OVX group, resection of bilateral ovaries), operation+BP group (group OVX+BP. After bilateral ovariectomy plus BP), with the classic model of osteoporosis to verify BP in vivo osteogenesis promoting role. Take micro computed tomography (micro CT, scanning and three-dimensional reconstruction, understand bergapten on mouse distal femoral bone microstructure and the influence of three groups of femoral specimens, and the mouse femur were observed by hematoxylin eosin staining), and osteocalcin and Runx2 immunohistochemical staining. The parameters of the micro CT scanning setup are:the scanning voltage is 70 KV, the power is 30 W, the scanning current is 429 mu A, and the thickness is 20 mu m. The parameters of bone metabolism in the post scan analysis include the following:small bone Liang Houdu (Tb.Th); BMD and trabecular bone volume (Tb.N).Results:1. Effect of BP concentration on the proliferation of bone marrow mesenchymal stem cellsBefore the effect of BP on bone marrow mesenchymal stem cells was observed, we needed a suitable concentration and had no toxic effect on the cells. Using CCK8 method to detect the optical density value of 0,0.1,1,10,100 groups. The results showed that the optical density value groups of0,0.1,1,10 groups did not differ statistically, indicating that the three concentrations of BP had no effect on cell viability, and obtained optical density value of 100 group decreased significantly, shows that the concentration of BP significantly inhibited the cell proliferation2、Effect of BP on osteogenic differentiation of bone marrow mesenchymal stem cells.We detected and stained by ALP activity showed that BP promoted the ALP activity of BMMSCS in the osteogenic induction medium, and the expression of ALP increased with the increase of the concentration. At the same time, from the OCN and RUNX2 protein immunoblotting can be seen, BP in osteogenic induction medium can also promote the expression of OCN and BMMSCS in RUNX2. By using OSX (Osterix) and immunofluorescence staining, BP can promote the osteogenic differentiation of bone marrow mesenchymal stem cells in osteogenic induction medium. Through the study on the mechanism of the, in the BMMSCS, joined BP’s experimental group A beta catenin and GSK-3 expression was significantly increased, as a downstream protein of the Wnt pathway, we can think BP activates the Wnt pathway so as to promote the BMMSCS to osteoblast differentiation.3. The study of BP promoting osteogenesis in vivoFirst, we found that in mice stained with H & E, and compared to the sham group, located in the distal femur of ovariectomized (OVX) and OVX+BP groups of mice bone trabecular sparse arrangement, trabecular and cortical bone thickness, suggesting that we in mice after surgery there is a manifestation of osteoporosis. This was caused by the decrease of estrogen secretion after the removal of the ovaries, but the OVX+BP group was significantly better than that of the OVX group, the trabecular bone was more dense, and the cortical thickness was thicker. In addition, the femur of mice found in CT scanning and Runx2 immunohistochemical staining and Osteocalcin (OCN) immune fluorescent color, we can find similar results, namely mice after surgery can find obvious osteoporosis, but after BP treatment of mice osteoporosis pine than operation group was significantly improved, further proved the BP in the animal body can also promote bone formation and prevent the osteoporosis, but we have reason to think BP may also promote BMMSCS to osteogenic differentiation in vivo.Conclusion:1.100 uM/L BP on bone marrow mesenchymal stem cell inhibited its proliferation significantly,0.1,1,10 uM/L BP does not affect the cell activity.2.BP can promote bone marrow mesenchymal stem cells to differentiate into the osteoblasts, related indicators such as ALP, OCN, Runx2, DSX,their expression will be increased, and the function of BP is through bone signal pathway of Wnt pathway activation.3.BP can promote bone formation in the mice, and it is proved that BP can promote the osteogenic differentiation of bone marrow mesenchymal stem cells into osteoblasts in vivo.