| Objective:With the development of modern medicine, at present, the level of clinical treatment of department of orthopedics has made considerable progress, many diseases can be cured, but some diseases, such as nonunion of fracture, osteoporosis, avascular necrosis of the femoral head, bone defect, still need to find new treatment means, with a view to further improve the level of clinical treatment. The discovery and application of bone marrow mesenchymal stem cells provide new ideas for clinical. The vascular regeneration of fracture site is a prerequisite for callus formation, during the vascular regeneration process, angiogenesis related genes play an important role; callus formation also need seed cells for bone callus formation, i.e., osteoblast, bone marrow mesenchymal stem cells differentiation into bone is the main source of osteoblasts, at present bone marrow mesenchymal stem cells, osteogenic differentiation has made great progress, the activation of the mitogen activated protein kinase(mitogen-activated protein kinase, MAPK) pathway play a role in the differentiation of bone marrow mesenchymal stem cells of bone forming process. Osteogenic growth peptide(OGP) is a kind of osteogenic growth factor, nearly ten years of research. Through the research of bone marrow mesenchymal stem cells differentiation induced by osteogenic growth peptide,to find the relationship between gene Angptl4, Fbln5 VEGFA expression and MAPK pathway in the way of bone growth, clarify the mechanism of OGP to promote osteogenic differentiation of bone marrow mesenchymal stem cell. There will lay the theoretical foundation for the clinical application of OGP in the treatment of osteoporosis and promote the healing of fracture. Includes four experiments: Experiment 1, rat bone marrow mesenchymal stem cells isolation and culture, through the experiment to grasp the technique and conditions of rat bone marrow mesenchymal stem cells how to separate and culture; Experiment two, rat bone marrow mesenchymal stem cells differentiate into bone by OGP.Through the experiment, verify the function of OGP how to induce bone marrow mesenchymal stem cells into bone, and find the best concentration of OGP osteogenic induction effect; Experiment three,To find the best concentration of fetal bovine serum,promoting effect of bone marrow mesenchymal stem cell proliferation and differentiation by fetal calf serum on osteogenic growth peptide. Experiment four,The role of MAPK pathway in the process of osteogenic differentiation of bone marrow mesenchymal stem cells induced by OGP,and the role of gene of vascular regeneration differentiation related gene expression,through the experiment, observed the expression changes of vascular regeneration related genes(Angptl4, Fbln5, VEGFA) and MAPK pathways during the proliferation process of bone marrow mesenchymal stem cells induced by OGP.Method:Experiment one: by whole bone marrow adherent culture method and culture of rat bone marrow mesenchymal stem cells, cell morphology was observed under inverted phase contrast microscope.Experiment two: P3 generation of bone marrow stem cells which is cultured in experiment one, divided into six groups: control group, OGP 10-10mol/L group, OGP10-9mol /Lgroup, OGP 10-8mol / L group, OGP 10-7mol / L group. The control group use serum-free DEEM medium, OGP group use serum-free DEEM culture medium added with corresponding concentration of OGP,Cultured for 12 consecutive days. Observe osteoblast morphology under inverted phase contrast microscope, Identify osreoblast with gomori alkaline phosphatase staining, alizarin red staining of calcium nodule.MTT detected the absorbance value(OD value)and the differences were compared between groups.Experiment three: Measure the proliferation of BMSCs under different conditions with colorimetric method of MTT thiazole blue:Second generations BMSCs which were typical type were inoculated on 6 pieces of sterile 96 well plate according 1×105/mL density with 10%FBS complete medium.Each hole add 200 uL cell liquid, 8h later cells completely adherent, change medium into serum free medium.12 h later, cell cycle will same as others.then start for subsequent grouping experiment. Each plate bearing six groups, respectively OGP10-10 mol / L group, OGP10-9mol /L group, OGP10-8mol / L group and control group, each group of cells at the same time with the concentration of FBS was 0, 2%, 5%, 8% and 10%, five gradient, each with three holes,change liquid each 3 days. MTT method was used to detect cell proliferation rate at 1D, 3D, 5D, 7d, 9D, 12 D. All the complex holes in the plate are reserved culture liquid of 100 uL, each hole put directly into MTT 10 uL, then put it back into the incubator,incubate for 4h on the same condition. After discarding the supernatant, join 100 uL 10% SDS in each hole, 370 C culture for one night. Enzyme mark instrument test each hole absorb degree on 570 nm, recording and analysis of each group of light absorption value. Determination of intracellular alkaline phosphatase activity in pNPP method: Second generations BMSCs which were typical type were inoculated on 6 pieces of sterile 96 well plate according 1 x 105/ml density,each hole 3mL. With 10%FBS inoculation for 8h,cells completely attached to the wall, the culture medium was replaced by serum free medium, 12 h later, cell cycle synchronization. Start for subsequent grouping experiment. Experimental group: OGP10-10mol/L group,OGP10-9mol / L group,OGP10-8mol / L group,control group: FBS content of 5%, 8% and 10% concentrations, each cell has 6 holes, change liquid each 3D, scraping cells on 9 days, 4000rpm/min,collected to 1.5mL centrifuge tube, then do alkaline phosphatase activity detection. compare the differences between each group.Experiment four: the P3 generation of pluripotent bone marrow stem cells, divided into: control group(OGP induced); OGP plusU0126;OGP plus SP600125 group;OGP plus SB203580group; OGP plus U0126 plus SP600125 plus SB203580 group, 5 groups together, with 1x104/ml density were inoculated in 96 holes plate, each group 6 holes, each group culture in the medium without serum for 24 hours, after cell cycle synchronization, different MAPK pathway inhibitors were added into 2 to 4 groups according corresponding concentration, 5 group include 2 to 4 groups pathway inhibitor concentration,pretreatment for 30 minutes, then add 10-8mol / L OGP, put culture plate in the 370 C, 5%CO2 incubator to culture. Cultured for 3 consecutive days. At the same time,the supernatant and cells were taken to detect the index. Detection of RT-PCR expression of c-myc, c-jun, ATF-2, Angptl4, Fbln5, VEGFA, Cbfa1 mRNA, Western-blot(Western blot assay) to detect expression of Angptl4, Fbln5, VEGFA. Compare the expression difference.ResultExperiment one: Under inverted microscope, bone marrow mesenchymal stem cells in cell morphology is uniform, mostly round, cell body translucent, size different, mixed with other miscellaneous cells in 24 h, after changing half of the medium, the visible cells attached to the wall, were round or polygonal, changed all liquid after 48 h, note many adherent cells, 72 h later adherent cells continue to increase, the morphology of the cells become uniform, a short fusiform, with the time prolonging, the cells were spindle shaped or fusiform, gradual integration into the film, swirl distribution. It has the typical characteristics of bone marrow mesenchymal stem cells.Experiment two: inverted phase contrast microscope. The morphology of the cells were spindle shaped or fusiform, presented fibroblast morphology, uniform distribution. Confluent cells, can be a cobblestone like changes, with bone cell morphology as a typical, cobalt by alkaline phosphatase staining, black particles, alizarin red staining orange red, indicated the cultured cells get into osteoblasts. MTT detection showed medium OGP concentration in 10-8mmol/l, cultured for 12 days, the light absorption degree are highest.Experiment three: MTT detection, the lower concentration of FBS, cell proliferation is slower. When the FBS concentration is 0 medium with OGP can only short-term promote the proliferation of BMSCs. When the concentration of FBS was 2% OGP 10-8mol /L in culture fluid in the group and the other groups,it obvious but short promote the proliferation of BMSCs cells, but eventually because of lack of nutrition growth factor and began to apoptosis. When the concentration of FBS was 5% in each group cells, OGP10-8mol / L group proliferation promoting effect was significantly higher than that of OGP10-9mol / L group(P<0.05), as the culture time prolonged to 9D, part of cell proliferation. When the FBS concentration 8% of each group were more obvious differences in proliferation(P<0.01), OGP10-8mol/L group proliferation effect is still greater than the OGP10-9mol / L group, control group and OGP10-10 mol / L group had no difference(P>0.05). OGP10-9mol / L group and OGP10-8mol/Lgroup decreased significantly, the difference was not statistically significant(P>0.05), OGP10-8mol / L group no longer show their proliferative advantage. Alkaline phosphatase activity showed: the control group with statistical significance between the three FBS concentration(P<0.05), that with the increase in content of serum culture system, ALP produces a corresponding change, the concentration of FBS can influence the osteogenic differentiation of BMSCs. The concentration of OGP with the concentration of FBS in plasma increased,ALP production also increased significantly(P<0.05), indicating that FBS increases the number of cell differentiation by inducing the proliferation of BMSCs. The 8%FBS concentration of group with OGP10-8mol / L group was significantly higher than that of OGP10-9mol / L group, but the 10%FBS concentration of with OGP10-8mol / L group and OGP10-9mol / L group showed no statistical significance(P>0.05), indicating that 10%FBS interferes with the differentiation promoting effect of OGP. In summary: we think the cultivation condition of 8%FBS is more beneficial to the detection of OGP induced BMSCs proliferation and osteogenic differentiation, established scientific and reasonable training conditions for further discussion on OGP induced BMSCs proliferation and osteogenic differentiation of signal mechanism.Experiment four:In the main pathway of MAPK, ERK1/2 、 JNK 、 P38 signal transduction pathways are involved in OGP promotion of bone marrow mesenchymal stem cells differentiation into bone. Angptl4, Fbln5, VEGFA in the bone marrow mesenchymal stem cells when induced into osteoblasts by OGP expressed. C-myc in bone marrow mesenchymal stem cells differentiating into osteoblasts in 0 days, 1 days, 3 days, 5 days, 7 days are expressed; c-jun in bone marrow mesenchymal stem cells into osteoblasts differentiated in 0 days, 1 days, 3 days, 5 days, 7 days, 9 days are expressed; ATF-2 in bone marrow mesenchymal stem cells differentiating into osteoblasts in 7 days, 9 days are expressed; VEGF in bone marrow mesenchymal stem cells into osteoblasts differentiated in 1 days, 3 days, 5 days are expressed; Fbln5 in bone marrow mesenchymal stem cells into osteoblasts in the seventh day expressed. Angptl4 in bone marrow mesenchymal stem cells into osteoblast in 1 days, 3 days, 5 days, 7 days are expressed; Cbfa1 in bone marrow mesenchymal stem cells into osteoblasts differentiated in 0 days, 1 days, 3 days, 5 days, 7 days, 9 days are not expressed. Therefore, adding inhibitor groups selected in the bone marrow mesenchymal stem cells into osteoblasts induced by seventh days of detection of c-jun, c-myc, ATF-2; selection in bone marrow mesenchymal stem cells into osteoblasts induced by fifth days of detection of VEGF, Angptl4. Group 2(10-8 mol/LOGP + U group) does not express the c-myc, it means ERK1/2 pathways involved in OGP induced bone marrow mesenchymal stem cells in the osteogenetic differentiation process;Group 3(10-8 mol/LOGP + SP) groups does not express c-jun, that means JNK pathway involved in OGP induced bone marrow mesenchymal stem cell in the osteogenetic differentiation process;4 groups(10-8 mol/L OGP + SB) group does not express atf-2, it means P38 pathways involved in OGP induced bone marrow mesenchymal stem cells in the osteogenetic differentiation process;Each group express VEGF, the 3 group express more than control group, 2 group, and 5 group express less than the control group, suggesting that ERK1/2 ways participate in the bone marrow mesenchymal stem cells osteogenetic differentiation process, not only this pathways involved in the expression of VEGF, there are other ways to attend;All groups express Fbln5, but 4 and 5 group express less than the control group, suggesting that P38 pathway participate in OGP induced bone marrow mesenchymal stem cells osteogenetic differentiation process,this pathways involved in Fbln5 expression;Angptl4 groups are expression in all groups, but the third and fifth group expression less than the control group, that means JNK pathway take part in OGP induced bone marrow mesenchymal stem cells osteogenetic differentiation process, this pathways involved in Angptl4 expression;Western-blot results confirmed that as the change of VEGF, Angptl4, Fbln5 mRNA expression, VEGF, Angptl4, Fbln5 protein changes accordingly. |
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