| Purpose:Investigative studies osteogenic growth peptide (OGP) on proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells, and to explore the cells of the bone marrow mesenchymal stem cells within the OGP concentration10-11-10-7mol/L range most suitable for the proliferation of concentration as well as the most appropriate concentration of osteogenic differentiation, into osteogenic growth peptide (OGP) to provide reliable theoretical basis for further laboratory studies and in vivo studiesMethods:Take to clean standard, a New Zealand white rabbits zoonotic diseases, Not limited to male and female, using the rabbit ear vein air embolism hair killed. Strict compliance with the principle of sterile remove bilateral femoral shaft. The DMEM/F12medium flushing the medullary cavity, the cell suspension. The application of whole bone marrow culture isolated from bone marrow mononuclear cells, suspension cells can be removed without adherent adherent method. Can be obtained after repeated subculturing a pure adherent cells, using immunohistochemical detection of adherent cell surface markers objects CD44can confirm that the resulting cells for bone marrow mesenchymal stem cells.Adherent were isolated rabbit bone marrow mesenchymal stem cells, passage through the medium was changed and purified cells. P2cell MTT assay, in order to determine OGP activity is concentrated on the proliferation of bone marrow mesenchymal stem cells Degrees; The transmission of the two generation of bone marrow mesenchymal stem cells, with2x104/ml density was inoculated into96well plates,each hole to join200ul cell suspension. Inoculated with a total of7plates, each inoculated with6holes, each plate inoculated with36holes. Divided into the control group and the experimental group, the details are as follows:divided into six groups: A group (control group), B group with10-11mol/L OGP, C group with10-10mol/L OGP, D group with10-9mol/L OGP, E group with10"8mol/L OGP, F group with10-7mol/L OGP. The control group culture medium without serum DMEM/F12.The transmission of the3generation of bone marrow mesenchymal stem cells by density inoculated with2x104/hole in6well plates, to determine to promote bone marrow mesenchymal stem cells into osteoblasts best OGP activity concentration. Divided into the control group and the experimental group, the details are as follows: Group A culture medium50μg/ml vitamin the C+10mmol/Lβ-glycerophosphate+10-7mol/LOGP; the B group culture medium50μg/ml vitamin C the+10mmol/Lβ-glycerophosphate+10-9mol/LOGP; the B group culture medium50μg/ml vitamin C the+10mmol/Lβ-glycerol sodium phosphate,+10-11mol/LogP; D group (control group) culture medium50μg/ml vitamin C+10mmol/Lβ glycerophosphate.2x104/hole was inoculated into6well plates. For a total of16samples, respectively, in fifth days, ten days, fifteen days, twenty days and four points in time to collect. Each time point6holes were used to detect the activity of alkaline phosphatase.In the most suitable bone marrow mesenchymal stem OGP concentration in bone differentiation of cultured bone marrow mesenchymal stem cells into the cell morphological observation, and alkaline phosphatase (ALP) in the Gomori calcium cobalt staining, Von Kossa staining, and MTB colorimetric assay for quantitative detection of calcium. Divided into two groups:group A, control group culture fluid group; B group with OGP group. ResultBone marrow mesenchymal stem cell isolation and culture after the initial training more ingredients in hematopoietic cells,with the extension of time, These impurities cell necrosis or with the change of liquid move slowly,2days later would be able to see some of the cells adherent growth, about4days later visible after a large number of adherent cells, into a colony, The cells begin fusiform shape of fibroblasts, radial growth. Cultured for9days, cell fusion be passaged more than90%, to train the3rd generation of bone marrow mesenchymal stem cells purely minimal impurities cells. Cultured cells by immunohistochemical analysis and found that bone marrow-derived mesenchymal stem cell-specific antibody CD44(+). Proof of cultured cells to bone marrow mesenchymal stem cells. Osteoblasts added to the culture medium induced bone marrow mesenchymal stem cells in culture, cell growth performance as there will be a longer growing plateau of approximately one to two days, the cells were slow-growing, At the same time the cell morphology mostly changed, We observed that cells slowly by the spindle gradually transformed into a cuboid, and then change the polygon, the cells became large, dense cytoplasmic side; digestion and passage cells were cultured and passaged to P3generation of cells after an incubation period of about a length of about24-48hours, nearly150to200hours, the cells through the platform of logarithmic growth remain in place after the transition to the plateau.MTT staining method for the determination of the the OGP maximum active concentration:Bone marrow mesenchymal stem cells grown in medium containing OGP, A high degree of light absorption values of the values in the degree of light absorption than that of the control group (without OGP medium)。 When the OGP concentration in culture medium was10-11mol/L, can get the most active cell proliferation. Alkaline phosphatase determination of bone marrow stem cell activation is the most appropriate OGP concentration:D group (control group) has been at a lower level of expression of alkaline phosphatase expression. Compared with the other groups, ALP expression was significantly increased in the group.While A, B, C group and in B group (OGP concentration was10-9mol/L) alkaline phosphatase activity was the highest.For a culture medium containing10-9mol/L GOP recharge bone marrow stem cells into bone-induced morphological observation, Alkaline phosphatase (ALP) Gomori calcium cobalt staining the Von Kossa improved contingent staining, as well as MTB colorimetric calcium quantitative detection:The first application of10-9mol/L cultured in GOP cells gradually changes from spindle to cubic shape, become polygonal, cell surface area increases, the nucleus, clear demarcation, cytoplasmic dense, cells in the stratum is growth, no contact inhibition, forming cells were widely distributed. The second alkaline phosphatase (ALP) Gomori calcium cobalt staining, in fourteenth to21days after induction was found:in the most weakly positive or negative expression of alkaline phosphatase, a strong positive gradually transformed into almost all the cells showed strong positive. And the cytoplasm can be seen everywhere in the black deposits. While the control group had no positive performance.Third Von Kossa modified natural dyeing deposition method for the determination of calcium, also in fourteenth to21days after induction found: gradually increased, color increase nodules. By dark brown gradually turned to black, and more and more, bigger. While the control group was no change.Fourth MTB colorimetric assay for quantitative detection of calcium, in fifth days,10days,15days,20days respectively using MTB colorimetric assay, in the presence of OGP (10-9mol/L) culture of bone marrow mesenchymal stem cells induced by bone was significantly higher than that of control group (without OGP) into. Statistics calculated P<0.05, with statistical significance.Conclusion1. OGP proliferative activity of bone marrow mesenchymal stem cells have a significant role in promoting the optimal concentration of10-11mol/L, in a certain range in the dose-effect relationship (10-7—10-1lmol/L)2. The the OGP to promote bone marrow mesenchymal stem cells ALP expression of the optimal concentration of10-9mol/L and added bone marrow the OGP in cultured bone marrow mesenchymal stem cells osteogenic are better than ordinary mineral mediummesenchymal stem cells. |
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