Anti-apoptosis And Pro-osteogenesis Effects Of The Dimethyloxalylglycine On Mouse BMSCs And Its Effects For Repairing The Steroid-induced Avascular Necrosis Of Femoral Head Of The Rabbits

ObjectivesProlyl hydroxylases(PHD) are the rate-limiting enzyme during the hypoxia inducible factor(HIF) degradation,while the proline hydroxylase inhibitors(PHI) can inhibit the PHD’s activities to avoid the degradation of HIF and to stabilize the HIF.In this way, we can artificially simulate the hypoxic atmosphere and we call this hypoxia as chemical hypoxia.Dimethyloxalylglycine(DMOG) belongs to the ketone glutaric acid analogues of the PHI,thus DMOG can stabilize the HIF.The incidence of the steroid-induced avascular necrosis of femoral head(SANFH) increased in recent years and the morbidity of the disease accounted the first place among the non-traumatic avascular necrosis of femoral head.Until now,we don’t have effective therapy to reverse the pathology of SANFH and at last the patients have to receive the total hip arthroplasty.In chapter1of the research, we focus on the effects of DMOG for decreasing BMSCs apoptosis induced by serum deprivation and the effects of DMOG for the expression of Bcl-2/Bax protein in vitro.;in the chapter2of the research, we studied the effects of DMOG for promoting BMSCs ostegenesis and we studied the the expression of key molecule protein involving the RhoA/ROCK pathway during the BMSCs ostegenesis.;in the chapter3of the research, we observed the effects of the complex of DMOG/bone marrow mesenchymal stem cells(BMSCs)/fibrin glue(FG) transplantation through decompression for curing SANFH Through the previous studies,we can lay the foundation of DMOG for further in vivo experiments and clinical application.MethodsChapter1:Isolating the mouse bone marrow mononuclear cells from the femoral and tibial marrow cavities,purifying the bone marrow mononuclear cells through the repeated medium changes and passaging by the adherence and the we identified the bone marrow mononuclear cells as BMSCs through flow cytometer and osteogenic,adipogenic,chondrogenic differentiation.We established the BMSCs apoptosis induced by serum deprivation in vitro and we observed the anti-apoptosis effects of DMOG on BMSCs through TUNEL staining of flow cytometry.Then,we also the morphologic changes of BMSCs nucleus through Hoechst staining.We detected the HIF-1α protein expression of BMSCs under normoxic condition,1%hypoxic condition,normoxic condition combined with DMOG using western blot.We also observed the effects of DMOG on BMSCs Bcl-2/Bax protein expression.Chapter2:we focused on the effects of DMOG on BMSCs osteogenic differentiation.We employed MTT to observe the effects of DMOG on BMSCs proliferation; we determined the most efficient concentration of DMOG to promote the BMSCs osteogenic differentiation by quantitative detection of BMSCs ALP;we determined the effects of the most efficient concentration DMOG(0.5mM DMOG) on the BMSCs RUNX2,Osterix mRNA expression through real-time PCR and RUNX2protein through western blot,ALP expression through AZO ALP staining,calcium deposition through Alizarin red S staining.Western blot was employed to determine the effects of the most efficient concentration DMOG(0.5mM DMOG)on the expression level of the key proteins(total-RhoA、active-RhoA、ROCK1、p-cofilin) involved in the RhoA/ROCK pathway;phalloidin staining was used to detect the BMSCs cytoskeleton changes influenced by the most efficient concentration DMOG(0.5mM DMOG);scratch assay was employed to assay the effects of DMOG on BMSCs migration ability.The ALP expression,Ca deposition,cells cytoskeleton changes were detected again through the same method above after the BMSCs were treated with RhoA/ROCK pathway specific inhibitor-10μM Y-27632.Chapter3:We employed the injection of twice E.coli endotoxin and three times of methylprednisolone to establish SANFH rabbits model and12weeks later after the last injection, we performed HE staining,X-ray,MRI to make sure that we establish the SANFH model successfully.24SANFH rabbits were divided into4groups random6in each group,group A:negative control;group B:core decompression only;group C:transplantation of BMSCs combined with FG through core decompression;group D:transplantation of BMSCs/FG/DMOG complex into the femoral head through core decompression12weeks later after the core decompression operation.we performed MRI,VEGF and RUNX2immunohistochemistry to understand the effects of the BMSCs/FG/DMOG complex to repair the femoral head.ResultsChapter1:the flow cytometry assay showed the bone marrow mononuclear cells presented CD90(+)、CD105(+)、CD44(+)、CD34(-),and the cells can undergo osteogenesis,adipogenesis,chondrogenesis.The TUNEL assay by flow cytometry showed that serum deprivation can induce cells apoptosis compared with control group(P<0.05) and DMOG can decrease cells apoptosis rate compared with serum deprivation only group(P<0.05).The Hoechst staining showed that DMOG can ameliorate the cells morphologic changes induced by serum deprivation.Western blot assay showed that1mM DMOG can up-regulate HIF-la expression compared with control group(P<0.05) and DMOG can up-regulate Bcl-2expression but down-regulate Bax expression compared with serum deprivation only group.Chapter2:MTT assay and ALP quantitative assay showed that compared with control group,0.5mM DMOG didn’t have obviously inhibiting effects on the BMSCs proliferation and0.5mM DMOG can obviously upregulate the BMSCs ALP expression(P<0.05).0.5mM DMOG can up-regulate the mRNA expression of RUNX2and Osterix,and up-regulate RUNX2protein expression.It also can enhance the ALP staining and alizarin red staining.The western blot assay showed that0.5mM DMOG can up-regulate total RhoA,active-RhoA,ROCK1,p-cofilin protein expression; phalloidin staining showed that0.5mM DMOG can change BMSCs cytoskeleton.After the BMSCs were treated with DMOG and Y-27632at the same time,the cells ALP expression was inhibited and the Ca deposition decreased,while the BMSCs cytoskeleton didn’t change obviously.Chapter3:12weeks later after the last methylprednisolone injection, the HE staining assay showed that the osteoblasts reduced;the X ray showed that the hip joint space narrowed and the MRI showed the signal of the femoral head weaked.The MRI examination showed that the signal intensity decreased abvious and the femoral head collapsed slightly in group A;in group B the signal intensity also decreased but better than that in group A and the femoral head didn’t collapse;in group C the signal intensity also decreased but better than that in group B and the appearance of femoral head didn’t change;the signal intensity in group D decreased slightly but abvious better than that in group B and in group C.The immunohistochemistry of VEGF and RUNX212weeks later after the operation showed that the staining intensity of group A was negative;weak positive of group B and group C;strong positive of group D.Conclusions1. DMOG can inhibit the BMSCs apoptosis induced by serum deprivation;to regulate the expression of Bcl-2/Bax may be the important mechanism involving of the anti-apoptosis effects of DMOG on BMSCs.2. DMOG can inhibit inhibited the BMSCs proliferation in a dose-dependent manner;DMOG can promote BMSCs osteogenic differentiation;. Activating RhoA/ROCK signal pathway may be the important mechanism of the osteogenesis promotion on BMSCs induced by DMOG.3. We successfully established the rabbits SANFH model through twice injection of E.coli endotoxin combined with three times injection of methylprednisolone;transplantation of DMOG/BMSCs/FG complex through can repair the femoral head of rabbits SANFH through osteogenesis and angiogenesis.