| Objective:An animal model of type 2 diabetes mellitus(T2DM)was established by intraperitoneal injection of streptozotocin(STZ),and on this basis,an animal model of closed tibial fracture was established to study the effect of T2 DM on fracture healing.Rat bone marrow mesenchymal stem cells(BMSCs)were extracted,and the constructed CXCL13 overexpression plasmid was transfected into BMSCs.On this basis,the effect of CXCL13 overexpression on the proliferation,apoptosis and osteoblast differentiation of BMSCs in high glucose environment was explored.On the basis of the established T2 DM rat fracture model,BMSCs overexpressed CXCL13 were injected into the fracture end to study the effect of CXCL13 overexpressed BMSCs on fracture healing in T2 DM rats.Methods:(1)Founding an effective diabetic fracture model in Wistar rats.In this experiment,T2 DM rat model was established by intraperitoneal injection of streptozotocin,and on this basis,a diabetic rat fracture model was further established by tibial osteotomy.During the experiment,the general state of the rats was observed,the blood glucose,blood ALP,blood calcium and blood phosphorus levels of the rats were detected,the bone mineral density at the fracture site of the rat was detected,and the biomechanical strength of the fracture site was detected by a biomechanical measuring instrument.H&E staining of tissue sections was used to observe the healing of fractures,and ALP staining was used to observe the proliferation and differentiation of osteoblasts at the fractures.The effect of diabetes on fracture healing in rats was comprehensively analyzed.(2)Effect of chemokine CXCL13 transfection on bone marrow mesenchymal stem cells.Rat mesenchymal stem cells(BMSCs)were extracted,isolated and cultured,CXCL13 plasmid was constructed,and CXCL13 plasmid was transfected into BMSCs cells by biotransfection technology.The transfection efficiency of CXCL13 was detected by Western Blot and q RT-PCR;the effect of high glucose environment on the expression of CXCL13 was detected by Western Blot and q RT-PCR;the cell viability was detected by MTT assay;the effect of CXCL13 on BMSCs apoptosis was detected by flow cytometry;The effect of CXCL13 on the expression of OPC and OC in BMSCs was detected by Western Blot and q RT-PCR;the effect of CXCL13 on the differentiation of BMSCs into osteoblasts was observed by ALP staining.(3)Effect of BMSCs transfected with CXCL13 on fracture repairing in diabetic rats.In the established diabetic rat fracture model,BMSCs transfected with CXCL13 were injected into the fracture site of diabetic rats,the bone mineral density of the fracture site was detected,and the biomechanical strength of the fracture site was detected by a biomechanical measuring instrument.After observation and sectioning,H&E staining was used to observe the tissue healing.To comprehensively analyze the effect of CXCL13-transfected BMSCs on bone healing in diabetic fracture rats.Results:(1)After successful model establishment of T2 DM rats,obvious symptoms of polydipsia,polyphagia,and polyuria were observed;in the established fracture model of T2 DM rats,dual-energy X-ray detection showed that the bone mineral density at the fracture end of T2 DM rats was significantly reduced;It was found that the biomechanical strength of the fracture end of the T2 DM rats was significantly lower;the general morphology of the fracture end and H&E staining of the tissue sections showed that the callus at the fracture end of the T2 DM rat was reduced,indicating that the bone tissue at the fracture site was poorly healed;ALP staining of the fracture end showed that the fracture of the T2 DM rat was fractured.The terminal ALP staining became lighter,suggesting that osteoblast differentiation was reduced;in addition,serum calcium,osteoprotegerin(OPG),and osteocalcin(OC)expressions in T2 DM fractured rats were significantly decreased.(2)Rat BMSCs cells were extracted and isolated.Through gross morphological observation,the isolated cells were identified as BMSCs by flow cytometry after labeling with BMSCs-specific antibodies.The CXCL13 plasmid was constructed and then transfected into BMSCs.Western Blot and q RT-PCR verification showed that CXCL13 was successfully transfected.Western Blot and q RT-PCR detection showed that high glucose environment could inhibit the expression of CXCL13 in BMSCs;Western Blot and q RT-PCR detection showed that CXCL13 transfection could increase the expression of OPG and OC in BMSCs cells;MTT assay showed that CXCL13 transfection could inhibit the expression of CXCL13 in BMSCs.Reverse high glucose-induced BMSCs proliferation slowdown;apoptosis experiments found that CXCL13 transfection can reverse the high glucose environment-induced BMSCs apoptosis;ALP staining found that CXCL13 transfection can reverse the high glucose environment-induced BMSCs differentiation weakened.(3)The BMSCs cells transfected with CXCL13 were constructed to construct the fracture model of T2 DM rats.BMSCs cells transfected with CXCL13 were injected into the fracture ends of T2 DM rats in the treatment group.Dual-energy X-ray detection showed that the bone mineral density at the fracture site of the treated group was significantly higher than that of the non-injection group,and biomechanical testing showed that the biomechanical strength of the fracture site of the treatment group was significantly higher than that of the non-injection group.The bone callus at the fracture end of the rats in the group was more abundant,and the injection of BMSCs transfected with CXCL13 showed the effect of promoting bone healing in the rats with T2 DM fractures.Conclusions:1.The T2 DM animal model can be successfully established by intraperitoneal injection of STZ,and the animal model is divided into the disease characteristics of the human body;after bone tissue imaging observation,tissue morphology observation,biomechanical testing,and tissue section staining observation,it is found that T2 DM has a significant effect on fracture healing.significant delay.This can provide more sufficient research clues for further experiments.2.CXCL13 transfection can promote the proliferation of BMSCs in high glucose environment in vitro,and at the same time promote their differentiation into osteoblasts.CXCL13 transfection also inhibits the apoptosis of BMSCs in high glucose environment in vitro.This suggests that CXCL13 may act as an important promoter of BMSCs proliferation and differentiation under high glucose environment.3.The injection of CXCL13-transfected BMSCs into the fracture end of diabetic rats can effectively promote the formation of bony callus at the fracture end of diabetic rats,and increase the biomechanical strength of the fracture end.It plays a pro-osteogenic effect,suggesting that the injection of CXCL13-transfected BMSCs at the fracture end of T2 DM can promote fracture healing.This study provides new ideas for the treatment of diabetic fractures in the future. |
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