| Background and objective:Steroid-induced Avascular Necrosis of the Femoral Head(SANFH),as a refractory disease in orthopedics,often develops irreversibly and the disability rate is extremely high.However,its exact pathogenesis is currently unclear.It is known that bone marrow-derived mesenchymal stem cells(BMSCs)may be closely related to abnormal osteogenic and adipogenic differentiation.Previous studies have shown that Intraflagellar Transport Proteins(IFT80)It is clearly related to the Hedgehog signaling pathway,and the downstream protein of the latter can regulate the osteoblast differentiation of BMSCs.The activation of Hedgehog signal can promote the activation of downstream related proteins,thereby activating Gli,entering the nucleus,and triggering the expression of osteogenic genes through the classic Hedgehog / Gli pathway.At the same time,Hedgehog is also a key regulator of adipogenesis,and activated Hedgehog can inhibit the ability of adipocyte differentiation through the non-classical pathway of Smo / Lkb1 / Ampk axis.However,the role of IFT80 /Hedgehog in regulating the osteogenic and adipogenic differentiation of BMSCs in SANFH has not been reported.We hypothesized that the IFT80 / Hedgehog signaling pathway is involved in regulating BMSCs’ osteogenic and adipogenic differentiation balance and plays an important role in SANFH.Preliminary detection of the expression changes of osteogenic related factors,adipogenic related factors,IFT80,and key protein Shh of Hedgehog signaling pathway in the femoral head of SANFH patients.For further verification,a rabbit model of hormonal femoral head necrosis was established,and the above relevant indicators were dynamically detected to more clearly verify their expression in hormonal femoral head necrosis.Initially explore the role of IFT80 / Hedgehog signal in SANFH,lay the foundation for further progress to clarify its mechanism of action,and then provide a new target for SANFH diagnosis and treatment.Research methods:Collect the femoral head of hip arthroplasty patients,a total of 63 cases,including 34 cases of hormonal femoral head necrosis(experimental group)and29 fresh femoral neck fracture specimens(control group);(immunohistochemistry,IHC)to detect the expression of IFT80 and Shh proteins in the femoral head;real-time-polymerase chain reaction(RT-PCR)to detect IFT80 and Sonic Hedgehog(Shh)in the femoral head of the two groups m RNA expression;extract the total protein of the femoral head of the patient,and quantitatively detect the IFT80,Shh,Runt-related transcription factor 2(Runt-related transcription factor 2,Runx2),peroxisome proliferator-activated receptor γ,PPAR-γ of the femoral head by Western blot)Expression to extract total RNA from the patient ’s femoral head.2.Take 72 healthy New Zealand adult white rabbits and divide them into groups A and B by simple randomization,divided into model group(group A)n= 36 and control group(group B)n = 36.In the model group,endotoxin(lipopolysaccharide,LPS,10 ug / kg,5ug / kg)+ Methylprednisolone sodium succinate for injection(MPS,20 mg / kg)injection was used to construct a hormonal femoral head necrosis model;the same batch The control group was injected with the same amount of normal saline at the same time.Each batch of rabbits was examined by magnetic resonance imaging(MR1)and hematoxylin and eosin(HE)staining at week 4,week 8,and week 16 during the model building process.Verify the effectiveness of the model.RT-PCR was used to dynamically detect the expression of IFT80 and Shh m RNA in two groups of femoral heads;Western blot was used to dynamically detect the expression of IFT80,Shh,Runx2 and PPAR-γ protein.Research result:1.The difference between the experimental group and the control group in terms of gender composition,age and body weight wasn’t statistically significant(P>0.05);immunohistochemical staining showed that IFT80 protein was positively expressed in osteoblasts and trabecular bone at the edge of trabecular bone In intraosseous cytoplasm and in bone marrow cavity intercellular stroma;Shh protein was positively expressed on the trabecular bone edge osteoblasts and bone cell membranes in trabecular bone;RT-PCR results showed hormonal femoral head necrosis group The expression levels of IFT80 and Shh m RNA were lower than those of femoral neck fracture group,the difference was statistically significant(P <0.05).Western blot results showed that the expression of IFT80,Shh,Runx2 protein in the hormonal femoral head necrosis group was significantly lower than that of the femoral neck fracture group,and the expression of PPAR-γ protein was higher than that of the femoral neck fracture group,the difference was statistically significant(P <0.05)2.MRI examination results showed that the T1 WI signal of the bilateral femoral heads in group A was reduced,spotted or line-like,and the T2 WI showed an uneven high signal;the B1 rabbit femoral heads had no obvious abnormal signals on T1 WI and T2WI;HE stained tissue sections Microscopic observation showed that the trabecular bone in the femoral head of the group A was disordered,thinned or even broken,the adipocytes in the medullary cavity increased and increased,and the hollow bone filling fossa of the trabecular bone was more common;The structure is basically normal,no disorder or fracture is found,and empty bone lacuna is rare;RT-PCR results show that the expression of IFT80 m RNA in the femoral head of group A is lower than that of group B(p<0.05),and the expression of Shh m RNA in group A is also lower than that of group B cut back.Western blot results showed that the expression of IFT80 protein,Shh and Runx2 protein in group A were lower than that in group B,while the expression of PPAR-γ protein increased,the difference was statistically significant(p <0.05).In conclusion:1.The expression of Runx2 protein is down-regulated and the expression of PPAR-γ protein is up-regulated during hormonal femoral head necrosis,suggesting an imbalance of osteogenic and adipogenic differentiation.2.In hormonal femoral head necrotic bone tissue,IFT80 expression is down-regulated,and Shh expression is also down-regulated,suggesting that IFT80 / Hedgehog(Shh)may be involved in the development of SANFH disease. |
PDF Full Text Request