| BackgroundFemoral head necrosis is caused by a combination of factors,resulting in a decrease in blood supply to the femoral head and death of subchondral bone cells.The mechanical strength of the femoral head is then reduced and eventually becomes a disease in which the femoral head collapses and the hip joint becomes dysfunctional.The existing research has done a lot of research on the etiology,pathology and pathogenesis of avascular necrosis of the femoral head,but its exact mechanism and related regulatory genes are still unclear,so it is necessary to find the regulatory genes and targets for the regulation of bone necrosis.It is important.At present,relevant studies suggest that the main causes of femoral head necrosis are:lipid metabolism disorders,including abnormal expression of PPARy and HDAC9c-S.PPARy is an adipokinetic transcription factor widely present in human adipose tissue.Glucocorticoids can activate a pro-regulatory transcription factor,which can specifically bind to the corresponding site in the promoter region of PPAR? gene and up-regulate the expression level of PPARy.When the expression of PPAR? is increased,the bone marrow mesenchymal cells can be regulated to differentiate into a large number of fat cells,resulting in a large accumulation of fat cells in the femoral head,resulting in a large volume of fat cells,an increase in pressure in the femoral head,and compression of the artery.Insufficient blood supply at the femoral head,which in turn oppresses the vein,interferes with the venous return of the femoral head,resulting in insufficient differentiation of osteoblasts,which ultimately leads to necrosis of the femoral head.BMP-2 is an important factor in bone growth and mineralization.It has strong osteoinductive activity and is ubiquitous in vertebrate bone matrix,which has obvious promotion effect on bone repair.OPG is a member of the tumor necrosis factor receptor superfamily.It is synthesized by osteoblasts and stromal cells and has the function of regulating osteoclast differentiation and activity.It can directly affect the differentiation and efficacy of osteoclasts and regulate bone remodeling.The secretion system next to the horizontal axis is involved in bone resorption.SERPINE1 is associated with intravascular disorders of the femoral head.The SERPINE1 gene located on human chromosome 7 encodes the PAI-1 protein,which is released by platelets and vascular endothelium when stimulated to form a thrombus,mainly acting on tissue plasminogen activator and uricase plasminogen activator.In turn,it acts to inhibit the activation of plasminogen,promotes blood clotting,and causes thrombosis or femoral head necrosis.This study will study the correlation between various related genes such as BMP-2,PPAR-?,OPG,HDAC9c-S,SERPINE1 and femoral head necrosis in the tissues of patients with femoral head necrosis by various experimental techniques.The relationship between the genetic polymorphism of SERPINE1 and the necrosis of the femoral head,and finally whether the overexpression of OPG has a protective effect on the apoptosis of chondrocytes.ObjectivesPart ?:To detect the expression levels of BMP-2,PPAR?,OPG,HDAC9c-S and SERPINE1 in the femoral head tissue of normal group,steroid-induced and alcoholic femoral head necrosis,and to investigate whether the occurrence of femoral head necrosis is between the above genes.There is a relationship.Part ?:Using the gene chip to detect the correlation between SERPINE1 rs1 1178 and rs2227631 polymorphisms and SERPINE1 haplotype analysis in peripheral blood and femoral head necrosisPart ?:According to the results of the first part of the study,OPG was overexpressed in human chondrocytes and its effect on tBHP-induced apoptosis of human chondrocytes was examined.MethodsPart ?:A total of 106 femoral skull tissues were collected from hospitalized patients who were admitted to our hospital from October 2012 to October 2015 and diagnosed as steroid-and alcohol-induced femoral head necrosis by clinical diagnosis and histopathological examination.The study subjects recorded a series of clinical features such as gender,age,duration of disease,and ARCO staging.109 patients who underwent femoral neck fractures in our hospital and volunteered to donate femoral skull tissue were used as control group.The pathological changes of the femoral head were observed.The expression of BMP-2,PPARy,OPG and HDAC9c-S in the femoral head was detected by qPCR.The BMP-2,PPARy,OPG and HDAC9c-in tissues were detected by western blot.S protein expression is detected.The second part:using gene chip polymorphism to detect SERPINE1rs11178 and rs2227631 polymorphism analysis,linkage disequilibrium and other methods to calculate the frequency difference of genotypes.Part ?:Application of lentiviral transfection in human chondrocytes to overexpress OPG in cells.Under oxidative stress conditions of tBHP,cell viability was detected by MTT assay,ROS and apoptosis were detected by flow cytometry,TUNEL The apoptosis was detected by Western blot and the expression of apoptosis-related signaling pathway protein was detected by western blot.ResultsPart ?1.Histopathological observation of the femoral head:In the normal control group,the femoral head tissue was full and round,the articular cartilage was intact,the surface was smooth and the color was white.In patients with steroid-induced femoral head necrosis,the shape of the femoral head is not full and round,flat collapse,and there is rupture and wear in the articular cartilage.After the dissection,the necrotic area is filled with gray-yellow granular sand-like necrotic tissue without bone trabecular structure.In patients with alcoholic femoral head necrosis,the femoral head tissue is not full,flat collapse,and there are ruptures and defects in the articular cartilage.After the dissection,large necrotic bones in the necrotic area can be seen,and there is a gray-yellow granular sand sample with or without trabecular structure.Necrotic tissue,surrounded by normal bone tissue separated by taupe bands.2.qPCR technology to detect genes in femoral head specimens:Compared with the normal group,the expression of BMP in patients with steroid-induced femoral head necrosis was significantly decreased(P<0.05);BMP expression level and normal group in patients with alcoholic femoral head necrosis There was no significant difference in patients with steroid-induced femoral head necrosis(P>0.05).Compared with the normal group,the expression of PPARy in patients with steroid and alcoholic femoral head necrosis was significantly increased(P<0.05).Compared with patients with steroid-induced femoral head necrosis,the expression of PPAR? was significantly increased in patients with alcoholic femoral head necrosis.(all P<0.05).Compared with the normal group,the expression of OPG in patients with steroid and alcoholic femoral head necrosis was significantly decreased(P<0.05).Compared with the normal group,the expression of HDAC9c-S was significantly increased in patients with steroid and alcoholic femoral head necrosis(both P<0.05).Compared with patients with steroid-induced femoral head necrosis,HDAC9c-S expression in patients with alcoholic femoral head necrosis The level was significantly increased(P<0.05).Compared with the normal group,the expression level of SERPINE1 was significantly increased in patients with steroid and alcoholic femoral head necrosis(both P<0.05).3.Western Blot detected protein expression in femoral head specimens:compared with the normal group,the expression of BMP-2 and OPG in the steroid-induced femoral head necrosis group and alcoholic femoral head necrosis group decreased significantly(P<0.05).Compared with the normal group,the expression of HDAC9c-S protein in the alcoholic femoral head necrosis group and the steroid-induced femoral head group decreased significantly(both P<0.05),and the steroid-induced femoral head necrosis was compared with the alcoholic femoral head necrosis group.There was a significant decrease in the group(P<0.05).In PPARy,the alcoholic femoral head necrosis group and the steroid-induced femoral head necrosis group were significantly higher than the normal group(both P<0.05),and the alcoholic femoral head necrosis group Compared with the steroid-induced femoral head necrosis group,the group increased significantly(P<0.05).Compared with the steroid-induced necrosis group and the alcoholic necrosis group,the normal group showed a significant decrease in the expression of SERPINE1 protein(P<0.05).Part ?1.There was no significant difference between the case group and the control group by observing the rs11178 genotype and allele frequency(P>0.05).Although the genotype frequency of rs2227631 AG was different between the case group and the control group,the difference was not obvious.The frequency of rs2227631 AA genotype was significantly increased,suggesting a significant correlation with the onset of ANFH(P=0.024,OR= 2.363,95%,Cl=1.110-5.028).The mutation of base A on rs2227631 is closely related to ANFH,which indicates an increased risk of ANFH(P=0.016,OR=1.606,95%CI=1.090-2.367).2.There was no linkage disequilibrium at the rs11178 and rs2227631 loci(D =0.765,R2 = 0.506).Four haplotypes consisting of two single nucleotide polymorphisms.T-A and C-A haplotypes were found to be more frequent in the case group than in the control group.However,compared with the T-G haplotype,the T-A haplotype was significantly increased,and the control group was significantly different from the femoral head necrosis group(P=0.016,OR=2.271,95%CI=1.154-4.469).Part ?Overexpression of OPG by lentiviral transfection in human chondrocytes showed that overexpression of OPG significantly inhibited the decrease of human chondrocyte activity and ROS induced by oxidative stress-inducing factor tBHP,mainly through the overexpression of OPG.Late apoptosis and early apoptosis of chondrocytes,TUNEL method further confirmed this effect;detection of apoptosis-related signaling pathway protein expression found that OPG over-expressed chondrocytes mainly promote the phosphorylation of Akt,promote Bcl-2 It expresses and inhibits the cleavage of Caspas-3 and inhibits the elevated expression of Bax to exert its anti-apoptotic and anti-oxidative stress effects.ConclusionsPart ?By comparing 106 femoral head tissues with femoral head necrosis and 109 patients with normal femoral head tissue,it was found that compared with normal femoral head tissue:BMP-2 expression down-regulation,PPARy expression up-regulation,OPG expression down-regulation,HDAC9c-S Up-regulation of expression,up-regulation of SERPINE1 expression;significant differences in PPARy and HDAC9c-S expression between patients with steroid-induced femoral head necrosis and alcoholic femoral head necrosis may be related to the pathogenesis of both types of disease,but further development is needed the study.The up-regulation of SERPINE1 and the possible regulation of BMP-2,PPAR?,OPG and HDAC9c-S may require further study.Part ?The SERPINE1 gene rs2227631 polymorphism was associated with femoral head necrosis,while the haplotypes rs11178 and rs2227631 were associated with femoral head necrosis;the up-regulation of SERPINE1 may be associated with the rs2227631 polymorphism.Part ?Overexpression of OPG in human chondrocytes can significantly inhibit the decrease of cell activity induced by oxidative stress-inducing factor tBHP and the increase of ROS,and this effect and its overexpression of OPG inhibit late apoptosis and early apoptosis,while TUNEL method This result was further confirmed.Through the study of apoptosis-related signaling pathways,it was found that OPG overexpressing chondrocytes exert their anti-apoptosis and anti-apoptosis mainly by promoting phosphorylation of Akt,promoting Bcl-2,inhibiting the cleavage of Caspas-3 and the expression of Bax. |
PDF Full Text Request