Research For The Role Of Leptin And Inflammation On Heterotopic Ossification

Objective To detect partial expression of leptin in achillotomy mice and to study the heterotopic ossification model of mice.To study the effect of parecoxib and celecoxib on partial tissue inflammation and heterotopic ossification in achillotomy wild type mice.To study the different effection on partial tissue inflammation and heterotopic ossification in achillotomy ob/ob mice which respectively received injection of NS,LEP or LEP+ Parecoxib.To study the the associations between NAT2 gene polymorphisms and osteosarcoma risk and metastasis in young Chinese population.Methods Achilles tenotomy surgery was carried out in groups to establish heterotopic ossification model.Mice in each group were respectively given each kind of medicine.ELISA was used to detect the concentration of TNF-α protein at different time points.X-ray and Histomorphology staining were used to observe the formation of heterotopic bone and the partial tissue morphology.Micro-CT was used to observe the parameters of newly formed bone.We carried out a case-control study to investigate seven NAT2 SNPs on the risk and prognosis of osteosarcoma.The Taq Man method was used to determine genotypes.Clinical characteristics of osteosarcoma were used to further investigate the relationship between SNPs and the incidence of osteosarcoma.Results In wild-type mice groups,LEP protein concentration and m RNA expression in the experimental group was significantly higher than the control group at 3 weeks,5weeks and 10 weeks.10 weeks after surgery,X-ray and Micro-CT showed that every mouse in experimental group acquired HO,but none in control group.At day 1 and day 3 after surgery,TNF-α concentration was significantly higher in both groups compared to untreated WT mice and was remarkably higer in celecoxib group than parecoxib group.At day 15,TNF-α concentration in both celecoxib group and parecoxib group decreased to the same level of untreated group.At 10 weeks after surgery,X-ray demonstrated that the occurrence rate was much higher in celecoxib group than parecoxib group.H&E staining showed that the number of chondrocytes and newly formed bone tissue was higher in celecoxib group.At day 1 and day 3 after surgery,TNF-α concentration was significantly higher in LEP group compared to the other two groups.At 15 days,TNF-α concentration decreased in all groups.At 3 weeks after surgery,H&E staining showed that the number of inflammatory cells were much higher in LEP group.Micro-CT also demonstrated that the bone volume and bone mineral density of newly formed bone were higher in LEP group when compared to the other two groups.Statistical significance was revealed in genotype frequencies of NAT2 rs1799931 G>A SNP between the experimental cases and the controls.The genotype CT frequency of rs1041983 in metastasis cases was greater when compared to cases without tumor metastasis,and the difference in frequency distribution showed statistical significance.Conclusions Achillotomy is an effective way to establish a HO model in mice.LEP m RNA and LEP protein was highly increased during the formation of heterotopic ossification.The suppression of inflammation and heterotopic ossification is higher in parecoxib than celecoxib.LEP can accelerate the inflammatory reaction after tissue damage.Exogenous leptin could significantly elevate the formation of heterotopic ossification in ob/ob mice.And the inflammation can be suppressed by parecoxib which also can decraese the formation of heterotopic ossification.LEP promotes heterotopic ossification by accelerating inflammatory response.NAT2 rs1799931 polymorphism played a key role in osteosarcoma resistance in young Chinese individuals.NAT2 rs1041983 polymorphism was related to metastasis of osteosarcoma,predicting poor prognosis.PART 1:PARTIAL EXPRESSION OF LEPTIN IN HETEROTOPIC OSSIFICATION ANIMAL MODEL AND EFFECTION OF LEPTIN ON OB/OB MICE HETEROTOPIC OSSIFICATION Objective To detect partial expression of leptin in achillotomy mice and to study the heterotopic ossification model of mice.Methods 24 C57BL/6 wild-type male mice of 8 weeks old were randomly divided into two groups:control group(n=12)and experimental group(n=12),Achilles tenotomy surgery was carried out in experimental group to establish heterotopic ossification models.ELISA were used to detect the concentration of leptin protein at different time points.Hematoxylin and Eosin staining were used to observe the tissue morphology.X-ray and Micro-CT were used to observe the occurrence of heterotopic bone formation.Results In wild-type mice groups,LEP protein concentration in the experimental group was significantly higher than the control group at 3 weeks,5weeks and 10 weeks.10 weeks after surgery,X-ray and Micro-CT showed that every mouse in experimental group acquired HO,but none in control group.Conclusions Achillotomy is an effective way to establish a HO model in mice.LEP protein was highly increased during the formation of heterotopic ossification.PART 2:THE EFFECTION OF PARECOXIB AND CELECOXIB ON THE TRAUMATIC INFLAMMATORY REACTION AND HETEROTOPIC OSSIFICATION Objective To study the effect of parecoxib and celecoxib on partial tissue inflammation and heterotopic ossification in achillotomy wild type mice.Methods 38 wild-type male mice of 8 weeks old were randomly divided into two groups(n=19): parecoxib group and celecoxib group,Achilles tenotomy surgery wascarried out in both groups to establish heterotopic ossification model.Mice in each group were respectively given each kind of medicine.ELISA was used to detect the concentration of TNF-α protein at different time points.H&E staining and X-ray were used to observe the formation of heterotopic bone and the tissue morphology.Results At day 1 and day 3 after surgery,TNF-α concentration was significantly higher in both groups compared to untreated WT mice and was remarkably higer in celecoxib group than parecoxib group.At day 15,TNF-α concentration in both celecoxib group and parecoxib group decreased to the same level of untreated group.At 10 weeks after surgery,X-ray demonstrated that the occurrence rate was much higher in celecoxib group than parecoxib group.H&E staining showed that the number of chondrocytes and newly formed bone tissue was higher in celecoxib group.Conclusions The suppression of inflammation and heterotopic ossification is higher in parecoxib than celecoxib.PART 3:THE EFFECTION OF PARECOXIB ON THE INFLAMMATION AND HO FORMATION OF ACHILLOTOMY OB/OB MICE WHICH RECEIVED LEP Objective To study the different effection on partial tissue inflammation and heterotopic ossification in achillotomy ob/ob mice which respectively received injection of NS,LEP or LEP+ Parecoxib.Methods 45 male ob/ob mice of 8 weeks old were randomly divided into three groups(each n=15): NS group,LEP group and LEP+Parecoxib group,Achilles tenotomy surgery was carried out in both groups to establish heterotopic ossification model.Mice in each group were respectively given each kind of medicine.ELISA was used to detect the concentration of TNF-α protein at different time points.At 3,5 and 10 weeks,histomorphology staining was used to observe the partial tissue morphology.At 10 weeks,Micro-CT was used to observe the parameters of newly formed bone ineach group.Results At day 1 and day 3 after surgery,TNF-α concentration was significantly higher in LEP group compared to the other two groups.At 15 days,TNF-α concentration decreased in all groups.At 3 weeks after surgery,H&E staining showed that the number of inflammatory cells were much higher in LEP group.Micro-CT also demonstrated that the bone volume and bone mineral density of newly formed bone were higher in LEP group when compared to the other two groups.Conclusions LEP can accelerate the inflammatory reaction after tissue damage.Exogenous leptin could significantly elevate the formation of heterotopic ossification in ob/ob mice.And the inflammation can be suppressed by parecoxib which also can decraese the formation of heterotopic ossification.LEP promotes heterotopic ossification by accelerating inflammatory response.PART 4:ASSOCIATIONS OF POLYMORPHISMS IN NAT2 GENE WITH RISK AND METASTASIS OF OSTEOSARCOMA IN YOUNG CHINESE POPULATION Objective To study the the associations between NAT2 gene polymorphisms and osteosarcoma risk and metastasis in young Chinese population.Methods We carried out a case-control study to investigate seven NAT2 SNPs on the risk and prognosis of osteosarcoma.This study included 260 newly diagnosed osteosarcoma cases under the age of 20 years and 286 cancer-free controls,during the period between February 2007 and March 2012.The Taq Man method was used to determine genotypes.Clinical characteristics(location,stage,operation,and metastasis)of osteosarcoma were used to further investigate the relationship between SNPs and the incidence of osteosarcoma.Results When the CC homozygote genotype was used as the reference group,the NAT2 rs1041983 CT genotype was associated with a decreased risk of osteosarcoma(CT versus CC: crude OR =0.67,95% CI =0.47–0.96,P=0.029;adjusted OR =0.68,95% CI =0.47–0.97,P=0.033).Statistical significance was revealed in genotype frequencies of NAT2 rs1799931 G>A SNP between the experimental cases and the controls(P=0.035).The genotype CT frequency of rs1041983 in metastasis cases(59.57%)was greater when compared to cases without tumor metastasis(37.09%),and the difference in frequency distribution showed statistical significance(P=0.010).Conclusions NAT2 rs1799931 polymorphism played a key role in osteosarcoma resistance in young Chinese individuals.NAT2 rs1041983 polymorphism was related to metastasis of osteosarcoma,predicting poor prognosis.