| Objective:To investigate the level of tumor necrosis factor-? in necrotic femoral head and the effect of TNF-? on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats.Methods:1.The femoral heads of patients with femoral head necrosis were obtained from the operation,the contents of TNF-? in normal bone,sclerotic bone and necrosis bone of the femoral head were determined by enzyme-linked immunosorbent assay.The differences of the contents of TNF-? in the three kinds of bone and the correlation between the contents of the sclerotic bone,age,sex,etiology,syndrome,pain time,ARCO staging and JIC classification were analyzed.2.SD rat bone marrow mesenchymal stem cells were extracted and cultured in vitro,and passaged to the 4th generation for experiments.To observe the effect of different concentrations of TNF-? on osteogenic differentiation of bone marrow mesenchymal stem cells,the ALP staining,ALP activity assay,alizarin red staining and calcium deposition quantitative determination of bone marrow-derived mesenchymal stem cells were performed on the 14 th day and 21 st day respectively.The optimum concentration of TNF-? was determined according to the staining results,and then real-time PCR and Western-blot were performed after the bone marrow mesenchymal stem cells were intervented with the optimum concentration.Genomic DNA was extracted on the fifth day after intervention.DNA methylation level in Runx2 promoter region was analyzed before and after intervention.On the fifth day after intervention,chromatin immunoprecipitation experiment was used to analysis changes of histone H3K4 and H3K27 methylation level.Results:1.A total of 60 femoral head osteonecrosis patients were obtained,including 45 males and 15 females,with an average age of 48.88±12.98 yearsold.The level of TNF-? were 90.66±40.29 pg/ml in normal bone and 122.61±45.84 pg/ml in sclerotic bone and 65.79±34.79 pg/ml in necrotic bone.In 45 male patients,the TNF-? contents of normal bone,sclerotic bone and necrotic bone were 88.74±36.89 pg/ml,128.02±46.67 pg/ml,and 65.91±35.55 pg/ml,respectively,and the TNF-? contents of normal bone,sclerotic bone and necrotic bone were 96.42±50.16 pg/ml,106.39±40.48 pg/ml,and 65.43±39.53 pg/ml respectively in 15 cases of female patients.The TNF-? contents of normal bone,sclerotic bone and necrotic bone were 90.87±39.54 pg/ml,104.93±35.11 pg/ml,and 61.27±35.39 pg/ml respectively in 14 cases of age from 18 to 35 years old.The TNF-? contents of normal bone,sclerotic bone and necrotic bone were 88.65±34.29 pg/ml,126.44±31.03 pg/ml and 69.67±28.99 pg/ml respectively in 18 cases of age from 36 to 50 years old,and the TNF-? contents of normal bone,sclerotic bone and necrotic bone were 91.84±45.27 pg/ml,128.99±56.38 pg/ml,65.55±38.62 pg/ml,respectively in the 28 cases who were older than 50 years old.Eight patients were idiopathic necrosis,liver and kidney deficiency,and the TNF-? contents of normal bone,sclerotic bone and necrotic bone were 99.18±40.45 pg/ml,130.87±43.29 pg/ml,77.00±31.13 pg/ml,respectively.There were 18 cases with hormonal necrosis,kidney deficiency and blood stasis,and their level of TNF-? were 93.10±42.68 pg/ml in normal bone,129.34±54.82 pg/ml in sclerotic bone,and 70.19 ± 37.37 pg/ml in necrotic bone.29 cases were alcoholic necrosis with phlegm and blood stasis type,and the contentss TNF-? were 88.87±39.02 pg/ml in normal bone,121.82±41.51 pg/ml in sclerotic bone,and 64.73±35.11 pg/ml in necrotic bone.Five patients were traumatic necrosis with qi stagnation and blood stasis,the contents TNF-? were 78.62±47.92 pg/ml in normal bone,89.74±34.47 pg/ml in sclerotic bone,and 38.19±17.11 pg/ml in necrotic bone.There were 33 patients with less than 12 months hip pain,the contents TNF-? were 91.24±42.93 pg/ml in normal bone,124.87±51.05 pg/ml in sclerotic bone,and 67.92±37.73 pg/ml in necrotic bone.There were 11 patients with hip pain time between 12 and 24 months,the contents TNF-? were 105.19±37.57 pg/ml in normal bone,115.21±45.55 pg/ml in sclerotic bone,and 68.76±34.44 pg/ml in necrotic bone.Patients with hip pain more than 24 months was 16,and the contents TNF-? were 79.48±34.96 pg/ml in normal bone,123.05±35.66 pg/ml in sclerotic bone,and 59.35±29.53 pg/ml in necrotic bone.Six cases were ARCO IIIA,their TNF-? contents were 95.36±34.22 pg/ml in normal bone,49.77±20.50 pg/ml in sclerotic bone,73.81±31.09 pg/ml in necrotic bone.31 patients were ARCOIIIB,their TNF-? contents were 92.27±43.04 pg/ml in normal bone,128.79±50.32 pg/ml in sclerotic bone,68.84±37.87 pg/ml in necrotic bone.Nine patients were ARCOIIIC,and their TNF-? contents were 104.11±43.99 pg/ml in normal bone,117.43±45.69 pg/ml in sclerotic bone,69.99±38.82 in necrotic bone.14 cases were ARCO IV,and their TNF-? contents were 76.42±32.99 pg/ml in normal bone,100.62±35.74 pg/ml in sclerotic bone,52.89±25.32 in necrotic bone.There 35 cases with JIC C1 type,their TNF-? contents were 96.96±42.00 pg/ml in normal bone,126.16±47.94 pg/ml in sclerotic bone,71.14±36.35 pg/ml in necrotic bone.25 cases were JIC C2 type,and their TNF-? contents were 81.82±36.76 in normal bone,117.64±43.21 pg/ml in sclerotic bone,58.29±31.67 pg/ml in necrotic bone.One-way analysis of variance(ANOVA)showed that the levels of TNF-? in normal bone,sclerotic bone and necrotic bone were positively correlated with the gender,age,etiopathogenisis,syndrome type,hip pain time,ARCO staging and JIC classification(P>0.05).According to the binary logistic regression analysis of multiple factors,the level of TNF-? in sclerotic bone had no correlation with the gender,age,etiopathogenisis,syndrome type,hip pain time and JIC classification(P>0.05),but had correlation with ARCO staging(P=0.049<0.05).The odds ratio was 0.381,which meant that for each additional stage of ARCO staging,the contents of TNF-? in femoral sclerosis bone will decrease 0.381 times.2.Extracted stem cells had the shape of long fusiform,with multidirectional differentiation potential,and can differentiate into osteoblasts,chondroblasts and lipoblasts.Bone marrow mesenchymal stem cells were extracted specifically;Different concentrations of TNF-?(10pg/ml,100pg/ml,1000pg/ml and 10000pg/ml)did not affect the activity of stem cells;ALP staining and alizarin red staining results showed that the effect of 10pg/ml and 100pg/ml on osteogenic differentiation was not obvious.While 1000pg/ml and 10000pg/ml TNF-? inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells,and the inhibitory effect of 10000pg/ml was the most obvious.Real-time PCR results showed that 10000pg/ml TNF-? significantly down-regulated the expression of Runx2,ALP and OPN.Western-blot showed that TNF-? at 10000pg/ml significantly inhibited Runx2 expression;The percentage of methylated CpG loci in the total CpG loci in Runx2 promoter of contral group was 3.33% and the percentage was 9.05% of TNF-?treated group.The results of chromatin immunoprecipitation showed that intervention of TNF-? at a concentration of 10000 pg/ml could inhibit the trimethylation of histone H3K4 and significantly promote histone H3K27 trimethylation at the same time.Conclusions:The level of NTF-? in normal bone,sclerotic bone and necrotic bone of femoral head in patients with necrosis of femoral head were obviously different.The contents of TNF-? in sclerotic bone were the highest,and its contents was not closely related to the gender,age,etiology,syndrome type,hip pain time,ARCO staging and JIC classification.High concentrations of TNF-?(1000pg/ml and 10000pg/ml)inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells by increasing the methylation level of CpG island in Runx2 promoter,promoted the trimethylation of histone H3K27 and inhibited the trimethylation of histone H3K4,this may be one of the mechanisms of TNF-? inhibiting osteogenic differentiation of bone marrow mesenchymal stem cells. |
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