CaMKⅡδ基因沉默对MAPKs、CREB信号及破骨细胞分化的影响

发布时间：2018-06-16 12:46

本文选题：破骨细胞 + 钙调蛋白依赖性激酶Ⅱδ　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的研究CamkⅡδ基因沉默对丝裂原激活的蛋白激酶(mitogen-activated protein kinases,MAPKs)、c AMP应答元件结合蛋白(CREB)以及活化T细胞核因子1(NFATc1)蛋白活性和破骨细胞分化的影响及分子机制。方法1 CamkⅡδ基因沉默对破骨细胞分化、功能的影响:将RAW264.7细胞分为3组:对照组、阴性载体组和干扰组。采用表达绿色荧光的阴性载体病毒转染细胞,确定最适的病毒转染MOI值以及转染效率。重组慢病毒转染12 h后加入50ng/ml的核因子-κB受体活化因子配体(RANKL)诱导培养5 d,通过抗酒石酸酸性磷酸酶(TRAP)染色、牛骨磨片吸收陷窝检测来评价破骨细胞生成及骨吸收情况;Real-time PCR和免疫印迹法检测CamkⅡδ的m RNA及蛋白表达水平;免疫荧光细胞化学、免疫印迹法检测NFATc1蛋白表达水平。2 CamkⅡδ基因沉默对CREB蛋白活化的影响:RAW264.7细胞分为阴性载体组和干扰组,转染3 d后加入50ng/ml RANKL诱导0 h、1h、3h、6h、12h、24h,免疫印迹法检测CREB蛋白磷酸化水平。3 CamkⅡδ基因沉默对MAPKs信号活化的调节机制:RAW264.7细胞分为阴性载体组和干扰组,转染3 d后加入50ng/ml RANKL诱导0 min、5min、10 min、15 min、30 min、60 min,免疫印迹法检测细胞外信号调节激酶(ERK)1/2、氨基末端激酶(JNK)、丝裂原激活蛋白激酶(p38)蛋白磷酸化水平;4 MAPKs信号阻断对破骨细胞分化及相关因子NFATc1表达的影响:RAW264.7细胞分为4组:对照组、ERK1/2抑制剂(PD98059)组、JNK抑制剂(SP600125)组、P38抑制剂(SB203580)组,每组用RANKL诱导破骨细胞分化的同时,应用相应因子的抑制剂处理,3d后免疫印迹法检测NFATc1的蛋白表达水平,5d后TRAP染色评价破骨细胞生成。结果1阴性载体病毒转染细胞,结果显示最适病毒转染滴度MOI值为30,转染效率为74.9%。RANKL诱导5d后,三组细胞均出现TRAP+多核破骨细胞(胞核≥3个)和骨吸收陷窝,但干扰组破骨细胞数目、牛骨磨片骨吸收陷窝的数目和面积分别为10.8±2.1、8.5±1.1和5463±884.5μm2,显著低于阴性载体组的24.7±2.2、24.8±1.9、10491±635.3μm2和对照组的27.6±3.9、25.8±2.3、11447±710.4μm2(P0.05);而对照组、阴性载体组之间无显著性差异(P0.05)。Real-time PCR结果显示,与对照组相比,干扰组中CamkⅡδ的m RNA表达量显著降低,干扰效率为77.2%(P0.05),而对照组和阴性载体组之间无显著差异(P0.05)。免疫印迹结果显示,与阴性载体组(1.11±0.12)相比CamkⅡδ蛋白表达水平在干扰组(0.33±0.04)中显著下调,差异均具有统计学意义(P0.01)。与对照组、阴性载体组相比,干扰组NFATc1的荧光强度明显减弱;免疫印迹法显示,干扰组NFATc1蛋白水平为1.52±0.04,较阴性载体组的2.25±0.12显著下调约72.3%(P0.01),而对照组与阴性载体组无显著差异(P0.05)。2 RANKL诱导0 h、1h、3h、6h、12h、24h,免疫印迹结果显示,干扰组中CREB磷酸化水平较阴性载体组下调了21%~56%,差异具有统计学意义(P0.05)。3 RANKL诱导0min、5 min、10 min、15 min、30 min、60 min,免疫印迹结果显示CamkⅡδRNA干扰组各时间点p-ERK1/2、p-JNK和p-p38活化均显著下调;与阴性载体组比较,p-ERK1/2、p-JNK、p-p38分别下调了55%~64%(P0.05)、12%~40%(P0.01)和25%~52%(P0.01),差异均具有统计学意义。4与对照组比较,ERK1/2抑制剂PD98059、JNK抑制剂SP600125和P38抑制剂SB203580均能够显著抑制RANKL诱发的NFATc1蛋白表达上调;三种抑制剂分别使NFATc1蛋白表达水平下调了37.3%(P0.01)、42.1%(P0.05)和38.2%(P0.05),差异具有统计学意义。ERK1/2抑制剂组、JNK抑制剂组和P38抑制剂组中TRAP+多核破骨细胞数目分别为9.7±1.5、10.0±2.0、8.7±2.1,较对照组中的16.0±1.0分别下降了39.4%、37.5%和45.6%,结果具有显著性差异(P0.01)。结论CamkⅡδ基因沉默可显著抑制破骨细胞的分化和生成,CREB和MAPKs信号分子ERK1/2、JNK、P38参与CamkⅡδ对破骨细胞分化和骨吸收功能的调控作用。
[Abstract]:Objective to study the effects and molecular mechanisms of Camk II delta gene silencing on mitogen activated protein kinase (mitogen-activated protein kinases (MAPKs), C AMP response element binding protein (CREB) and activated T nuclear factor 1 (NFATc1) protein activity and osteoclast differentiation. Methods 1 Camk II delta gene silencing on osteoclast differentiation and function Effect: the RAW264.7 cells were divided into 3 groups: control group, negative carrier group and interference group. The transfection of the most suitable virus transfection MOI and transfection efficiency were determined by the negative carrier virus expressing green fluorescence. The recombinant lentivirus was transfected to 12 h, and the nuclear factor kappa B receptor activator ligand (RANKL) was added to the culture of 5 d after transfection of the recombinant lentivirus. The acid phosphatase (TRAP) staining was used to detect the formation of osteoclast and bone resorption by bovine bone graft. Real-time PCR and immunoblotting were used to detect the m RNA and protein expression level of Camk II Delta; immunofluorescence cytochemistry and Western blot detection of NFATc1 egg white expression level.2 Camk II delta gene silencing on CREB protein activation RAW264.7 cells were divided into negative carrier group and interference group. After 3 D transfection, the cells were added to 50ng/ml RANKL to induce 0 h, 1H, 3h, 6h, 12h, 24h, and the immunoblotting method was used to detect the activation mechanism of CREB protein phosphorylation level II delta gene silencing. KL induced 0 min, 5min, 10 min, 15 min, 30 min, 60 min. Immunoblotting was used to detect the level of phosphorylation of extracellular signal regulated kinase (ERK), amino terminal kinase (JNK), mitogen activated protein kinase (p38) protein, and the effect of 4 MAPKs signals on osteoclast differentiation and related factors: 4 groups: control group 2 inhibitor (PD98059) group, JNK inhibitor (SP600125) group and P38 inhibitor (SB203580) group. Each group used RANKL to induce osteoclast differentiation. The protein expression level of NFATc1 was detected by immunoblotting in 3D after 3D, and osteoclast formation was evaluated by TRAP staining after 5D. Results the result of 1 negative carrier virus transfected cells was found. The result was the result of transfection of cells. The result was the result of transfection of cells. The result was the result of transfection of cells. The result was the result of the transfection of cells. The result was the result of transfection of cells. The result was the result of the transfection of cells. The result was the result of transfection of the cells. The result was the result of the transfection of the cells. The result was the result of the transfection of the cells. The MOI value of the optimal virus transfection titer was 30. After the transfection efficiency was 74.9%.RANKL induced 5D, the three groups of cells all appeared TRAP+ polynuclear osteoclasts (nuclei more than 3) and bone resorption lacunae, but the number of osteoclast cells in the interference group and the number and area of the bone resorption lacunae were 10.8 + 2.1,8.5 1.1 and 5463 + 884.5 micron M2 respectively, significantly lower than negative The carrier group was 24.7 + 2.2,24.8 + 1.910491 + 635.3 Mu m2 and 27.6 + 3.9,25.8 + 2.311447 + 710.4 M2 (P0.05) in the control group, but there was no significant difference between the control group and the negative carrier group (P0.05).Real-time PCR results. Compared with the control group, the m RNA expression of Camk II Delta in the interference group was significantly reduced, and the interference efficiency was 77.2% (P0.05), while the interference efficiency was 77.2% (P0.05), and There was no significant difference between the group and the negative carrier group (P0.05). The results of immunoblotting showed that compared with the negative carrier group (1.11 + 0.12), the expression level of Camk II delta protein decreased significantly in the interference group (0.33 + 0.04), and the difference was statistically significant (P0.01). Compared with the control group, the fluorescence intensity of NFATc1 in the interference group was significantly reduced. The immunoblotting showed that the level of NFATc1 protein in the interference group was 1.52 + 0.04, which was significantly lower than that of the negative carrier group by 2.25 + 0.12 (P0.01), but there was no significant difference between the control group and the negative carrier group (P0.05).2 RANKL induced 0 h, 1H, 3H, 6h, 12h, 24h, and the immunoblotting results showed that the phosphorylation level of the CREB group was lower than that of the negative carrier group. The difference has statistical significance (P0.05).3 RANKL induced 0min, 5 min, 10 min, 15 min, 30 min, 60 min. The difference was statistically significant.4 compared with the control group, ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and P38 inhibitor SB203580 could significantly inhibit the up regulation of NFATc1 protein expression induced by RANKL; the three inhibitors reduced the expression level of NFATc1 protein by 37.3% (P0.01), 42.1% (P0.05) and 38.2%, with statistical significance. The number of TRAP+ polynuclear osteoclasts in the.ERK1/2 inhibitor group, the JNK inhibitor group and the P38 inhibitor group were 9.7 + 1.5,10.0 + 2.0,8.7 + 2.1, respectively, and decreased by 39.4%, 37.5% and 45.6% in the control group, respectively. The results showed significant difference (P0.01). Conclusion Camk II delta gene depression could significantly inhibit the differentiation and formation of osteoclast, CREB and MAPKs signaling molecules ERK1/2, JNK and P38 participate in the regulation of Camk II Delta on osteoclast differentiation and bone resorption function.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R78

【参考文献】