本文選題：破骨細胞 + 鈣調(diào)蛋白依賴性激酶Ⅱδ��； 參考：《華北理工大學》2017年碩士論文

【摘要】：目的研究CamkⅡδ基因沉默對絲裂原激活的蛋白激酶(mitogen-activated protein kinases,MAPKs)、c AMP應(yīng)答元件結(jié)合蛋白(CREB)以及活化T細胞核因子1(NFATc1)蛋白活性和破骨細胞分化的影響及分子機制。方法1 CamkⅡδ基因沉默對破骨細胞分化、功能的影響:將RAW264.7細胞分為3組:對照組、陰性載體組和干擾組。采用表達綠色熒光的陰性載體病毒轉(zhuǎn)染細胞,確定最適的病毒轉(zhuǎn)染MOI值以及轉(zhuǎn)染效率。重組慢病毒轉(zhuǎn)染12 h后加入50ng/ml的核因子-κB受體活化因子配體(RANKL)誘導培養(yǎng)5 d,通過抗酒石酸酸性磷酸酶(TRAP)染色、牛骨磨片吸收陷窩檢測來評價破骨細胞生成及骨吸收情況;Real-time PCR和免疫印跡法檢測CamkⅡδ的m RNA及蛋白表達水平;免疫熒光細胞化學、免疫印跡法檢測NFATc1蛋白表達水平。2 CamkⅡδ基因沉默對CREB蛋白活化的影響:RAW264.7細胞分為陰性載體組和干擾組,轉(zhuǎn)染3 d后加入50ng/ml RANKL誘導0 h、1h、3h、6h、12h、24h,免疫印跡法檢測CREB蛋白磷酸化水平。3 CamkⅡδ基因沉默對MAPKs信號活化的調(diào)節(jié)機制:RAW264.7細胞分為陰性載體組和干擾組,轉(zhuǎn)染3 d后加入50ng/ml RANKL誘導0 min、5min、10 min、15 min、30 min、60 min,免疫印跡法檢測細胞外信號調(diào)節(jié)激酶(ERK)1/2、氨基末端激酶(JNK)、絲裂原激活蛋白激酶(p38)蛋白磷酸化水平;4 MAPKs信號阻斷對破骨細胞分化及相關(guān)因子NFATc1表達的影響:RAW264.7細胞分為4組:對照組、ERK1/2抑制劑(PD98059)組、JNK抑制劑(SP600125)組、P38抑制劑(SB203580)組,每組用RANKL誘導破骨細胞分化的同時,應(yīng)用相應(yīng)因子的抑制劑處理,3d后免疫印跡法檢測NFATc1的蛋白表達水平,5d后TRAP染色評價破骨細胞生成。結(jié)果1陰性載體病毒轉(zhuǎn)染細胞,結(jié)果顯示最適病毒轉(zhuǎn)染滴度MOI值為30,轉(zhuǎn)染效率為74.9%。RANKL誘導5d后,三組細胞均出現(xiàn)TRAP+多核破骨細胞(胞核≥3個)和骨吸收陷窩,但干擾組破骨細胞數(shù)目、牛骨磨片骨吸收陷窩的數(shù)目和面積分別為10.8±2.1、8.5±1.1和5463±884.5μm2,顯著低于陰性載體組的24.7±2.2、24.8±1.9、10491±635.3μm2和對照組的27.6±3.9、25.8±2.3、11447±710.4μm2(P0.05);而對照組、陰性載體組之間無顯著性差異(P0.05)。Real-time PCR結(jié)果顯示,與對照組相比,干擾組中CamkⅡδ的m RNA表達量顯著降低,干擾效率為77.2%(P0.05),而對照組和陰性載體組之間無顯著差異(P0.05)。免疫印跡結(jié)果顯示,與陰性載體組(1.11±0.12)相比CamkⅡδ蛋白表達水平在干擾組(0.33±0.04)中顯著下調(diào),差異均具有統(tǒng)計學意義(P0.01)。與對照組、陰性載體組相比,干擾組NFATc1的熒光強度明顯減弱;免疫印跡法顯示,干擾組NFATc1蛋白水平為1.52±0.04,較陰性載體組的2.25±0.12顯著下調(diào)約72.3%(P0.01),而對照組與陰性載體組無顯著差異(P0.05)。2 RANKL誘導0 h、1h、3h、6h、12h、24h,免疫印跡結(jié)果顯示,干擾組中CREB磷酸化水平較陰性載體組下調(diào)了21%~56%,差異具有統(tǒng)計學意義(P0.05)。3 RANKL誘導0min、5 min、10 min、15 min、30 min、60 min,免疫印跡結(jié)果顯示CamkⅡδRNA干擾組各時間點p-ERK1/2、p-JNK和p-p38活化均顯著下調(diào);與陰性載體組比較,p-ERK1/2、p-JNK、p-p38分別下調(diào)了55%~64%(P0.05)、12%~40%(P0.01)和25%~52%(P0.01),差異均具有統(tǒng)計學意義。4與對照組比較,ERK1/2抑制劑PD98059、JNK抑制劑SP600125和P38抑制劑SB203580均能夠顯著抑制RANKL誘發(fā)的NFATc1蛋白表達上調(diào);三種抑制劑分別使NFATc1蛋白表達水平下調(diào)了37.3%(P0.01)、42.1%(P0.05)和38.2%(P0.05),差異具有統(tǒng)計學意義。ERK1/2抑制劑組、JNK抑制劑組和P38抑制劑組中TRAP+多核破骨細胞數(shù)目分別為9.7±1.5、10.0±2.0、8.7±2.1,較對照組中的16.0±1.0分別下降了39.4%、37.5%和45.6%,結(jié)果具有顯著性差異(P0.01)。結(jié)論CamkⅡδ基因沉默可顯著抑制破骨細胞的分化和生成,CREB和MAPKs信號分子ERK1/2、JNK、P38參與CamkⅡδ對破骨細胞分化和骨吸收功能的調(diào)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.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R78

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本文編號：2026748