CaMKⅡγ信号干扰对破骨细胞分化及相关基因NFATc1、TRAP、c-Src表达的影响

发布时间：2018-04-24 02:21

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

【摘要】：目的研究钙调蛋白依赖性激酶Ⅱ(Calmodulin-dependent kinase Ⅱ,CaMKⅡ)γRNA干扰对破骨细胞生成、骨吸收功能以及下游信号分子活化T-细胞核因子c1(Nuclear factor of activated T-cells cytoplasmic 1,NFATc1)、非受体酪氨酸激酶(cell-sarcoma receptor coactivator,c-Src)、抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)基因表达的影响,以证实CaMKⅡγ在破骨细胞分化中的关键调控作用和分子机制。方法1破骨细胞分化中CaMKⅡγ基因表达规律研究。应用50 ng/ml受体活化核因子-κB配体(Receptor activator of nuclear factor-κB ligand,RANKL)诱导小鼠RAW264.7细胞向破骨细胞分化,并于诱导第0d、1d、3d、5d四个时间点收获细胞,采用实时荧光定量PCR、蛋白质印迹法、免疫荧光细胞化学法检测破骨细胞分化中CaMKⅡγ基因的表达规律。2唑U;膦酸对破骨细胞分化及相关基因表达影响的研究。小鼠RAW264.7细胞分为两组:A组为对照组,B组为唑来膦酸处理组。两组细胞均用50 ng/ml RANKL诱导细胞向破骨细胞分化;B组在培养1d后,加用1×10-6mol/L的唑来膦酸处理2d,然后撤掉唑来膦酸,继续培养。于第5d、7d收获细胞进行相关检测。应用TRAP染色、牙本质磨片吸收陷窝检测评价两组细胞破骨细胞生成及骨吸收情况;并通过实时荧光定量PCR、蛋白质印迹法、免疫荧光细胞化学检测两组细胞CaMKⅡγ、NFATc1、TRAP、c-Src基因表达情况。3唑来磷酸对破骨细胞分化中CaMKⅡ和Colmodulin蛋白结合的影响。小鼠RAW264.7细胞分为A、B两组:A组为对照组,B组为唑来磷酸处理组。两组均用50ng/ml RANKL诱导,B组在1 d后加用1×10-6 M唑来膦酸处理2 d。应用免疫共沉淀(Co-Immunoprecipitation,Co-IP)及反向Co-IP对CaMKⅡ与Colmodulin蛋白结合进行分析。4 CaMKⅡγRNA干扰对破骨细胞分化及相关基因表达的影响。应用携带绿色荧光蛋白(GFP)的慢病毒载体构建三个CaMKⅡγ重组RNA干扰载体。阴性载体转染RAW264.7细胞,确定最适的病毒转染滴度MOI值和转染效率。在最佳MOI值下用CaMKⅡγ重组RNA干扰载体转染RAW264.7细胞,通过实时荧光定量PCR及蛋白质印迹法检测破骨细胞向分化中三个CaMKⅡγ重组载体的干扰效果,确定干扰效果最佳的重组干扰载体,用于下一步实验。实验分为A、B、C三组:A组为对照组、B组为阴性载体组、C组为干扰载体组。细胞转染12小时后,换用含50 ng/ml RANKL的培养基诱导细胞向破骨细胞分化,并于诱导5天后收获细胞。通过TRAP染色及牙本质磨片吸收陷窝检测评价三组细胞破骨细胞生成及骨吸收情况;通过实时荧光定量PCR、蛋白质印迹法、免疫荧光细胞化学检测三组细胞CaMKⅡγ及其下游相关基因NFATc1、TRAP、c-Src基因表达情况。结果1破骨细胞分化第0d、1d、3d、5d,CaMKⅡγmRNA水平分别为1.067±0.179、1.840±0.070、9.493±0.453和30.767±0.573;蛋白水平分别是494.567±20.121、663.533±38.741、858.600±19.367和980.367±23.403;与第0d比较,除第1d蛋白水平外(P0.05),各时间点m RNA水平(P0.01)及蛋白水平(P0.01)均呈时间依赖性表达增强。免疫荧光检测显示第0d和1d蛋白表达较弱,而在第3d和第5d蛋白表达强度明显增加,并有多核破骨细胞形成。2唑U;膦酸处理下B组多核破骨细胞数、牙本质吸陷窝数目和面积分别为11.33±1.52(个)、8.66±2.08(个)和5034.35±775.42μm2,较对照组的37.66±5.68(个)、23.00±4.00(个)和15042.71±1906.03μm2显著减少(P0.01),下降幅度分别为69.91%、62.60%和66.53%。唑来膦酸对破骨细胞分化过程中CaMKⅡγ及其下游因子NFATc1、TRAP和c-Src m RNA及蛋白水平也产生了抑制作用;与A组比较,B组上述4个基因m RNA水平分别下降了44.603%、54.126%、58.942%和51.546%(P0.01);蛋白水平分别下降了46.127%、36.799%、27.140%和32.060%(P0.01);免疫荧光细胞化学也证实B组蛋白水平明显下降。3 Co-IP及反向Co-IP检测显示,B组CaMKⅡ与Calmodulin蛋白结合较A组显著降低,分别下降了59.75%和50.87%(P0.01);在总蛋白中,B组Calmodulin与CaMKⅡ蛋白较A组也显著下降,分别降低了52.12%和51.49%(P0.01)。4本实验成功构建了CaMKⅡγ重组慢病毒干扰载体。最适病毒滴度MOI值是30,其转染效率80%。经实时荧光定量PCR和蛋白质印迹法检测,#3重组载体对CaMKⅡγ干扰效果最佳,干扰效率在m RNA及蛋白水平分别为78.158%和62.226%;因而应用#3重组干扰载体进行下面实验。经转染后,三组细胞中C组(干扰载体组)多核破骨细胞数、牙本质吸陷窝数目和面积在分别为10.670±1.52(个)、87.330±1.528(个)和4922.000±64.086μm2,显著低于B组(阴性载体组)的22.670±1.2528(个)、12.670±2.082(个)、0924.330±66.905μm2和A组(对照组)的26.670±1.528(个)、16.000±1.000(个)、11980.000±70.000μm2(P0.05);而A组和B组之间无显著性差异(P0.05)。CaMKⅡγRNA干扰显著抑制了CaMKⅡγ及其下游因子NFATc1、TRAP、c-Src的表达。实时荧光定量PCR检测表明C组CaMKⅡγ、NFATc1、TRAP、c-Src m RNA水平较A组分别下降了79.872%、49.856%、43.649%和53.567%(P0.01);蛋白质印迹法检测显示C组上述4个基因蛋白水平较A、B组也显著减弱(P0.01),下降幅度与B组比较分别为61.70%、54.22%、46.75%和45.86%;免疫荧光化学检测也证实C组上述基因荧光强度明显弱于A、B组。结论1.CaMKⅡγ在破骨细胞分化中呈时间依赖性表达增强,提示其在破骨细胞分化中可能发挥关键调控作用;2.唑来膦酸可显著抑制破骨细胞生成、骨吸收及CaMKⅡγ、NFATc1、cSrc、TRAP基因表达;上述基因可能参与了唑U;膦酸对破骨细胞的抑制;3.唑来膦酸可显著抑制破骨细胞分化中CaMKⅡ与Calmodulin的蛋白结合,这可能与其诱发的破骨细胞抑制有关;4.CaMKⅡγRNA干扰可显著抑制破骨细胞生成、骨吸收功能及下游基因NFATc1、c-Src、TRAP的表达;上述基因可能介导了CaMKⅡγ对破骨细胞分化的调控。
[Abstract]:Objective to study the formation of osteoclasts, bone resorption and the activation of T- nuclear factor C1 (Nuclear factor of activated T-cells cytoplasmic 1) by the interference of Calmodulin-dependent kinase II (CaMK II) and gamma RNA interference. -Src), the effect of tartrate resistant acid phosphatase (TRAP) on the expression of tartaric acid acid phosphatase (TRAP), in order to confirm the key regulatory role and molecular mechanism of CaMK II in osteoclast differentiation. Method Study on the expression of CaMK II gamma in the differentiation of 1 osteoclasts. The 50 ng/ml receptor activation of nuclear factor kappa B ligand (Receptor Activa) should be used. Tor of nuclear factor- kappa B ligand, RANKL) induced mouse RAW264.7 cells to differentiate into osteoclasts, and the cells were harvested at the four time points of 0d, 1D, 3D, 5D, using real time fluorescence quantification, protein blotting, and immunofluorescence cytochemistry to detect the expression regularity of the gene in osteoclast. Phosphonic acid was fine for osteoclast. RAW264.7 cells were divided into two groups: the A group was the control group, the B group was the zoledronic acid treatment group. The two groups were induced to differentiate into the osteoclast by 50 ng/ml RANKL; after the culture of 1D, the group B was added to the zoledronic acid at 1 * 10-6mol/L, and then the zoledronic acid was withdrawn and continued to be cultured. In 5D, 7d harvested cells were detected by TRAP staining. The formation of osteoclasts and bone resorption of two groups of cells were evaluated by the absorption of lacunae from dentin disc, and the expression of CaMK II gamma, NFATc1, TRAP, and c-Src was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence cytochemistry, the expression of.3 in.3 The effect of the binding of CaMK II and Colmodulin protein in the cell differentiation. The mouse RAW264.7 cells were divided into A, B two groups: the A group was the control group, the B group was the zoledronic acid treatment group. The two groups were all induced by 50ng/ml RANKL, and the B group was treated with 1 * 10-6 M zolidonic acid after 1 D. Colmodulin protein binding was used to analyze the effect of.4 CaMK II gamma RNA interference on osteoclast differentiation and related gene expression. Three CaMK II gamma recombinant RNA interference carriers were constructed with the lentivirus carrier carrying green fluorescent protein (GFP). Negative vectors transfected to RAW264.7 cells to determine the optimum virus transfection titer MOI value and transfection efficiency. RAW264.7 cells were transfected with CaMK II gamma recombinant RNA interference carrier under good MOI value. The interference effect of osteoclast to three CaMK II gamma recombinant vectors in differentiation was detected by real-time fluorescence quantitative PCR and Western blot, and the recombinant interference carrier with the best interference effect was determined for the next experiment. The experiment was divided into A, B, and C three groups: A group as control group, B The group was negative carrier group and group C was interference carrier. After 12 hours transfection, the cells were induced to differentiate into osteoclasts with 50 ng/ml RANKL, and the cells were harvested after 5 days. TRAP staining and dentin absorption lacunae were used to evaluate the formation of osteoclast and bone resorption in the three groups. PCR, Western blot and immunofluorescence cytochemistry were used to detect the expression of CaMK II gamma and its downstream related genes NFATc1, TRAP, c-Src gene. Results 1 osteoclast differentiation of 0d, 1D, 3D, 5D, CaMK II - y mRNA were 1.067 + + + 0.453 and 30.767 + 0.573, and protein levels were 494.567 +. 3.533 + 38.741858.600 + 19.367 and 980.367 + 23.403, compared with 0d, except for 1D protein level (P0.05), m RNA level (P0.01) and protein level (P0.01) were increased in time dependent expression. The expression of 0d and 1D protein was weak, while the expression intensity of 3D and 5D proteins increased significantly, and there was a multi nucleus rupture. The bone cells formed.2 azole U, and the number and area of dentine sucking lacunae in group B were 11.33 + 1.52, 8.66 + 2.08 and 5034.35 + 775.42 m2, respectively, 37.66 + 5.68 in the control group, 23 + 4 (1) and 15042.71 + 1906.03 M2 (P0.01), and the decrease was 69.91%, 62.60% and 66.53%. azole respectively. In the process of osteoclast differentiation, CaMK II gamma and its downstream factors NFATc1, TRAP and c-Src m RNA and protein levels were also inhibited. Compared with the A group, the m RNA levels of the 4 genes of the B group decreased by 44.603%, 54.126%, 58.942% and 51.546% (P0.01), and the protein levels decreased by 46.127%, 36.799%, 27.140% and 32.060% (P0.01), respectively. The immunofluorescent cytochemistry also confirmed that the level of.3 Co-IP and reverse Co-IP in the B group showed that the binding of CaMK II and Calmodulin protein in B group decreased by 59.75% and 50.87% (P0.01), and the Calmodulin and CaMK II protein in the total protein decreased by 52.12% and 51.49%, respectively. The CaMK II gamma recombinant lentivirus interference carrier was successfully constructed in this experiment. The optimal virus titer MOI value was 30. The transfection efficiency 80%. was detected by real-time fluorescence quantitative PCR and Western blot, and the #3 recombinant vector had the best effect on CaMK II gamma interference, and the interference efficiency was 78.158% and 62.226% respectively in m RNA and protein levels. Therefore, the #3 recombination interference load was applied. After transfection, the number and the number and area of the dentin sucking nest in the three groups of the three groups were 10.670 + 1.52, 87.330 + 1.528 and 4922 + 64.086 Mu respectively, significantly lower than that of the B group (negative carrier group) 22.670 + 1.2528 (), 12.670 + 2.082 (), 924.330 + 66.905 Mu m2 and A Group (control group) was 26.670 + 1.528 (Group), 16 + 1 (P0.05), 11980 + 70 M2 (P0.05), but there was no significant difference between group A and B group (P0.05).CaMK II gamma interference significantly inhibited the NFATc1, TRAP, c-Src expression of CaMK II gamma and its downstream factors. The group decreased by 79.872%, 49.856%, 43.649% and 53.567% (P0.01), and Western blot assay showed that the above 4 gene protein levels in group C were more than A, and the B group was also significantly decreased (P0.01), and the decrease was 61.70%, 54.22%, 46.75% and 45.86%, respectively, compared with the B group. The fluorescence intensity of the immunofluorescence assay also confirmed that the above gene fluorescence intensity of the C group was significantly weaker than that of A and B group. Conclusion 1.CaMK II - y has a time dependent expression in osteoclast differentiation, suggesting that it may play a key regulatory role in osteoclast differentiation, and 2. zoledronic acid can inhibit osteoclast formation, bone absorption and CaMK II gamma, NFATc1, cSrc, TRAP gene expression, and these genes may be involved in the inhibition of azolic U; phosphonic acid on osteoclasts; 3 Zoledronic acid can significantly inhibit the binding of CaMK II and Calmodulin protein in osteoclast differentiation, which may be related to the induced osteoclast inhibition; 4.CaMK II gamma RNA interference can significantly inhibit osteoclast formation, bone absorption and the expression of NFATc1, c-Src, and TRAP in the downstream gene; the above gene may mediate CaMK II gamma against osteoclast. Adjustment and control.

【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

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