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

【摘要】：目的研究鈣調(diào)蛋白依賴性激酶Ⅱ(Calmodulin-dependent kinase Ⅱ,CaMKⅡ)γRNA干擾對破骨細胞生成、骨吸收功能以及下游信號分子活化T-細胞核因子c1(Nuclear factor of activated T-cells cytoplasmic 1,NFATc1)、非受體酪氨酸激酶(cell-sarcoma receptor coactivator,c-Src)、抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)基因表達的影響,以證實CaMKⅡγ在破骨細胞分化中的關(guān)鍵調(diào)控作用和分子機制。方法1破骨細胞分化中CaMKⅡγ基因表達規(guī)律研究。應(yīng)用50 ng/ml受體活化核因子-κB配體(Receptor activator of nuclear factor-κB ligand,RANKL)誘導(dǎo)小鼠RAW264.7細胞向破骨細胞分化,并于誘導(dǎo)第0d、1d、3d、5d四個時間點收獲細胞,采用實時熒光定量PCR、蛋白質(zhì)印跡法、免疫熒光細胞化學(xué)法檢測破骨細胞分化中CaMKⅡγ基因的表達規(guī)律。2唑U;膦酸對破骨細胞分化及相關(guān)基因表達影響的研究。小鼠RAW264.7細胞分為兩組:A組為對照組,B組為唑來膦酸處理組。兩組細胞均用50 ng/ml RANKL誘導(dǎo)細胞向破骨細胞分化;B組在培養(yǎng)1d后,加用1×10-6mol/L的唑來膦酸處理2d,然后撤掉唑來膦酸,繼續(xù)培養(yǎng)。于第5d、7d收獲細胞進行相關(guān)檢測。應(yīng)用TRAP染色、牙本質(zhì)磨片吸收陷窩檢測評價兩組細胞破骨細胞生成及骨吸收情況;并通過實時熒光定量PCR、蛋白質(zhì)印跡法、免疫熒光細胞化學(xué)檢測兩組細胞CaMKⅡγ、NFATc1、TRAP、c-Src基因表達情況。3唑來磷酸對破骨細胞分化中CaMKⅡ和Colmodulin蛋白結(jié)合的影響。小鼠RAW264.7細胞分為A、B兩組:A組為對照組,B組為唑來磷酸處理組。兩組均用50ng/ml RANKL誘導(dǎo),B組在1 d后加用1×10-6 M唑來膦酸處理2 d。應(yīng)用免疫共沉淀(Co-Immunoprecipitation,Co-IP)及反向Co-IP對CaMKⅡ與Colmodulin蛋白結(jié)合進行分析。4 CaMKⅡγRNA干擾對破骨細胞分化及相關(guān)基因表達的影響。應(yīng)用攜帶綠色熒光蛋白(GFP)的慢病毒載體構(gòu)建三個CaMKⅡγ重組RNA干擾載體。陰性載體轉(zhuǎn)染RAW264.7細胞,確定最適的病毒轉(zhuǎn)染滴度MOI值和轉(zhuǎn)染效率。在最佳MOI值下用CaMKⅡγ重組RNA干擾載體轉(zhuǎn)染RAW264.7細胞,通過實時熒光定量PCR及蛋白質(zhì)印跡法檢測破骨細胞向分化中三個CaMKⅡγ重組載體的干擾效果,確定干擾效果最佳的重組干擾載體,用于下一步實驗。實驗分為A、B、C三組:A組為對照組、B組為陰性載體組、C組為干擾載體組。細胞轉(zhuǎn)染12小時后,換用含50 ng/ml RANKL的培養(yǎng)基誘導(dǎo)細胞向破骨細胞分化,并于誘導(dǎo)5天后收獲細胞。通過TRAP染色及牙本質(zhì)磨片吸收陷窩檢測評價三組細胞破骨細胞生成及骨吸收情況;通過實時熒光定量PCR、蛋白質(zhì)印跡法、免疫熒光細胞化學(xué)檢測三組細胞CaMKⅡγ及其下游相關(guān)基因NFATc1、TRAP、c-Src基因表達情況。結(jié)果1破骨細胞分化第0d、1d、3d、5d,CaMKⅡγmRNA水平分別為1.067±0.179、1.840±0.070、9.493±0.453和30.767±0.573;蛋白水平分別是494.567±20.121、663.533±38.741、858.600±19.367和980.367±23.403;與第0d比較,除第1d蛋白水平外(P0.05),各時間點m RNA水平(P0.01)及蛋白水平(P0.01)均呈時間依賴性表達增強。免疫熒光檢測顯示第0d和1d蛋白表達較弱,而在第3d和第5d蛋白表達強度明顯增加,并有多核破骨細胞形成。2唑U;膦酸處理下B組多核破骨細胞數(shù)、牙本質(zhì)吸陷窩數(shù)目和面積分別為11.33±1.52(個)、8.66±2.08(個)和5034.35±775.42μm2,較對照組的37.66±5.68(個)、23.00±4.00(個)和15042.71±1906.03μm2顯著減少(P0.01),下降幅度分別為69.91%、62.60%和66.53%。唑來膦酸對破骨細胞分化過程中CaMKⅡγ及其下游因子NFATc1、TRAP和c-Src m RNA及蛋白水平也產(chǎn)生了抑制作用;與A組比較,B組上述4個基因m RNA水平分別下降了44.603%、54.126%、58.942%和51.546%(P0.01);蛋白水平分別下降了46.127%、36.799%、27.140%和32.060%(P0.01);免疫熒光細胞化學(xué)也證實B組蛋白水平明顯下降。3 Co-IP及反向Co-IP檢測顯示,B組CaMKⅡ與Calmodulin蛋白結(jié)合較A組顯著降低,分別下降了59.75%和50.87%(P0.01);在總蛋白中,B組Calmodulin與CaMKⅡ蛋白較A組也顯著下降,分別降低了52.12%和51.49%(P0.01)。4本實驗成功構(gòu)建了CaMKⅡγ重組慢病毒干擾載體。最適病毒滴度MOI值是30,其轉(zhuǎn)染效率80%。經(jīng)實時熒光定量PCR和蛋白質(zhì)印跡法檢測,#3重組載體對CaMKⅡγ干擾效果最佳,干擾效率在m RNA及蛋白水平分別為78.158%和62.226%;因而應(yīng)用#3重組干擾載體進行下面實驗。經(jīng)轉(zhuǎn)染后,三組細胞中C組(干擾載體組)多核破骨細胞數(shù)、牙本質(zhì)吸陷窩數(shù)目和面積在分別為10.670±1.52(個)、87.330±1.528(個)和4922.000±64.086μm2,顯著低于B組(陰性載體組)的22.670±1.2528(個)、12.670±2.082(個)、0924.330±66.905μm2和A組(對照組)的26.670±1.528(個)、16.000±1.000(個)、11980.000±70.000μm2(P0.05);而A組和B組之間無顯著性差異(P0.05)。CaMKⅡγRNA干擾顯著抑制了CaMKⅡγ及其下游因子NFATc1、TRAP、c-Src的表達。實時熒光定量PCR檢測表明C組CaMKⅡγ、NFATc1、TRAP、c-Src m RNA水平較A組分別下降了79.872%、49.856%、43.649%和53.567%(P0.01);蛋白質(zhì)印跡法檢測顯示C組上述4個基因蛋白水平較A、B組也顯著減弱(P0.01),下降幅度與B組比較分別為61.70%、54.22%、46.75%和45.86%;免疫熒光化學(xué)檢測也證實C組上述基因熒光強度明顯弱于A、B組。結(jié)論1.CaMKⅡγ在破骨細胞分化中呈時間依賴性表達增強,提示其在破骨細胞分化中可能發(fā)揮關(guān)鍵調(diào)控作用;2.唑來膦酸可顯著抑制破骨細胞生成、骨吸收及CaMKⅡγ、NFATc1、cSrc、TRAP基因表達;上述基因可能參與了唑U;膦酸對破骨細胞的抑制;3.唑來膦酸可顯著抑制破骨細胞分化中CaMKⅡ與Calmodulin的蛋白結(jié)合,這可能與其誘發(fā)的破骨細胞抑制有關(guān);4.CaMKⅡγRNA干擾可顯著抑制破骨細胞生成、骨吸收功能及下游基因NFATc1、c-Src、TRAP的表達;上述基因可能介導(dǎo)了CaMKⅡγ對破骨細胞分化的調(diào)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.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R78

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