【作者】 周韶琼；

【导师】 管思明；

【作者基本信息】 华中科技大学 ， 老年医学， 2013， 博士

【摘要】 第一部分Notch1-RBP-Jκ信号通路与动脉中膜钙化的关系目的：明确Notch1-RBP-Jκ信号通路和血管平滑肌细胞钙化的相关性。方法：建立钙化模型,按钙化干预时间分为钙化0天、7天和14天三组。应用von kossa染色、钙离子含量测定、碱性磷酸酶(ALP)活性并检测Msx2表达以明确VSMCs成骨样转化；RT-PCR和western blot检测Notch1、RBP-Jκ mRNA和蛋白表达、免疫组化检测N1-ICD蛋白以明确Notch1—RBP-Jκ信号通路表达和激活。结果：von kossa染色显示VSMCs钙化模型中钙沉积随钙化干预时间延长而增加：钙化7天组可见少量散在钙沉积,钙化14天组有明显黑色钙沉积。随钙化干预时间增加钙离子含量表达上升：钙化14天组表达最高(P<0.05)。Notch1、RBP-Jκ mRNA和蛋白均随钙化程度增加表达上升,钙化14天组表达最高(P<0.05),Msx2表达在钙化14天组表达最高(P<0.05)。免疫组化结果显示N1-ICD蛋白表达也随钙化程度增加而增加。结论：动脉中膜钙化过程中,Notchl-RBP-Jκ信号通路表达增加并被激活,其表达同VSMCs钙化程度呈正相关。第二部分Notch1-RBP-Jκ信号通路对动脉中膜钙化的影响目的：探讨抑制Notch1信号通路对VSMCs钙化的影响。方法：建立VSMCs钙化模型和Notch1信号抑制模型(DAPT:Notch1信号抑制剂)。按干预时间分为六组,分别为钙化0天组、7天组和14天组,与DAPT0天组、7天组和14天组。检测Notchl、RBP-Jk mRNA和蛋白表达及Nl-ICD蛋白表达,明确Notchl—RBP-Jk信号通路激活情况。检测VSMCs钙盐沉积程度(von kossa染色、钙离子含量测定)和检测钙调节相关标志物(ALP活性、Msx2含量),明确VSMCs成骨样转化程度。结果：加入DAPT后,DAPT组与干预相同时间的钙化组相比,Notch1、RBP-Jκ mRNA和蛋白表达明显下降(P<0.05),免疫组化显示N1-ICD下调,Notchl信号通路受抑制。von kossa染色可见,同干预相同时间钙化组相比,DAPT14天组钙盐沉积程度明显减轻,钙离子含量和ALP活力下降(P<0.05),成骨样转化转录因子Msx2表达下降(P<0.05)结论：抑制Notchl信号表达和激活,动脉中膜钙化受抑制。第三部分骨保护素在动脉中膜钙化过程中对Notch1-RBP-Jκ信号通路的作用目的：骨保护素(OPG)在动脉中膜钙化过程中对Notch1-RBP-Jκ信号通路的作用。方法：本实验中VSMCs被分为四组：钙化组、OPG组1(0.1ng/ml)、OPG组2(1ng/ml)和OPG组3(10ng/ml)。干预14天后,应用von kossa染色、钙离子含量测定、碱性磷酸酶(ALP)活性并检测Msx2表达以明确VSMCs成骨样转化；QT-PCR和、vesterr blot检测Notch1、RBP-Jκ mRNA和蛋白表达、免疫组化检测Nl-ICD以明确Notch1-RBP-Jκ信号通路表达和激活。结果：同钙化组相比,OPG干预组von kossa染色黑色钙盐沉积明显减轻,钙离子含量、ALP活性和Msx2表达,均随OPG干预浓度的增加明显下降(P<0.05)。Notch1、 RBP-Jκ蛋白和mRNA水平表达也随OPG浓度增加而减少(P<0.05)。结论：OPG可抑制血管平滑肌细胞钙化发生,同时抑制Notch1-RBP-Jκ信号表达和激活,其效应呈现浓度依赖性。第四部分阿仑膦酸钠在动脉中膜钙化过程中对Notch1信号的作用目的：探讨阿仑膦酸钠(alendronate, ALN)在动脉中膜钙化过程中对Notch1-RBP-Jκ信号通路的作用。方法：VSMCs按照不同干预被分为七组：对照组、钙化组、DAPT组、ALN组1(10-8mmol/1)、ALN组2(10-6mmol/1)、ALN组3(10-4mmol/1)和ALN组4(10-3mmol/1)。干预14天后应用von kossa染色、钙离子含量测定、碱性磷酸酶(ALP)活性、Msx2表达以明确VSMCs成骨样转化；QT-PCR和western blot检测Notch1、 RBP-Jκ mRNA和蛋白表达、免疫组化检测N1-ICD以明确Notch1信号通路表达和激活。结果：同钙化组相比,von kossa染色证实钙盐沉积随ALN干预浓度增加明显减轻,钙离子含量、ALP活性和Msx2表达也随ALN浓度增加下降(P<0.05),并于10-4mmol/1时达到最大抑制程度。同时,细胞中Notch1、RBP-Jκ转录和翻译水平也随ALN浓度增加而下调(P<0.05)。结论：ALN具有抑制血管平滑肌细胞钙化,并抑制Notch1信号表达和激活,其效应呈现浓度依赖性。更多还原

【Abstract】 Part Ⅰ The relationship between Notchl signaling pathway and medial artery calcificationObjective:To investigate the role of Notchl-RBP-Jκ signaling pathway in the medial calcification.Methods:A vascular smooth muscle cell calcification model was set up. Modles were seperated into three groups according to their calcification intervention time:0day group,7days group and14days group. The degree of local calcium deposition and calcium-regulating markers, that includes von kossa staining, calcium content, alkaline phosphatase (ALP) activity, Msx2content, were tested to reflect the osteogenic transformation. Then the expression of moleculars in Notchl-RBP-Jκ signaling pathway were analysed by RT-PCR, western blot detection, and immunohistochemical detection.Results:VSMCs calcification model was successfully established.von kossa staining showed that positive correlation existed betweencalcium deposition and calcification intervention time:few scattered calcium deposits were displayed in the7days group, while obvious black calcium deposition wereobserved in14days group samples. Results of calcium content test proved that expression of calciumcontent were also increased along with the increase of intervention time, and reached its peak in14days group (P<0.05). The mRNA and protein level of Notchl and RBP-Jic,as well as the expression of Msx2, were found increased with the degree of calcification and reached its peak after14days intervention (P<0.05). Immunohistochemical results showed that activation levels of N1-ICD were also increased with theseverityof calcification.Conclusion:Notchl-RBP-Jκ signaling pathway is activated during the arterial medial calcification process. Its expression was positively correlated with the degree of VSMCs calcification. Part Ⅱ The effect of Notchl signaling pathway on medial artery calcificationObjective:To investigate the effect of Notchl signaling pathway on VSMCs calcification by inhibiting the signaling pathway.Methods:A blocker for Notchl signaling pathway--DAPT--was employed to inhibit the VSMCs calcification. It was also divided into6groups based onintervention time:0day group,7day group,14day group,0day group with DAPT,7day group with DAPT, and14day group with DAPT. The protein and mRNA expression of Notchl, RBP-Jκ and N1-ICD activation were tested to clearifythe effect of the Notchl signaling pathway.After inhibition of Notchl signaling pathway,von kossa staining, calcium content, ALP activity, MSX2content were adopted to clearifytheextent of osteogenic transformation.Results:After treated with DAPT, the transcription and translation level of Notchl, RBP-Jκ were down-regulated significantly when compared with calcification group at same intervention time point (P<0.05), Nl-ICD content was also decreased. All these proved that the Notchl signal pathway was inhibited, von kossa staining also revealed that the calcium deposition, the degree of calcium content and ALP activity of14-day with DAPT group was significantly decreased compared with14-day group(P<0.05).The expression of Msx2, an transcriptional factorof osteogenic transformation, was decreased at the same time (P<0.05).Conclusion:Inhibition of the expression and activation of Notchl signaling pathway can suppress the medial calcification. Part Ⅲ The effect of osteoprotegerinon the Notch1-RBP-Jκ signaling pathway in the medial artery calcificationObjective:To investigate the osteoprotegerin in the development of calcification of rat aortic vascular smooth muscle cells.Methods:The cells were divided into6groups based on intervention time:control group, calcified group, DAPT group, OPG group1with O.lng/ml OPG, OPG group2with lng/ml OPQand OPG group3with10ng/ml OPQ. The protein and mRNA expression of Notchl, RBP-Jk and N1-ICD activation were detected to clearify the effect of the Notchl signaling pathway. After inhibition of Notchl signaling pathway, von Kossa staining, calcium content, ALP activity, Msx2content were determined to clearify the extent of osteoblast transformation.Results:Based on the results of real-time qPCE and western blotting, we indentified that expression of Notchl and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both mRNA and protein levels, these effects were abolished by the treatment of OPG dose-dependently. Furthermore, we found that Msx2, a downstream target of Notchl/RBP-JK, was markedly down-regulated by the treatment of OPGConclusion:OPG could inhibit vascular calcification through the down regulation of Notchl-RBP-Jκ signaling pathway. Part Ⅳ The effect of alendronate on the Notchl-RBP-JK signaling pathway in the medial artery calcificationObjective:To investigate the effect of alendronate (ALN) on the Notch1-RBP-Jκ signaling pathway in the artery medial calcification.Methods:The cells were also divided into7groups based on intervention time:control group, calcified group, DAPT group, ALN group1with1×10-8mmol/1ALN, ALN group1with1×10-6mmol/1ALN, ALN group1with1×10-4mmol/l ALN, and ALN group1with1×10-3mmol/1ALN. The protein and mRNA expression of Notchl, RBP-Jk and N1-ICD activation were tested expression of the Notch1-RBP-Jκ signaling pathway. Von kossa staining, calcium content, ALP activity, Msx2content were determined to clearify the extent of osteoblast transformation.Result:Compared with calcification group, von kossa staining showed that calcium deposit was inhibited in the ALN groups in a does-dependent way. And calcium content, ALP activity and MSX2were inhibited in the ALN groups in a does-dependent way. The expressions of Notch1, RBP-Jκand N1-ICD were inhibited by alendronate does-dependently.Conclusion:alendronate could inhibit vascular calcification through the down regulation of Notchl-RBP-Jκ signal pathway.更多还原