【作者】 彭昱东；

【导师】 程龙献； 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 第一部分IFNy及CD40L诱导钙调神经磷酸酶依赖的VSMCs凋亡及其RANTES变化目的：观察γ干扰素(interferon-γ, IFNy)和CD40配体(CD40 ligand,CD40L)刺激下,大鼠血管平滑肌细胞(vascular smooth muscle cells, VSMCs)发生钙调神经磷酸酶(calcineurin, CaN)依赖的凋亡及其受激活调节正常T细胞表达和分泌因子(Regulated on Activated Normal T-Cell Expressed and Secreted, RANTES)表达水平的关系。方法：采用组织贴块法体外原代培养大鼠胸主动脉平滑肌细胞,并随机分为6组。A组：DMEM培养基培养24h；B组和C组：分别在培养基中加入大鼠重组INFy (rINFy, 20ng/ml)和重组CD40L (rCD40L, 100ng/ml)培养24h；D组：在培养基中同时加入rIFNy及rCD40L(剂量同前)培养24h；E组：给予CaN抑制剂FK506(10ng/ml)预培养1h后再按D组条件培养24h；F组：给予RANTES的小干扰RNA(siRNA)转染6h后再按D组条件培养24h。收集细胞用Western Blot检测CaN表达水平；酶化学法检测CaN活性,提取细胞RNA采用RT-PCR检测RANTES的：mRNA表达；ELISA法检测上清中RANTES浓度,流式细胞仪检测细胞表面CD40表达及VSMCs凋亡。结果：通过CD40-CD40L信号通路,rINFy和rCD40L的联合刺激强烈地诱导了VSMCs内CaN的合成和活力以及RANTES的转录和表达。但在上述炎症因子刺激下,与VSMCs凋亡率呈持续正相关的是RANTES转录和表达水平(P<0.01),而不是CaN活力(P>0.05)。结论：炎症因子IFNy和CD40L可能通过调节RANTES的表达及分泌,调控了CaN依赖的VSMCs凋亡。第二部分兔动脉粥样硬化斑块内VSMCs凋亡与RANTES水平的相关性研究目的：研究血浆和斑块局部受激活调节正常T细胞表达和分泌因子(Regulated on Activated Normal T Cell Expressed and Secreted, RANTES)表达水平与兔动脉粥样硬化斑块内血管平滑肌细胞(vascular smooth muscle cells, VSMCs)凋亡程度的相关性。方法：40只雄性家兔随机分为：(1)空白组(Blank组,10只)和(2)对照组(Control组,10只)：均普通饲料喂养16W；(3)稳定斑块组(AS组,10只)和(4)易损斑块组(VAP组,10只)：均高脂饮食(含胆固醇1%和猪油5%)喂养16W。Control和VAP组于16W处死前24和48h给予两次药物触发(蝰蛇毒±组织胺),Blank和AS组于16W处死前按上述时间、剂量、给药方法注射生理盐水。测定0W、16W各组血脂和血浆RANTES水平, Real Time-PCR和Western Blot检测主动脉RANTES转录和表达水平,TUNEL染色后计算各组斑块VSMCs凋亡率及其与RANTES水平的相关系数。结果：高脂喂养的家兔血脂较普通喂养组明显升高(P<0.01)。VAP组RANTES转录翻译水平和凋亡率明显高于AS组(P<0.01)、control组(P<0.0 1)和blank组(P<0.01);AS组明显高于control组(P<0.01)和blank组(P<0.01)；但AS组和control组间无显著性差异(P>0.05)。各组的VSMCs凋亡率与RANTES转录翻译水平均成正相关(P<0.05)。结论：RANTES过表达可能通过加重斑块局部的炎症反应而诱导了斑块内VSMCs凋亡第三部分兔动脉粥样硬化斑块易损性与RANTES水平的相关性研究目的：研究血浆和斑块局部受激活调节正常T细胞表达和分泌因子(Regulated on Activated Normal T Cell Expressed and Secreted, RANTES)水平与家兔动脉粥样硬化斑块易损性的关系。方法：40只雄性家兔随机分为：(1)空白组(Blank组,10只)和(2)对照组(Control组,10只)：均普通饲料喂养16W；(3)稳定斑块组(AS组,10只)和(4)易损斑块组(VAP组,10只)：均高脂饮食(含胆固醇1%和猪油5%)喂养16W。Control和VAP组于16W处死前24和48h给予两次药物触发(蝰蛇毒±组织胺),Blank和AS组于16W处死前按上述时间、剂量、给药方法注射生理盐水。测定0W、16W各组血脂和血浆RANTES水平,检测16W各组斑块局部易损指数和校正的斑块面积、核因子-κB(nuclear factor-KB, NF-κB)和RANTES的转录和表达水平,计算易损指数和校正的斑块面积与RANTES转录和表达水平的相关性。结果：高脂喂养组的血脂较普通喂养组明显升高(P<0.01); Blank和Control组间的RANTES及斑块内NF-κB转录和表达水平无显著性差异(P>0.05)；从Blank/Control组到AS、VAP组,RANTES及斑块内NF-κB转录表达水平同步逐渐升高；VAP组易损指数和校正的斑块面积明显高于AS组(P<0.01)；血浆和局部的RANTES水平与易损指数和校正的斑块面积正相关(P<0.05)。结论：血浆和斑块局部的RANTES可能通过NF-κB信号通路诱导炎症反应导致了斑块不稳定。更多还原

【Abstract】 PART I The role of RANTES as a crucial downstream cytokine in calcineurin-dependent VSMCs apoptosis stimulated by INFy and CD40LObjective:To evaluate the effects of RANTES (Regulated on Activated Normal T Cell Expressed and Secreted) on calcineurin-dependent vascular smooth muscle cells (VSMCs) apoptosis costimulated by IFN-y and CD40L (CD40 ligand).Materials and methods:VSMCs of SD rats cultured by tissue-sticking method were divided into six groups randomly. Group A was only incubated in DMEM medium for 24h; Group B and C were cultured with rat recombinant INFy (rINFy) at 20ng/ml and rCD40L at 100ng/ml for 24h, respectively; Group D was costimulated by rINFy (20ng/ml) and rCD40L (100ng/ml) for 24h;Group E was pretreated 1h with the CaN inhibitor FK506 (10ng/ml) before treatment according to group D; Group F was stimulated according to group D after transfected with the small interference RNA(siRNA) of RANTES for 6h. Expression of CD40 on the VSMC membranes in group A and B were investigated. The activity and level of CaN as well as the expression and transcription of RANTES were detected in all groups.Simultaneouly,the apoptosis rate was investigated in every group respectively.Results:The synthesis and activity of CaN as well as the expression and transcription of RANTES depended strongly on the costimulation of rINFy and rCD40L. A positive relationship was presented persistently between the rate of apoptosis and the synthesis of RANTES (P<0.01) but not CaN activity(P>0.05).Conclusion:CD40-CD40L interaction enhanced by IFN-y prestimulation induces a CaN-dependent apoptosis signal pathway in VSMCs. RANTES is a potential treating target for vulnerable atherosclerotic plaque due to its crucial downstream regulating role in the CaN-dependent VSMCs apoptosis. PART II The correlations between the expression of RANTES and apoptosis of VSMCs in atherosclerotic rabbitsObjective:To study the correlations between the expression of RANTES (Regulated on Activated Normal T Cell Expressed and Secreted) and apoptosis of vascular smooth muscle cells (VSMCs) in atherosclerotic rabbits.Materials and methods:40 male rabbits were divided into four groups randomly:(1) Blank group (n=10) and control group (n=10):received a standard diet for 16 weeks; (2)AS group (n=10) and VAP group (n=10):received a high-cholesterol diet(1% cholesterol,5% lard in rabbit food) for 16 weeks. Blank and AS groups were triggered by Russel’s viper venom (RVV) and histamine at 24 and 48h before execution, control and AS groups were administered with the same dosage of sterile saline injection according to the above delivery time and location. Rabbits were euthanized at 16 weeks, blood-fat and plasma RANTES were measured in 0, 16 weeks. The transcription and expression of RANTES were calculated with Real-time PCR and Wester Blot. VSMCs apoptosis rate in plaques was detected with TUNEL and its correlation coefficients with RANTES were measured.Results:Blood-fats of high-cholesterol diet groups were high than that of standard diet groups (P<0.01). The levels of RANTES and apoptosis rates of VSMCs in AS and VAP groups increased significantly compared with that of blank and control groups (P<0.01). The distinctions of RANTES and apoptosis rates were presented between VAP and AS group (P<0.01) but not presented between blank and control group (P>0.05). VSMCs apoptosis rates were related positively with RANTES in all groups(P<0.05).Conclusion:The progression of VAMCs apoptosis may correlate with the local inflammatory reaction which induced by the overexpression of RANTES. PARTⅢThe correlations between the levels of RANTES and vulnerability of rabbit atherosclerotic plaquesObjective:To study the correlations between the levels of RANTES (Regulated on Activated Normal T-Cell Expressed and Secreted) and the vulnerability of atherosclerotic plaques.Materials and methods:40 male rabbits were divided into four groups randomly:(1) Blank group (n=10) and control group (n=10):received a standard diet for 16 weeks; (2) AS group (n=10) and VAP group (n=10):received a high-cholesterol diet (1% cholesterol,5% lard in rabbit food) for 16 weeks. Blank and AS groups were triggered by Russel’s viper venom (RVV) and histamine at 24 and 48h before execution, control and AS groups were administered with the same dosage of sterile saline injection according to the above delivery time and location. Rabbits were euthanized at 16 weeks, blood-fats and plasma RANTES were measured in 0, 16 weeks. The vulnerability index and corrected plaque area were evaluated in high-cholesterol diet groups. The local transcription and expression of NF-κB and RANTES were calculated in all groups. The correlation coefficients of the vulnerability index and corrected plaque area with transcription and expression of RANTES were measured in high-cholesterol diet groups.Results:Blood-fats of high-cholesterol diet groups were high than that of standard diet groups (P<0.01). The distinction of transcriptions and expressions of RANTES and local NF-κB was not presented between blank and control groups (P>0.05). The transcriptions and expressions of RANTES and NF-κB increased gradually from Blank/Control to AS and VAP group. The vulnerability index and corrected plaque area in VAP group were high than that in AS group obviously (P<0.01). Transcription and expressions of RANTES were related positively with the vulnerability index and corrected plaque area in VAP and AS groups(P<0.05). Conclusion:RANTES levels in plasma and in lesions may promote the vulnerability of atherosclerotic plaques by NF-κB signaling pathway.更多还原