【作者】 赵立玲；

【导师】 廖二元；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 第一部分脂联素基因敲除小鼠动脉钙化病理特征性观察目的观察脂联素基因敲除小鼠组织病理学和组织化学的特性。方法SPF级6周龄雄性脂联素基因敲除小鼠纯合子(Adiponectin-/-)30只随机分为5组,第1、2、3组分别给予普通膳食喂养10、20、30周,第4组每2周经颈静脉注射空白腺病毒载体(即β-半乳糖甘酶腺病毒载体)并予普通膳食喂养30周,第5组每2周经颈静脉注射重组脂联素腺病毒载体并予普通膳食喂养30周；另随机选取SPF级6周龄雄性野生型小鼠(WT)6只为正常对照组,编为第6组,给予普通膳食喂养30周。Adiponectin-/-与WT小鼠均由具有C57BL／6J背景的脂联素杂合子(Adiponectin+/-)小鼠相互交配产生。安乐死处死动物,摘眼球取血,分离血浆,采用酶法测定血糖浓度,放射免疫法测定血浆胰岛素和脂联素水平。分离鼠胸主动脉置4%多聚甲醛固定,石蜡包埋,连续切片,行茜素红钙化染色,免疫组化方法检测动脉血管壁OPG和RANKL的表达。分离主动脉弓到髂骨分支的动脉,用比色法测定10%甲酸抽提的钙含量。超声破碎胸主动脉,用Bradford法测总蛋白含量,离心后取上清液采用对硝基苯酚法测定ALP活性。结果经普通膳食喂养的各组脂联素基因敲除小鼠与野生型小鼠在体重、血糖、血胰岛素水平方面无明显区别。与野生型小鼠、喂养10及20周的脂联素基因敲除小鼠相比,喂养30周的脂联素基因敲除小鼠出现了轻度的动脉钙化,其动脉的钙含量及ALP活性升高,动脉血管壁OPG蛋白表达下降。通过对脂联素基因敲除小鼠进行外源性脂联素的补充,抑制了动脉钙化的出现及ALP活性升高,并增加了血管壁OPG的表达。动脉血管壁未见RANKL蛋白表达。结论在普通膳食喂养30周后,脂联素基因敲除小鼠出现轻度的动脉钙化,其机制可能与动脉中升高的ALP活性和OPG蛋白表达下降有关；外源性脂联素的补充可抑制脂联素基因敲除小鼠动脉钙化的发生,提示脂联素为动脉钙化的保护因子。第二部分脂联素对钙化的血管平滑肌的影响目的探讨体外培养的钙化血管平滑肌细胞中脂联素受体和OPG/RANKL/RANK系统的表达及其用重组脂联素干预后对钙化血管平滑肌的影响。方法剖杀用普通饮食喂养30W脂联素基因敲除小鼠,分离主动脉行钙化动脉平滑肌细胞培养,应用免疫细胞化学方法鉴定血管平滑肌细胞。应用RT-PCR和Western-blot分别测定AdipoR、OPG、RANKL及RANK mRNA和AdipoR、OPG、RANKL、RANK蛋白在钙化血管平滑肌细胞中的表达情况。取不同浓度的(0,3,10,30μg/mL)脂联素干预钙化的血管平滑肌细胞48h或用30μg/mL脂联素干预12-48h,观察脂联素对血管平滑肌细胞的剂量及时间效应。采用实时定量PCR法检测OPG mRNA水平,ELISA法测定OPG蛋白的表达,对硝基苯酚法测定碱性磷酸酶(ALP)活性,放射免疫法检测骨钙素(OC)含量,Western-blot检测Runx2蛋白的表达。用30μg/mL的脂联素干预20d后,1%茜素红进行钙化结节染色。结果钙化血管平滑肌细胞α-肌动蛋白(a-actin)的免疫细胞化学染色证定培养细胞为血管平滑肌细胞,且该细胞能自发形成钙化结节。钙化的平滑肌细胞主要表达脂联素受体R1 mRNA和蛋白,可检测到OPG、RANK mRNA和蛋白,但未能检测到RANKL的mRNA和蛋白。脂联素干预能使钙化的血管平滑肌细胞OPG mRNA和蛋白表达增加,ALP活性降低,OC分泌减少,Runx2蛋白表达下降,并且呈剂量依赖和时间依赖关系。用30μg/mL脂联素干预20d后,钙化血管平滑肌细胞的钙化结节得到显著抑制。结论脂联素能抑制培养的钙化血管平滑肌细胞的钙化,其机制可能通过与钙化血管平滑肌细胞上的AdipoRl结合发挥作用,从而促进血管保护因子OPG的表达,并抑制血管平滑肌细胞向成骨细胞表型的转换。第三部分脂联素通过AdpoRl/p38信号途径抑制CVSMCs体外成骨样钙化目的探讨脂联素抑制脂联素基因敲除鼠钙化血管平滑肌细胞体外成骨样钙化的作用机制。方法用30μg/mL脂联素干预钙化血管平滑肌细胞,0、5、30、60min后抽提细胞总蛋白,用Western-blot方法检测细胞传导信号p-JNK、JNK、p-p38、p38、p-ERK、ERK蛋白在细胞内的表达。合成脂联素受体R1的小干扰RNA(AdipoR1-SiRNAs)抑制AdipoRl表达。加入p38信号传导抑制剂SB203580,联合AdipoRl-SiRNAs,用实时定量PCR法检测钙化血管平滑肌细胞OPG mRNA水平的变化,用ELISA法检测OPG蛋白的表达,用对硝基苯酚法测定钙化血管平滑肌细胞碱性磷酸酶(ALP)的活性。结果脂联素可诱导钙化血管平滑肌细胞p38磷酸化,但对JNK和ERK的激活无作用。p38抑制剂SB203580和SiRNAs-AdipoRl可减弱脂联素对钙化血管平滑肌细胞OPG表达的促进作用及ALP活性的影响。说明AdipoRl/p38信号通路在脂联素抑制钙化血管平滑肌细胞的钙化中起重要作用。结论脂联素可通过AdipoRl/p38信号途径促进OPG的表达,并抑制钙化血管平滑肌细胞体外成骨样钙化。更多还原

【Abstract】 Part one Observation on pathological character of arterial calcification in adiponectin knockout miceObjectiveto observe on histopathologic and histochemical characters in adiponectin knockout (Adipo-/-)mice.MethodsThirty-six of 6 weeks old mice were divided at random into 6 groups (n=6 per group).Group 1:Adiponectin-/-mice fed chow diet for 10 weeks.Group 2:Adiponectin-/-mice fed chow diet for 20 weeks.Group 3:Adiponectin-/- mice fed chow diet for 30 weeks. Group 4: Adiponectin-/-mice treated with P-galactosidase adenovirus for 30 weeks. Group 5:Adiponectin-/-mice treated with adiponectin adenovirus for 30 weeks.Group 6:Wild-type mice fed chow diet for 30 weeks.The mice were euthanized, blood was collected from retro-orbital venous plexus, and plasma concentrations of glucose were determined enzymatically. Insulin and adiponectin concentrations were determined by radioimmunoassay. The thoracic aortas were dissected, and then aortas were fixed in 4% paraformaldebyde.Alizarin Red S staining was used to detect calcification. Immunohistochemistry was used to detect the expression of OPG and RANKL protein.Aortic segments from aorta arch to the iliac bifurcation were removed, then calcium was extracted with 10% formic acid and the colorimetric quantification of calcium was achieved. The thoracic aorta was homogenized by ultrasounds, the ALP activity of supernatant was measured by spectrophotometric measurement of P-nitrophenol release.Total protein was determined using Bradford protein assay.ResultsThere was no significant difference in body weight, serum fasting glucose and insulin levels between Adiponectin-/- mice and wild type mice.Adiponectin-/-mice developed slight arterial calcification after fed with normal chow diet for 30 weeks,the aortic calcium contents and ALP activity increased, and the expression of OPG decreased. There were no visible arterial calcification observed in Adiponectin-/- mice with chow diet for 10 or 20 weeks.After adenovirus-mediated supplement of adiponectin for Adiponectin-/- mice,no arterial calcification were shown. The aortic calcium and ALP activity contents also decreased, and the expression of OPG increased. It showed that adenovirus-mediated supplement of adiponectin attenuated arterial calcification. RANKL protein wasn’t detected in these mice.ConclusionOur findings demonstrated that Adiponectin-/-mice developed arterial calcification, and this could be attributed to the elevated ALP activity and the decreased expression of OPG, and adenovirus-mediated supplement of adiponectin attenuated arterial calcification.It suggested that adiponectin play a protective role against arterial calcification. Part two The effects of adiponectin on cultured calcifying vascular smooth muscle cellsObjectiveTo investigate the expression of adiponectin receptor and OPG/RANKL/RANK system in culured calcifying vascular smooth muscle cells (CVSMCs) in vitro and the effects of adiponectin on CVSMCs.Methods6 weeks old male adiponectin knockout mice (Adipo-/-) were fed cow diet for 30 weeks, then these mice were sacrificed, the aorta removed, and the CVSMCs were cultured. The culured CVSMCs were identifed by their positive staining with monoclonal antibody a-actin.The expression of adiponectin receptor and OPG/RANKL/RANK mRNA and protein in CVSMCs were detected using RT-PCR and western-blot.When the cells were treated with adiponectin at 3μg/mL、10μg/mL、30μg/mL for 48h, or treated with 30μg/mL adiponectin for 12-48h, to investigate the dose effect and time effect of adiponectin, the OPG mRNA was measured by Real-time quantitative PCR and OPG protein was measured by ELISA; alkaline phosphatase (ALP) activity was measured by spectrophotometric measurement of P-nitorphenol release, osteocalcin(OC) was detected by radioimmunoassay, and Runx2 expression was analyzed by Western-blot. The calcified nodules were stained by 1% Alizarin Red S in the presence of 30μg/mL for 20 days.ResultsThe immunocytochemical stain for smooth muscleα-actin confirmed vascular smooth muscle cells (VSMCs) pheotype and the multicellular nodules spontaneously appeared in VSMCs culture. Adiponectin receptor R1 (AdipoR1)and Adiponectin receptor R2 (AdipoR2) mRNA were expressed in CVSMCs, but only AdipoRl Protein was detected; OPG、RANK mRNA and protein was detected,but RANKL mRNA and protein was not detected;and Adiponectin elevated the level of OPG mRNA and protein, but suppressed ALP activity, OC secretion and Runx2 protein expression in a dose-dependent and time-dependent manner. Adiponectin also decreased calcified nodules formation in at 30μg/mL concentration for 20 days.ConclusionThese data indicated that adiponectin significantly inhibited cultured CVSMCs calcification in vitro may mediate by AdipoR pathway, thereby promoted the expression of vessel protecting factor OPG and blocked the transform from VSMCs to osteoblasts phenotype. Part three Adiponectin inhibited osteoblastic calcification of CVSMCs via AdipoRl/p38 signaling pathway in vitroObjectiveTo investigate mechanisms of adiponectin inhibited osteoblastic calcification of cultured calcifying vascular smooth muscle cells (CVSMCs) in vitro.MethodsAfter in the presence of 30μg/mL adiponectin for 0,5,30,60min, the total protein were extracted. The protein of P-JNK, JNK, P-p38,p38, P-ERK, ERK were detected using Western-blot method. Small interfering RNA (SiRNAs) was used to down-regulate the expression of AdipoRl in CVSMCs.The OPG mRNA was measured by Real-time quantitative PCR and OPG protein was measured by ELISA, and the ALP activity was measured by spectrophotometric measurement of p-nitrophenol release when SB 203580 blocked p38 or SiRNAs blocked AdipoRl.ResultsAdiponectin induced activation of p38,but not JNK and ERK in CVSMCs.Furthermore,pretreatment of CVSMCs with p38 inhibitor (SB203580) or SiRNAs-AdipoRl abolished adiponectin induced the expression of OPG and ALP activity. These date suggested that p38 signaling pathway played an important role in inhibiting the calcification of CVSMCs. ConclusionOur date indicated adiponectin induced the expression of OPG and inhibited osteoblastic calcification of CVSMCs via AdipoR1/p38 signaling pathway.更多还原