【作者】 王敏；

【导师】 罗湘杭；

【作者基本信息】 中南大学 ， 内科学， 2008， 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鼠组织病理学和组织化学的特性。方法40只6W龄小鼠随机分为5组(n=8),包括16只脂联素基因敲除鼠和野生型鼠24只。分别给予(前4组)高脂高糖膳食饲料和普通膳食,第5组腹腔注射链脲菌素1次(30mg/kg)再高脂高糖膳食喂养构建2型糖尿病模型鼠。10W后安乐死处死动物,摘眼球取血,分离血浆,采用酶法测定血糖浓度,放射免疫法测定血胰岛素水平。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。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,离心后取上清液采用对硝基苯酚法测定ALP活性。用Bradford法测总蛋白含量。Western-blot检测Run x2蛋白表达。结果高脂高糖膳食喂养条件下,脂联素基因敲除鼠与链脲菌素干预鼠的血糖、血胰岛素水平明显高于野生型鼠。敲除鼠出现了严重的动脉钙化。脂联素基因敲除鼠动脉钙含量明显高于野生型鼠和链脲菌素干预鼠。与普通膳食身高喂养的小鼠比较,高脂高糖膳食组鼠ALP活性升高,敲除鼠升高更为明显。Runx2蛋白的表达脂联素基因敲除鼠明显高于野生鼠。结论在高脂高糖膳食喂养条件下,脂联素基因敲除鼠出现严重的2型糖尿病性动脉钙化,其机制可能与动脉中升高的ALP活性和Runx2蛋白的表达有关。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。方法剖杀用高脂高糖饮食喂养10W脂联素基因敲除鼠,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PT-PCR和Western-blot分别测定脂联素受体mRNA和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,采用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。脂联素干预能使钙化的血管平滑肌细胞ALP活性降低,OC分泌减少,Runx2蛋白表达下降,并且呈剂量依赖关系。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表达AdipoR1结合发挥作用。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 (AdipoRl-SiRNAs)抑制AdipoRl表达,观察AdipoR1、p-p38和p38蛋白在钙化血管平滑肌细胞的表达。加入p38信号传导抑制剂SB203580,联合AdipoRl-SiRNAs,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。SiRNAs-AdipoRl转染可阻断AdipoRl表达,可抑制p38磷酸化。p38抑制剂SB203580和SiRNAs-AdipoR1可减弱脂联素对钙化血管平滑肌细胞ALP活性的影响。说明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AdipoR1/p38信号途径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 miceObjective to observe on histopathologic and histochemical characters in adiponectin knockout (Adipo-/-) mice.MethodsForty of 6 weeks old mice were divided at random into 5 groups (n=8 per group). Group 1:wild type (WT) mice fed high-fat/high-sucrose (HF/HS)diet. Group 2:Adipo-/-mice fed HF/HS diet. group3:WT mice fed normal chow diet. Group 4:Adipo-/-mice fed normal diet. Goup 5: WT mice intraperitoneal injected streptozotocin (30mg/kg body weight) 1 time and then fed HF/HS diet developing a model of type 2 diabetes mellitus. After 10 weeks, the mice were euthanized.Blood was collected from retro-orbital venous plexus, and plasma concentrations of glucose were determined enzymatically.Insulin 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. 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.Runx2 protein expression was analyzed by Western-blot.ResultsAdipo-/-mice and the model mice of type 2 diabetes mellitus from the HF/HS diet and displayed high fasting glucose levels, and high fasting plasma insulin levels, these were significantly higher than those in other mice. Severe aortic calcification could be seen in Adipo-/-mice, and aortic calcium contents in Adipo-/-mice with HF/HS diet increased more obviously than WT mice and streptozotocin injected mice compared with the normal diet.WT mice with HF/HS showed significantly elevated ALP activity, but Adipo-/-mice with HF/HS diet increased more obviously. The expression of Runx2 protein in aorta of Adipo-/-mice with HF/HS diet was higher than that of WT diabetic mice (group 5).ConclusionOur work demonstrated that Adipo-/-mice developed severe diabetic arterial calcification, and the mechanism was associated with the elevated ALP activity and Runx2 protein expression in the arota. Part two The effects of adiponectin on cultured calcifying vascular smooth muscle cellsObjectiveTo investigate the expression of adiponectin receptor 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 high-fat/high-sucrose (HF/HS) diet diet for 10 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 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, the alkaline phosphatase (ALP) activity was measured by spectrophotometric measurement of P-nitorphenol release, osteocalcin(OC) was detected by radioimm-unoassay, 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 muscle a-actin confirmed vascular smooth muscle cells (VSMCs) pheotype and the multicellular nodules spontaneously appeared in VSMCs culture. Adiponectin receptor R1 (AdipoRl) and Adiponectin receptor R2 (AdipoR2) mRNA were expressed in CVSMCs, but only AdipoRl Protein was detected. Adiponectin suppressed ALP activity, OC secretion and Runx2 protein expression in a dose-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. 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, then AdipoRl, p-p38, p38 were analysed. 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. These effects were blocked by suppression of AdipoRl with SiRNA. Furthermore, pretreatment of CVSMCs with p38 inhibitor (SB203580) or SiRNAs-AdipoRl abolished adiponectin induced ALP activity. These date suggested that p38 signaling pathway played an important role in inhibiting the calcification of CVSMCs. ConclusionOur date indicated adiponectin inhibited osteoblastic calcification of CVSMCs via AdipoR1/p38 signaling pathway.更多还原