【作者】 李长江；

【导师】 张梅；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 背景随着研究的深入，发现脂肪组织不仅是静态的能量贮存器官，还是高度活跃的内分泌器官。脂肪细胞分泌的细胞因子主要包括：脂联素(adiponectin，APN)，抗素(resistin)，肿瘤坏死因子(tumor necrosis factorα，TNFα)等。目前，在脂肪细胞因子的研究中，发现脂联素与动脉粥样硬化关系密切。脂联素能够减少脂质斑块的面积，抑制新生内膜的增厚及平滑肌细胞的增殖与迁移，抑制血管粘附分子的表达。通过这些作用，发挥抗动脉粥样硬化的保护性效应。脂联素是由脂肪组织分泌的胶原样蛋白质，从分子结构上看，脂联素与补体Clq相似。脂联素在脂肪细胞的分化过程中被诱导，其分泌受胰岛素调控。脂联素影响机体糖和脂质的代谢。在肥胖和2型糖尿病患者，血浆脂联素浓度降低，与胰岛素抵抗和2型糖尿病的危险性相关，而高血浆脂联素则能够降低2型糖尿病的危险。在脂联素基因敲除的小鼠表现出明显的胰岛素抵抗和糖耐量降低。在对动脉粥样硬化的研究中，新西兰大白兔是一种合适的动物模型，由于兔脂联素的序列尚未见报道，限制了利用该模型对脂联素的抗动脉粥样硬化作用进行深入研究。这些问题构成了本研究的设计思路和研究目的。研究目的1．克隆新西兰大白兔脂联素cDNA并完成测序。2．体外表达新西兰大白兔脂联素蛋白质，明确克隆的基因含完整的开放性阅读框架(ORF)。3．探讨不同年龄组的新西兰大白兔脂联素基因表达的变化，明确年龄与脂联素表达的关系。4．探讨高胆固醇饲料喂养后新西兰大白兔脂联素基因表达的变化，明确高脂血症与脂联素的关系。研究方法1．新西兰大白兔脂联素cDNA序列的测定(1)取普通饲料喂养的3月龄正常雄性新西兰大白兔颈背部皮下脂肪组织约100mg用于RNA的提取。(2)脂肪组织RNA提取：使用Trizol试剂从组织中提取总RNA。(3)兔脂联素基因片段的克隆：按照M-MLV逆转录酶试剂盒说明书配逆转录反应体系。根据人和小鼠脂联素高度保守序列设计PCR反应引物。上游引物：5′-ACACCTCCAGGGCTCAGGATGCT-3′，下游引物：5′-TCAGTTGGTATCATGGTAGAGAAG-3′。PCR反应后产物经琼脂糖凝胶电泳检测，割胶回收。并将其克隆入pGEM-T载体内，按试剂盒说明书操作。终产物命名为pGEM-T-APN。(4) pGEM-T-APN连接反应产物转化大肠杆菌JM109，筛选阳性重组体。(5)阳性重组体的菌落37℃摇床扩增，送上海英骏公司测序。2．新西兰大白兔脂联素蛋白质的体外表达新西兰大白兔脂联素蛋白质的体外表达与纯化采用pGAPZ_αPichia Expression Kit(Invitrogen)试剂盒。(1)以脂肪组织总cDNA为模板，用含EcoR I和Not I酶切位点的引物重新克隆脂联素DNA，并将产物插入pGEM-T载体，产物命名为pGEM／APN，经测序证实联结正确。(2)产物pGEM／APN与pGAPZ_αPichia酵母双酶切(EcoRI和Not I)，将脂联素DNA插入pGAPZ_αPichia酵母表达载体中，得到产物pGAPZ_α-APN。(3)将pGAPZ_α-APN转化大肠杆菌DH5α，在低盐LB培养基中扩增，抽提质粒，电转入GS115酵母菌株，培养，挑取阳性单克隆。(4)阳性单克隆在YPD培养基中培养，脂联素蛋白质分泌入上清液中。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质，Western blot分析蛋白质分子量大小。(5)酵母培养液中脂联素浓度分析利用大鼠抗人脂联素ELISA试剂盒分析酵母上清液中脂联素浓度。3．年龄因素对新西兰大白兔脂联素蛋白质表达的影响选取2周龄、1月龄、3月龄、6月龄、12月龄雄性新西兰大白兔各10只，3％戊巴比妥钠(30mg／kg)静脉麻醉后，各取颈背部皮下脂肪组织约100mg用于RNA的提取。荧光实时定量PCR检测脂肪组织mRNA的表达，ELISA试剂盒分析血清中脂联素浓度。4．高胆固醇饲料喂养后新西兰大白兔脂联素蛋白质表达的变化为研究高胆固醇饲料喂养后新西兰大白兔脂联素蛋白质表达的变化，3月龄雄性新西兰大白兔12只，体重1.5～2kg，1％高胆固醇饲料喂养，由山东省农科院购买。分别于高胆固醇饲料喂养前及喂养后第1、2、3、4月时间点取脂肪组织和静脉血标本。荧光实时定量PCR检测脂肪组织mRNA的表达，ELISA试剂盒分析血清中脂联素浓度。5．统计学分析：数据采用均数±标准误(mean±SEM)表示。组间比较采用方差分析，所有数据均用SPSS 13.0软件包处理，P＜0.05为统计学有显著性差异。结果1．新西兰大白兔脂联素cDNA序列测定经RT-PCR方法克隆出兔脂联素cDNA，测序证实含完整的开放性阅读框架(ORF)，即包含起始密码子(atg)和终止密码子(tga)。核苷酸序列分析显示兔脂联素cDNA序列与人、小鼠同源性分别为86.39％和81.45％，氨基酸序列的同源性分别为85.66％和85.25％。2．新西兰大白兔脂联素蛋白质的体外表达Western blot分析证实，GS115酵母上清液中含脂联素蛋白质，分子量约30 kDa。ELISA分析，上清液中蛋白质浓度为0.29±0.02μg／ml。3．年龄因素对新西兰大白兔脂联素蛋白质表达的影响荧光实时定量PCR检测结果显示，不同年龄组的新西兰大白兔皮下脂肪组织脂联素mRNA表达有显著性差异(P＜0.01)，在1月龄组最高，然后随年龄增加而呈现出与年龄相关的下降趋势。血清脂联素浓度ELISA分析结果显示，不同年龄组的新西兰大白兔血清脂联素浓度有显著性差异(P＜0.01)，在1月龄组最高，然后随年龄增加而呈现出与年龄相关的下降趋势。4．高胆固醇饲料喂养后新西兰大白兔脂联素蛋白质表达的变化高胆固醇饲料喂养后，脂肪组织脂联素基因mRNA转录与血清HDL-C浓度成正相关(Pearson Correlation=0.469，P=0.001)，在对照组无此相关性。在高脂组和对照组，不同的时间点脂肪组织脂联素基因mRNA转录均不同(Repeated Measurements Analysis of Variance，P＜0.01)：在高胆固醇饲料喂养前，2组之间脂联素基因mRNA转录无显著性差异(P＞0.05)；在高胆固醇饲料喂养后1、2、3、4月固定的时间点上，2组之间脂联素基因mRNA转录有显著性差异(P＜0.01)。高胆固醇饲料喂养对脂联素血清浓度有相似的影响：脂联素血清浓度与血清HDL-C浓度成正相关，在对照组无此相关性。在高脂组和对照组，不同的时间点脂联素血清浓度均不同；在高胆固醇饲料喂养前，2组之间脂联素血清浓度无显著性差异(P＞0.05)；在高胆固醇饲料喂养后1、2、3、4月固定的时间点，2组之间脂联素血清浓度有显著性差异(P＜0.01)。结论1．兔脂联素cDNA序列、氨基酸序列与人、小鼠均有高度的同源性，脂联素基因在兔、人、小鼠具有相似的功能。2．在不同的年龄组脂联素基因表达存在显著性差异，在1月龄组最高，随年龄增加而呈现出与年龄相关的下降趋势。3．脂质代谢异常对脂联素的基因表达有显著性影响，脂联素与血清高密度脂蛋白胆固醇成正相关。背景越来越多的研究表明，脂联素能够减少脂质斑块的面积，抑制新生内膜的增厚及平滑肌细胞的增殖与迁移，抑制血管粘附分子的表达。通过这些作用，发挥抗动脉粥样硬化的效应。通过抑制核转录因子κB(nuclear factorκB，NF-κB)，脂联素抑制TNFα诱导的粘附分子的表达，抑制丝裂原活化蛋白(mitogen activated protein，MAP)通路进而抑制生长因子诱导的平滑肌细胞的增殖。在巨噬细胞，脂联素抑制清道夫受体的表达和泡沫细胞的形成。因此，脂联素积聚在内皮细胞损伤的部位，发挥抗动脉粥样硬化的保护性效应。目前国内、国际对脂联素与胰岛素抵抗、动脉粥样硬化的关系及作用机制方面做了大量的研究，但有许多问题尚未明了：1．在动物实验中观察到了脂联素减轻动脉内膜的增厚，降低动脉平滑肌细胞的增殖和迁移。脂联素是通过什么途径实现这种作用的?2．血管周存在脂肪组织，局部脂肪组织分泌的脂联素能否通过血管外膜影响动脉粥样硬化斑块的发生与发展?3．在动脉粥样硬化血管壁，通过转染携带脂联素基因的腺病毒，在体观察斑块的形态学改变如何，目前尚未见相关报道。以上这些问题，构成了本研究的设计思路和研究目的。研究目的1．对比脂联素通过内膜与外膜对动脉粥样硬化斑块的作用。2．观察脂联素对斑块易损性的影响。研究方法1．动脉粥样硬化新西兰大白兔动物模型的构建3月龄雄性新西兰大白兔70只，经右侧股动脉置入球囊导管，充盈球囊拉伤腹主动脉；1％高胆固醇饲料喂养，造成腹主动脉粥样硬化模型。2．腺病毒转染及血管内超声(intravascular ultrasonography，IVUS)检查高胆固醇喂养3月时，首先行IVUS检查。然后，将兔子随机分为4组，将携带脂联素基因的腺病毒载体(Ad-APN)或表达β半乳糖酐酶的腺病毒载体(Ad-βgal)转染到腹主动脉内膜和外膜。3．血清脂联素浓度分析利用大鼠抗人脂联素ELISA试剂盒分析普通饲料喂养大白兔、高胆固醇饲料喂养大白兔转染腺病毒前后血清脂联素浓度。4．组织学分析：腺病毒转染14天后，IVUS检查，处死，取局部转染部位腹主动脉标本。8μm厚冰冻切片做HE染色，天狼猩红染色，油红O染色。Image-Pro Plus 5.0图像分析软件计算粥样硬化斑块面积。并做黏附分子VCAM-1(vascular cell adhesion molecule-1)、ICAM-1(intercellular adhesion molecule-1)，Ⅰ型胶原，Ⅲ型胶原、RAM11、α-actin的免疫组化分析，计算斑块易损指数。5．荧光实时定量PCR分析：荧光实时定量PCR方法检测腹主动脉黏附分子VCAM-1、ICAM-1，Ⅰ型胶原，Ⅲ型胶原mRNA表达的变化。6．统计学分析：组间比较采用方差分析(ANOVA)，所有数据均采用SPSS 13.0软件包处理，P＜0.05为统计学有显著性差异。数据采用均数±标准误(mean±SEM)表示。结果1．血管局部转染Ad-APN对动脉粥样硬化斑块的影响与转染前比较，血管局部转染Ad-APN后，血清脂联素浓度增加约9倍(P＜0.01)。与对照组转染Ad-βgal比较，血管局部转染Ad-APN后，血清脂联素浓度增加约10倍(P＜0.01)。血管局部转染Ad-APN后，与没有腹主动脉损伤正常胆固醇饲料喂养的新西兰大白兔比较，血清脂联素浓度增加约4倍(P＜0.01)。血管内膜局部转染Ad-APN后，动脉粥样硬化斑块面积与转染前相比明显缩小达35.17％(P＜0.01)，管腔面积狭窄率减小32.71％(P＜0.01)，与对照组(转染Ad-βgal)相比斑块面积缩小35.82％(P＜0.01)，管腔面积狭窄率减小24.09％(P＜0.01)。血管外膜转染Ad-APN与转染前相比，斑块面积明显缩小达28.92％(P＜0.01)，管腔面积狭窄率减小23.57％(P＜0.01)，与外膜转染Ad-βgal相比斑块面积缩小25.64％(P＜0.01)，管腔面积狭窄率减小19.43％(P＜0.05)；内膜或外膜转染Ad-βgal前后斑块面积均无统计学意义(P＞0.05)。油红O染色结果：内膜或外膜转染Ad-APN与对照组相比斑块面积缩小(P＜0.01)，斑块最大厚度减小(内膜转染与对照组相比：P＜0.01；外膜转染与对照组相比：P＜0.05)。内膜和外膜转染Ad-APN斑块面积、最大厚度两组间无统计学差异(P＞0.05)。2．脂联素对血管壁黏附分子表达的影响内膜转染Ad-APN与转染Ad-βgal比较：VCAM-1mRNA的表达降低18.5％(P＜0.05)，ICAM-1 mRNA的表达显著降低40.7％(P＜0.01)。外膜转染Ad-APN与转染Ad-βgal比较：VCAM-1 mRNA的表达降低26.9％(P＜0.01)，ICAM-1mRNA的表达降低30.7％(P＜0.01)。3．脂联素对血管壁胶原分子表达的影响内膜转染Ad-APN与转染Ad-βgal比较：血管壁Ⅰ型胶原mRNA的表达为1.82±0.26 versus 1.55±0.17％，P＞0.05，Ⅲ型胶原mRNA的表达为26.96±5.12 versus25.15±3.67％，P＞0.05。外膜转染Ad-APN与转染Ad-βgal比较：血管壁Ⅰ型胶原mRNA的表达为2.51±1.07 versus 1.86±0.28％，P＞0.05，Ⅲ型胶原mRNA的表达为19.98±3.20 versus23.07±4.07％，P＞0.05。4．脂联素对动脉粥样硬化斑块易损性的影响腹主动脉内膜局部转染Ad-APN组血管壁血管壁天狼猩红染色阳性百分比无显著性差异(P＞0.05)，α-actin表达增加28.88％(P＜0.01)，油红O染色阳性百分比降低24.48％(P＜0.01)，RAM11染色阳性百分比降低28.89％(P＜0.01)，易损指数显著降低33.78％(P＜0.01)。腹主动脉内膜局部转染Ad-APN组血管壁血管壁天狼猩红染色阳性百分比无显著性差异(P＞0.05)，α-actin表达增加23.88％(P＜0.01)，油红O染色阳性百分比降低25.54％(P＜0.01)，RAM11染色阳性百分比降低32.11％(P＜0.01)，易损指数显著降低27.03％(P＜0.01)。结论1．动脉粥样硬化病变血管内膜或外膜局部转染Ad-APN后，斑块的面积缩小，同时，血清脂联素浓度明显升高，脂联素可通过抑制血管壁黏附分子VCAM-1和ICAM-1的表达发挥抗动脉粥样硬化的作用；脂联素通过血管外膜或内膜对动脉粥样硬化的作用是一致的。2．血管内膜或外膜局部转染Ad-APN后，脂联素对血管壁Ⅰ型胶原和Ⅲ型胶原的表达没有影响。3．局部转染脂联素基因后，动脉粥样硬化斑块内胶原的含量虽然无明显变化，而斑块内平滑肌细胞量的增加，脂质含量和巨噬细胞的数量降低，可使斑块的易损性降低。更多还原

【Abstract】 BackgroundsTraditionally, adipose has been regarded as a simple energy storage organ, but mounting evidence suggests it produces and secretes many bioactive substances, collectively referred to adipocytokines such as adiponectin （APN）, resistin, TNFα, etc. Adiponectin has been found to play an important role in preventing atherosclerosis in which it reduces the size of atherosclerotic lesions, inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, and suppresses expression of vascular adhesion molecules.Secreted by adipocytes, adiponectin is a collagen-like protein whose encoding gene is located in chromosome 3q27 and named as apM 1 gene in human beings. There are 17 kbp in apM 1 gene including 3 exons and 2 introns and the complete apM 1mRNA has 4517 bp. The molecular structure of adiponectin is like complement C1q. Adiponectin is induced in the process of adipocytes differentiation and its secretion is regulated by insulin.Adiponectin has significant roles in regulating metabolism of glucose and fatty acids. Low plasma adiponectin levels are associated with insulin resistance and risk of type 2 diabetes and high plasma adiponectin levels decrease the risk of type 2 diabetes. The adiponectin-knockout mice exhibit significant insulin resistance and decreased carbohydrate tolerance.New Zealand rabbit is an ideal model in the research on atherosclerosis, but there are some limitations due to the rabbit adiponectin gene sequence has not been reported at present. With this consideration in our mind, the present study was designed. Research Objectives1. Cloning and sequencing of New Zealand rabbit adiponectin gene.2. Expressing in vitro and sequencing of New Zealand rabbit adiponectin protein.3. Investigating the relationship between New Zealand rabbit adiponectin gene expression and age.4. Investigating the relationship betwene New Zealand rabbit adiponectin gene expression and high-cholesterol diet.Methods1. Cloning of the rabbit adiponectin gene fragments（1） To clone the gene fragments, cervical back fat （brown fat） was obtained from 3-month-old male rabbits freely consuming a standard diet.（2） Total RNA was extracted from adipocytes by use of Trizol reagent following the manufacturer’s instruction.（3） Degenerative primers were designed from highly conserved regions of mouse and human adiponectin sequences and used to amplify adiponectin cDNA （forward primer, 5’-ACACCTCCAGGGCTCAGGATGCT-3’; reverse primer, 5’-TCAGTTGGTATCATGG TAGAGAAG-3’）. The PCR product was separated and purified with use of the EZNA Gel Extraction Kit according to the manufacturer’s protocol. The PCR product was cloned into a pGEM-T Vector System I and named pGEM-T-APN.（4） The pGEM-T-APN was transformed into E. coli JM109 and the positive transformants were selected.（5） The positive transformants were amplified at 37℃ with shaking and then sequenced （Invitrogen, Shanghai, China）.2. Protein expression in Pichia pastorisExpression and purification of the protein were carried out with a pGAPZα Pichia Expression Kit （Invitrogen） following the manufacturer’s instruction.（1） The cDNA containing the coding region of mature rabbit adiponectin was amplified by PCR （containing EcoR I and Not I sites）, then inserted into the pGEM-T vector by T/A cloning strategy, leading to a subcloning vector pGEM/APN （containing adiponectin gene）. By sequencing, the pGEM/APN was confirmed to contain adiponectin gene.（2） The pGEM/APN was then inserted between the EcoR I and Not I sites of pGAPZα （containing an amino-terminal His-tag） leading to pGAPZα-APN.（3） The pGAPZα-APN was transformed into E. Coli DH5α.（4） After the above transformation, plate transformation was mixed onto low salt LB plates with 25 μg/ml Zeocin^TM （Invitrogen） and Zeocin^YM resistant colonies were selected. The pGAPZα-APN was transformed into the GS115 yeast by electroporation.（5） The recombinant yeasts were cultured in YPD medium and grown in flask at 30 °C with shaking overnight. The supernatant was transferred to a separate tube and stored at -80 °C until ready to assay. Sample proteins were separated by SDS-PAGE and analyzed with Western blot.（6） Measurement of adiponectin level in the supernatant of the recombinant yeasts medium was performed by using a rat anti-human adiponectin ELISA kit.3. The effect of age on the expression of New Zealand rabbit adiponectin gene About 100mg subcutaneous adipose tissue was obtained for extraction of totaladipocytic RNA from each rabbit of 2-week old, 1-month old, 3-month old, 6-month old and 12-month old. Adipose tissue mRNA transcription levels were measured by a real-time PCR procedure and the plasma adiponectin concentrations were determined by using a rat anti-human ELISA kit.4. The effect of age on the expression of New Zealand rabbit adiponectin gene Twelve New Zealand rabbits of 3-month old were fed on diet containing 1%cholesterol. The subcutaneous adipose tissues and bood samples were harvested on different timepoint （before high-cholesterol diet, 1-month, 2-month, 3-month, and 4-month after high-cholesterol diet）. As above, real-time PCR and ELISA assay were carried out.5. Statistical analysesData are present as mean±SEM and were analysed by using ANOVA procedure. A value of P<0.05 was considered as significant. Results1. Cloning of rabbit adiponectinTo investigate whether rabbit adiponectin gene might have the same function as that in humans and mice, we used RT-PCR and adipocytes RNA to clone rabbit adiponectin with translation start code （atg） and stop code （tga） （GenBank DQ334867）. The open reading frame for rabbit adiponectin is 735 bp long, and when translated, yields a protein of 244 amino acids. Rabbit adiponectin is highly homologous to that of humans （86.4%; GenBank NM004797） and mice （81.5%; GenBank NM009605）, with 85.7% and 85.3% identity at the amino acid level, respectively.2. Expression of rabbit adiponectin in vitroWestern blotting with the anti-His tag monoclonal antibody or adiponectin polyclonal antibody indicated a single predominant band at approximately 42 kDa （containing 9.3 kDa α-factor signal peptide and 2.5 kDa C-terminal His-tag）. Aiponectin level in the supernatant of cultured Pichia pastoris was 0.29±0.02 μg/ml.3. Effect of age on the expression of rabbit adiponectinThe results of real-time PCR showed that adiponectin mRNA expression of aidpose tissue in different age rabbits was significantly different （P<0.01）. It was highest in 1-month old group and a decending trend relevant to age could be seen. The adiponectin plasma levels exhibited an alike result as the mRNA expression.4. Effect of high-cholesterol diet on rabbit adiponectin expressionThe high-cholesterol diet played a significant role on the expression of rabbit adiponectin gene. Adiponectin mRNA transcription was positively correlated with the serum high cholesterol concentrations （Pearson Correlation=0.469,P=0.001） and no such relationship was seen in the control rabbits fed on normal diet. The mRNA transcription levels were significantly different among different timepoint no matter in the high-cholesterol diet rabbits or in the control （Repeated Measurements Analysis of Variance, P<0.01）. No significant difference was observed in adiponectin gene expression before diet intervention between the two groups （P>0.05）. The mRNA transcription of adipose tissue adiponectin on fixed timepoint were significantly different between high-cholesterol diet rabbits and the control （P<0.01）. The adiponectin plasma levels exhibited an alike result as the mRNA expression. Conclutions1 The rabbit adiponectin cDNA sequence shared a high homology with those of human beings and mouse.2 Rabbit adiponectin gene expression among different age rabbits was significantly different.3 The high-cholesterol diet played a significant role on the expression of rabbit adiponectin gene. Adiponectin mRNA transcription was positively correlated with the serum high cholesterol concentrations. BackgroundsAdiponectin has been found to play an important role in preventing atherosclerosis in which it reduces the size of atherosclerotic lesions, inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, and suppresses expression of vascular adhesion molecules.Through inhibiting nuclear factor KB, adiponectin inhibits the expression of adhesion molecules induced by TNF a and inhibits mitogen activated protein pathway. And then adiponectin plays a role of repressing smooth muscle cells induced by growth factors. Adiponectin suppresses the formation of foam cells by inhibiting the expression of scavenger receptor in macrophagus. Therefore, adiponectin assembles in injured arteries and plays a protective role of anti-atherosclerosis.Although there are a lot of researches on the relationship of adiponectin to insulin resistance and atherosclerosis, many problems need to elucidate such as:1. Adiponectin inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, but how does it produce this effect?2. Is there a pathway that adiponectin prevents atherosclerosis by adventitia?3. What in vivo morphological changes of blood vessels could be found after local transfer of adiponectin-producing adenovirus?All above questions are main design route and objective of our present study. Research Objectives1. To investigate the role and mechanism of adiponectin in preventing atherosclerosis. 2. To investigate the effect and mechanism of adiponectin in preventing atherosclerosis via adventitia. Methods1. Construction of atherosclerotic rabbit modelsWe created the atherosclerotic model as described. Briefly, a balloon catheter was inserted into the abdominal aorta of rabbits （n=70） through the right femoral artery, and the aorta was injured by engorged balloons. All rabbits were fed on 1% high-cholesterol diet.2. Local transfer of adenovirus and intravascular ultrasonography analysesAfter received 3 months of high-cholesterol diet, all rabbits were performed intravascular ultrasonography （IVUS） analyses and then were randomly divided into the following 4 groups: 1) Ad-APN transfer via intima, 2) Ad-βgal （adenovirus expressing β galactosidase gene） transfer via intima, 3) Ad-APN transfer via adventitia and 4) Ad-βgal transfer via adventitia. Ad-APN or Ad-βgal was transferred to abdominal aortic intima or adventitia.3. ELISA assayAiponectin levels in rabbit serum and in the supernatant of cultured Pichia pastoris were quantitatively determined via immunoassays. A kit consisting of rat anti-human adiponectin enzyme was applied and the measurements were performed following the manufacturer’s instructions.4. Histochemical analysesFor the histochemical analyses, the 3 sections （100μm apart） from each rabbit on the 14th day after adenovirus transfer were stained with hematoxylin and eosin, Oil Red O （ORO）, and picrosirius red. The lesion size was quantified with use of Image-Pro Plus 5.0 software. For immunohistochemical analyses, paraffin-imbedded cross-sections （5 μm thick） were incubated with either rabbit adiponectin polyclonal antibody, mouse vascular cell adhesion molecule-1 （VCAM-1） monoclonal antibody, mouse intercellular adhesion molecule-1 （ICAM-1） monoclonal antibody,a-actin monoclonal antibody, RAM 11 monoclonal antibody, mouse collagen I monoclonal antibody, or mouse collagen III monoclonal antibody. 5. Real-time PCR analysesVCAM-1, ICAM-1 and type I and III collagen cDNA was cloned with total RNA of abdominal aortas by RT-PCR. The relative quantitation of target gene expression was determined by use of LightCycler following the manufacturer’s protocol. To quantify the expression of VCAM-1 and ICAM-1, real-time PCR involved use of the SYBR Green kit according to the manufacturer’s instructions. For quantification of type I and III collagen level, TaqMan hydrolysis probes were utilized.6. Statistical analysesData were analyzed using an ANOVA procedure. A value of P<0.05 was considered statistically significant. Data are presented as mean±SEM. SPSS for Windows Version 13.0 was used for statistical analysis. Results1. Atherosclerosis was reduced by regional transfer of Ad-APNSerum samples were collected from rabbits before aorta injury, before and after adenovirus transfer, and subjected to ELESA assay. Serum adiponectin concentrations in rabbits after Ad-APN intima and adventitia transfer increased about 9 times as much as those before transfer （intima: 9.17±0.61 versus 1.02±0.06 μg/ml, P<0.01; adventitia: 9.27±0.23 versus 1.02±0.06 μg/ml, P<0.01）. After transfer, the serum adiponectin concentrations elevated to a level about 10 times higher than the level of endogenous adiponectin in Ad-βgal treated rabbits （intima: 9.17±0.61 versus 0.92±0.05 μg/ml, P<0.01; adventitia: 9.27±0.23 versus 0.90±0.05 μg/ml, P<0.01）, and about 4 times higher than those in aorta non-injured rabbits on normal cholesterol diet （2.45±0.17μg/ml, P<0.01）.Ultrasonography revealed the atherosclerotic plaque area in abdominal aortas of rabbits infected via intima with Ad-APN was significantly reduced, by 35.2% compared with the area before treatment （3.98±0.32 versus 2.58±0.12 mm², P<0.01）, and by 35.8% compared with that in Ad-βgal-treated rabbits （4.02±0.31 versus 2.58±0.12 mm², P<0.01）. The lumen area stenosis （LAS） was also reduced, by 32.7% （35.07±2.43 versus 23.60±1.97%, P<0.01） and 24.1% （31.09±2.03 versus 23.60±1.97%, P<0.01）, respectively. In rabbits infected via adventitia, Ad-APN treatment reduced plaque area by 28.9% as compared with the area before treatment （4.08±0.41 versus 2.90±0.23 mm², P<0.01） and 25.6% compared with that in Ad-βgal-treated rabbits （3.90±0.16 versus 2.90±0.23 mm², P<0.01）. Likewise, the LAS was reduced, by 23.6% （34.19±2.12 versus 26.13±1.88%, P<0.01） and 19.4% （32.43±1.54 versus 26.13±1.88%, P<0.05）, respectively.In rabbits with Ad-APN infection via intima, the atherosclerotic plaque area as seen on ORO staining was reduced significantly, by 35.8% （2.57±0.30 versus 1.65±0.15 mm², P<0.01） and the maximal thickness of plaque was reduced, by 26.7% （0.75±0.04 versus 0.55±0.03 mm, P<0.01） as compared with before treatment. In rabbits infected via adventitia, the plaque area and maximal thickness of plaque was reduced by a similar, significant level.2. Adiponectin inhibits mRNA expression of VCAM-1 and ICAM-1 Immunohistochemical analyses revealed that adenovirus-derived rabbit adiponectinabundantly adhered to cells in the atherosclerotic plaque of rabbits via intima transfer. In rabbits treated via adventitia, adiponectin adhered to cells among adventitia.Compared with Ad-βgal treatment, infection with Ad-APN via intima showed significantly suppressed mRNA expression of VCAM-1, by 18.5% （20.70±1.29 versus 16.87±0.88%, P<0.05） and expression of ICAM-1 by 40.7% （2.70±0.68 versus 1.60±0.19%, P<0.01）. Rabbits infected via adventitia showed significantly suppressed mRNA expression of VCAM-1 by 26.9% （21.70±0.79 versus 15.87±0.58%, P<0.01） and expression of ICAM-1 by 30.7% （2.70±0.21 versus 1.87±0.19%, P<0.01） compared with Ad-βgal treated rabbits. However, rabbits infected via intima or adventitia showed no significant difference in atherosclerotic plaque area, LAS, plaque maximal thickness or mRNA level of VCAM-1 and ICAM-1 in the abdominal aorta.3. Local transfer of Ad-APN had no effect on expression of collagen Ad-APN-treated and Ad-βgal-treated rabbits did not differ in expression of type Iand III collagen, regardless of infection via intima or adventitia.Compared with Ad-βgal treatment, infection with Ad-APN via intima showed no significant difference in mRNA expression of type I collagen （1.82±0.26 versus 1.55±0.17%, P>0.05） and type III collagen （26.96±5.12 versus 25.15±3.67%, P>0.05）. As well, compared with Ad-βgal treatment, infection with Ad-APN via adventitia showed no significant difference in mRNA expression of type I collagen （2.51± 1.07 versus 1.86±0.28%, P>0.05） and type III collagen （19.98±3.20 versus 23.07±4.07%, P>0.05）.4. Effect of adiponectin on the vulnerobility index of atherosclerotic plaqueCompared with Ad-βgal treatment, the area of α-actin in abdominal aortas of rabbits infected via intima with Ad-APN was significantly incresed by 28.88 （P<0.01）, that of Oil Red staining reduced by 24.48% （P<0.01）, and RAM 11 by 28.29% （P<0.01）. So the vulnerability index of plaque was reduced by 33.78% （P<0.01）.Compared with Ad-βgal treatment, the area of α-actin in abdominal aortas of rabbits infected via intima with Ad-APN was significantly incresed by 23.88 （P<0.01）, that of Oil Red staining reduced by 25.54% （P<0.01）, and RAM 11 by 32.119% （P<0.01）. So the vulnerability index of plaque was reduced by 27.03% （P<0.01）. Conclusions1 After adenovirus transfer, the serum adiponectin concentrations of Ad-APN-treated rabbits notably increased and the atherosclerotic area was significantly reduced by inhibiting the expression of adhesion molecules （VCAM-1 and ICAM-1） in the vascular walls.2 Local transfer of Ad-APN had no effect on the expression of type I and III collagen in the vascular walls.3 Local transfer of Ad-APN reduced the vulnerability of plaque by increasing the amount of smooth muscle cells and decreasing the amount of lipids and macrophage cells in vascular walls.更多还原