全反式维甲酸对兔颈动脉粥样硬化病灶VSMC增殖及AKT1表达影响
作者：张青青 董果雄 张社华
【摘要】 目的 探讨全反式维甲酸(atRA)对兔颈动脉内膜损伤后内膜增生以及蛋白激酶B(AKT1)、增殖细胞核抗原（PCNA）表达的影响和机制。方法 新西兰雄性大白兔36只，随机分为6组：假手术A、B组,手术A、B组,手术并予atRA治疗A、B组（治疗A、B组），每组各6只。各组均给予高脂饮食14 d后，手术组作颈动脉内膜损伤模型。治疗组于术前3 d开始每天给予atRA灌胃，其余操作同手术组。假手术组手术但不损伤内膜。于术后7 d处死A组动物， 28 d处死B组动物。采取颈动脉标本，对血管粥样硬化病变进行大体形态学观察，用免疫组化法检测粥样硬化病灶中AKT1、PCNA的表达水平。结果 手术组术后7 d内膜开始增生， 28 d时增生明显，粥样斑块形成，管腔狭窄，AKT1、 PCNA阳性表达明显。治疗组内膜增生较轻， 28 d时，内膜面积及斑块厚度低于手术组(t=3.797、3.704,P<0.01)，AKT1、PCNA阳性表达指数亦低于手术组(t=4.741、2.890,P<0.05)。结论atRA能抑制兔颈动脉内皮损伤后AKT1、PCNA的表达，抑制平滑肌细胞增殖，从而抑制内膜增生及动脉粥样硬化病变进程。
【关键词】 维甲酸；肌细胞,平滑肌；冠状动脉再狭窄；原癌基因蛋白质c?akt
［ABSTRACT］ Objective To investigate the influence of all?trans retinoic acid (atRA) on intima proliferation and expressions of AKT1 and PCNA in rabbit carotid artery after endomembrane injury. Methods Thirty?six New Zealand rabbits wereevenly randomized to six groups: sham?operation group A and group B; operation group A and group B; and treatment group A and group B. All the rabbits were fed with high fat diet for 14 days. A model of carotid artery endomembrane injury was created in operation group. In treatment groups, rabbits were lavaged with atRA daily for 3 days before operation. Those in sham?operation groups underwent surgery without injuring the endomembrane. Rabbits in groups A were killed 7 days after operation and those in groups B, 28 days. Carotid specimens were taken for gross examination and immunohistochemistry study used for detection of the expressions of AKT1 and PCNA proteins. Results Seven days after operation, neointima formation was found in carotid artery in the operation group, and it became obvious afetr 28 days with atherosclerosis plaque, narrowing of arterial lumen and expressions of AKT1 and PCNA. In the treatment group, there was mild intima proliferation, and, 28 days after surgery, the intima area was smaller and the plague was thinner than that of the operation group (t=3.797, 3.704;P<0.01). The index of positive expressions of AKT1 and PCNA in the treatment group was also lower than that of the operation group (t=4.741, 2.890;P<0.05). Conclusion atRA can suppress the expressions of AKT1 and PCNA in injured endomembrane of rabbit carotid artery, inhibit the proliferation of vascular smooth muscle cells, and therefore inhibit neointima proliferation and progression of atherosclerosis.
［KEY WORDS］ tretinoin; myoblasts, smooth muscle; coronary restenosis; proto?oncogene proteins c?akt
经皮冠状动脉成形术(PTCA)及支架植入术已被广泛用于冠状动脉粥样硬化性心脏病的治疗，然而其术后的再狭窄（RS）问题影响了其远期疗效。再狭窄是受损内膜增生的表现,其主要病理机制则是血管平滑肌细胞（VSMC）的过度增殖。全反式维甲酸(atRA)是维生素A的天然衍生物，近年来研究发现它可抑制VSMC的表型转化、迁移，调节细胞内信号转导通路及细胞周期进程，从而影响新生内膜形成的多个环节[1?2]。蛋白激酶B(AKT1)是细胞内信号转导通路中重要的.信号分子，调控细胞多种基本功能，尤其在细胞增殖和存活方面发挥着关键作用。本文旨在进一步探讨atRA抑制VSMC增生、预防RS发生的机制，为临床选药提供依据。
1 材料和方法
1.1 动物分组及模型制作
新西兰雄性大白兔36只，青岛市动物实验中心提供，4月龄，体质量（2.50±0.12）kg。随机分为6组：治疗A组，治疗B组，手术A组，手术B组，假手术A组，假手术B组。治疗组和手术组每日进食150 g高脂饲料（每100 g饲料含猪油 6.0 g,胆固醇1.5 g,基础饲料92.5 g。），假手术组每日进食150 g基础饲料，均自由饮水。各组动物饲养14 d后在无菌操作下进行手术：手术组行颈动脉内膜空气干燥术，治疗组于术前3 d开始给予atRA(重庆华邦制药公司) 6 mg·kg-1 ·d-1灌胃，其余操作同手术组。假手术组分离颈动脉但不损伤内膜。各组术后每天肌注青霉素40万单位，连续3 d。分别于术后7 d时处死A组动物，28 d时处死B组动物，取病变部位血管等距切分4～5段，40 g/L中性缓冲甲醛固定，石蜡包埋，血管标本横截面切片(3 μm)。
1.2 血管形态学及动脉粥样硬化病变检测
术后取出血管段，先进行大体肉眼观察。蜡块标本切片行苏木精?伊红染色（HE染色），显微镜下观察血管结构，采用图像分析软件Image?Pro Plus(IPP)进行图像分析，测定各个血管截面的内膜面积(IA)、中膜面积(MA)，计算内膜/中膜面积比（I/M)、管腔狭窄度(LSD)。LSD =内膜面积/内弹力板下面积×100％，取平均值。取术后28 d切片，显微镜下观察动脉粥样硬化病变情况，根据AHA对动脉粥样硬化的分期(Ⅰ、Ⅱ、Ⅲ期)计数每期发生动物例数，以最重病变为准，测量动脉粥样硬化病变最厚处内弹力板至腔面垂直距离，作为该动物的最大斑块厚度。
1.3 AKT1和增殖细胞核抗原(PCNA)表达检测
采用免疫组织化学二步法行AKT1（即用型兔抗兔AKT1，武汉博士德公司)免疫组化反应，工作液未做稀释，二抗采用PV?6001试剂盒(北京中杉公司)，DAB显色，苏木精复染，胞质呈棕黄、棕褐色为阳性。以PBS代替一抗作阴性对照。用IPP进行图像分析，随机测量每张切片中6个高倍视野的阳性部位平均吸光度（A）和阳性面积百分比，取其平均值。AKT1阳性表达指数=平均吸光度（A）×平均阳性面积百分比×100。用二步法进行鼠抗兔PCNA（北京中杉公司）染色，一抗稀释度为1∶100，显色步骤同AKT1。采用IPP进行图像分析，随机测量每张切片6个高倍视野的阳性胞核占总胞核比例及阳性胞核平均光吸光度（A），计算PCNA阳性表达指数。PCNA阳性表达指数=平均阳性胞核比×平均吸光度（A）×100。