血管外膜脂肪组织源性脂联素通过磷脂酰肌醇-3-激酶/蛋白激酶B信号通路促进血管内膜损伤后内皮细胞增生的研究

目的 研究血管外膜脂肪组织分泌的脂联素对血管内膜损伤后内皮细胞增生的影响及可能的分子机制.方法 将C57BL/6J野生型小鼠72只完全随机分为A组（仅剪开颈部皮肤后再缝合）、B组（给予颈动脉套管结扎）、C组（剪开颈部皮肤后于颈动脉周围移植脂联素基因敲除小鼠脂肪组织）及D组（颈动脉套管结扎同时于颈动脉周围移植脂联素基因敲除小鼠脂肪组织）,每组18只.手术8周后处死小鼠,取各组小鼠左颈总动脉切片,采用苏木精-伊红染色,观察各组小鼠颈动脉内膜增生情况并测定内膜增厚程度,以内膜面积与中膜面积的比值表示.应用酶联免疫吸附试验检测各组小鼠颈动脉组织内脂联素水平.采用蛋白质印迹法检测各组小鼠颈动脉组织内磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（AKT）及其磷酸化产物的水平.体外实验将小鼠内皮细胞以5＆#215;104个/ml接种于内皮细胞生长培养基中,将细胞分为对照组、脂联素组（在对照组基础上加入重组脂联素100 mg/L）及PI3K抑制组（在对照组基础上加入重组脂联素100 mg/L和PI3K抑制剂LY29400 250 μg/L）,利用标记5-溴脱氧尿嘧啶核苷（BrdU）的微小RNA（miR-21）模拟物转染小鼠内皮细胞,使其在细胞中呈过表达状态,通过荧光显微镜观察各组内皮细胞的增生情况,比较BrdU阳性细胞百分率.结果 B组小鼠颈动脉内膜增厚程度明显大于A组、C组和D组,差异均有统计学意义（1.37±0.09）比（0.82±0.18）、（0.71±0.05）、（1.03±0.06）,P值分别为0.023、0.001、0.010].B组小鼠脂联素的表达水平明显高于A组、C组和D组,差异有统计学意义（95±6） ng/L比（77±5）ng/L、（55±5）ng/L、（70±7）ng/L,P=0.049、0.021、0.027].C组与D组小鼠颈动脉组织内AKT及PI3K磷酸化产物水平较A组与B组降低.体外实验脂联素组内皮细胞的BrdU阳性细胞率较对照组明显增加,差异有统计学意?

Adiponectin from perivascular adipose tissue promotes endothelial cells proliferation after endovascular injury by regulating phosphatidylinositol-3-kinase/protein kinase B signaling pathway

Objective To investigate the effects of adiponectin from perivascular adipose tissue on endothelial cell proliferation in the mouse model of endovascular injury.Methods Seventy two male wild-type （C57BL/6J） mice were randomly divided into group A,group B （undergoing cuff operation on common carotid artery）,group C （undergoing perivascular adipose tissue transplant from adipose tissue of adiponectin gene knockout mice） and group D （undergoing cuff operation as well as perivascular adipose tissue transplantation from adipose tissue of adiponectin gene knock-out mice）.Vascular intimal hyperplasia was compared 8 weeks after operation.Expression levels of adiponectin,phosphorylation of phosphatidylinositol-3-kinase （PI3K） and protein kinase B （AKT） in focal intima tissue of the injury were measured by enzyme-linked immunosorbent assay and western blot.In vitro experiments,endothelial cells were incubated with adiponectin with or without PI3K inhibition to assess the cell proliferation by immunostaining for BrdU.Results Morphometric analysis found that neointimal hyperplasia was significantly enhanced in group B compared with that in group A,group C,group D （1.37 ± 0.09） vs.（0.82 ± 0.18）,（0.71 ± 0.05）,（1.03 ± 0.06）].Adipose tissue transplantation significantly attenuated the intimal hyperplasia in the injury groups （P =0.023,P =0.001,P =0.010）.The expression of adiponectin was significantly increased in vascular tissues in group B compared with that in group A,group C,group D （95 ± 6） ng/L vs.（77 ± 5） ng/L,（55 ± 5） ng/L,（70 ± 7） ng/L,P =0.049,0.021,0.027].After injury,the phosphorylation of PI3K and AKT was remarkably lower in vascular tissue in group D and group C than in group B and group A.After incubation with adiponectin,the endothelial cells showed increasing proliferation compared with that in control group （30.0 ± 1.7） % vs.（10.5 ± 1.4） %,P 〈 0.01] ; but this response was inhibited by PI3K inhibitor（13.9 ± 1.8） % vs.（30.0