ADRP、PKC在动脉硬化斑块的表达及与稳定性关系的研究
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摘要
研究目的:通过比较人离体腘动脉粥样硬化斑块中稳定斑块与不稳定斑块内脂肪分化相关蛋白(ADRP)、蛋白激酶C(PKC)、CD36的表达情况的差异,探讨这些因子与斑块稳定性的关系及在不稳定斑块形成中的作用机制。研究方法:取动脉硬化闭塞症行手术治疗患者的腘动脉18份为动脉硬化组,于斑块中心处把标本纵向剖开截为两份,一份用10%甲醛溶液固定,行HE染色及免疫组化分析;另一份冻存于-80℃冰箱中用于PCR方法检测。取无动脉硬化病史及危险因素行肠道手术患者肠系膜动脉10例为正常对照组,标本同样截为两份分别行甲醛固定及-80℃冰箱冻存。甲醛固定标本先行HE染色及免疫组化分析。根据HE染色结果把动脉硬化组分为稳定斑块组及不稳定斑块组两个亚组。应用免疫组化方法检测ADRP、PKC及CD36在正常对照组、稳定斑块组、不稳定斑块组中的表达,应用统计学方法检验各组中这三种物质表达量是否不同。再于冻存标本中每组随机抽取相同数量样本按正常对照组、稳定斑块组、不稳定斑块组分别行RT-PCR检测ADRPmRNA表达,并通过计算机图像分析系统进行分析,应用统计学方法检验各组标本中ADRPmRNA表达量是否不同。研究结果:1.HE病理染色显示:正常对照组血管壁内膜光滑,管腔侧内皮细胞排列整齐,核蓝染,中膜平滑肌细胞核呈梭形,末见增生。实验组血管壁破坏严重,均有典型的粥样斑块形成,内膜增厚、纤维化,血管内膜向管腔突出,斑块内可见大量泡沫细胞浸润及脂质沉积,内膜可见炎性细胞和增生的平滑肌细胞,中膜平滑肌细胞明显增生、紊乱。不稳定斑块可见大的脂质核心、炎细胞浸润及胆固醇结晶。2.免疫组化半定量分析显示: ADRP、PKC及CD36在动脉硬化组斑块中大量表达,不稳定斑块中ADRP、PKC、CD36表达量略高于稳定斑块。3.RT -PCR检测结果:ADRP在动脉硬化斑块中表达增加,不稳定斑块中的ADRP表达高于稳定斑块。结论:1.动脉粥样硬化斑块中的ADRP、PKC及CD36表达上调。2.不稳定性斑块中ADRP、PKC及CD36表达量高于稳定斑块。3、ADRP、PKC及CD36在斑块中高表达与斑块不稳定性相关。
For many years, modification and accumulation of lipid in arterial endothelium has been considered the beginning of atherosclerosis. Recent studies have found that adipose differentiation-related protein (ADRP, adipophilin) is an important role in the pathogenesis of atherosclerotic disease. ADRP is a lipid droplets around the main protein, which distributed a large number of lipid droplets in the presence of cells. It is a specific marker of lipid accumulation.In early differentiation in the fat cells, it has high expression, but in mature fat cells its expression was decreased. ADRP involved in adipocyte lipid metabolism, lipid droplets and liver triglyceride (TG) synthesis and metabolism. Also promote macrophage, smooth muscle cell foam, and promote the long-chain fatty acid intake, milk secretion. ADRP expression was associated with the pathological processes of atherosclerosis, insulin resistance and tumor closely. Studies have confirmed that ADRP expression in animal models of atherosclerosis increased, but the mechanism in the formation of atherosclerosis is not fully understood. Recent years` study found, individe the plaque into stable plaque and unstable plaque based on anatomical characteristics atherosclerotic is more reasonable. Because of its tendency to thrombosis and rapid development of acute vascular events can lead to "criminal patch", how to effectively identify unstable plaques and inhibit plaque formation and development becomes more important. At present, the research of ADRP expression in unstable plaques is relatively less. This experiment has done research on this, and to explore the expression of protein kinase C in unstable atherosclerotic plaques, which may participate the pathogenesis of atherosclerosis.
This study based on deriving atheroscleros sample from lower limb artery. Limited in clinical conditions the control group failed to obtain lower limb artery, take the mesenteric artery as control group, they are all medium-sized artery. By HE staining the plaque lesion group are divided into two subgroups :stable and unstable plaque group. Observe the ADRP expression in atherosclerotic plaques by immunohistochemistry and reverse transcription polymerase chain reaction, and observe the expression of both PKC and CD36 in atherosclerotic plaques with different stability by immunohistochemistry experiments for they may have association with the Regulation of ADRP`s expression.
Objective: To evaluate the expressions of adipophilin, PKC and CD36 in atherosclerotic lesion plaque, and to further explore the relationship between plaque stability and their expression.
Methods: Take the popliteal artery in 18 patients of arteriosclerosis obliterans who take treatment of amputation or prosthetic vessel replacement as the experimental group, Individe the samples at prominent plaque place into two part, one fixed with 10% formaldehyde solution for HE staining and immunohistochemical analysis, the other freese in -80℃refrigerator for PCR. Take mesenteric artery from pations ofgastrectomy and without risk factors of atherosclerosis as normal control group, samples were also divided into two parts for formaldehyde fixed and frozen in -80℃refrigerator. Take all of formaldehyde fixed paraffin embedded samples, sections, first HE staining. Select the HE stained specimens with clear structure for the study. Then divided the tissues into stable plaque and unstable plaque group in accordance with the reference criteria Currently widely used for the classification of plaque instability(lipid core plaque area of more than 40%). Frozen samples of atherosclerosis group were divided into a stable plate group and an unstable plaque group according to the corresponding mark. Specimens of each group take ADRP、PKC and CD36 immunohistochemistry respectively. Select five clear stain specimens from stable plaque group and unstable plaque group respectively ,observe each specimen under a 200 optical microscope of consecutive detection of 4-5 fields of vision. Images collected under the same conditions and analyzed the relative average gray of the observation site. Frozen samples of atherosclerosis group were divided into a stable plate group and an unstable plaque group according to the corresponding mark. Randomly selected six samples from unstable plaque group and control group respectively.Take all the samples in the stablegroup, determination for PT-PCR detection of ADRP expression. Analyze the PCR products by agarose gel electrophoresis, and analyze the optical density of electrophoresis strip by gel electrophoresis image ,determine their content with the ratio of the target gene and internal reference GAPDH . The experimental data were expressed with mean±standard deviation ( x±S) ,comparisons between groups were analyzed using analysis of variance and t test, completed by the software SPSS11.0, determined significant difference by P<0.05.
Results: 1.HE pathological staining: intima of vascular wall is smooth in normal control group, luminal side of endothelial cells stained blue, arranged in neat rows ,medial smooth muscle cells were spindle, no hyperplasia. Experimental group are of severely damaged vessel wall and typical of formation plaque, intimal thickening, fibrosis, vascular intimal the lumen prominent, visible plaque lipid deposition and foam cells can be seen. There are large number of inflammatory cells and intimal proliferation of smooth muscle cells in endomembrane, endocardium and calcium deposition in necrotic material, significant proliferation of medial smooth muscle cells and calcification were spotty or flake.2. Semi-quantitative immunohistochemical analysis showed that: ADRP、PKC and CD36 expressed high in the experimental group , they are slightly higher in unstable plaques than in stable plaques. 3.RT-PCR test results: ADRP expression increase in atherosclerotic plaques, it is higher in unstable plaques than in stable plaques.
Conclusion: 1. ADRP、PKC and CD36 in the atherosclerotic plaque are upregulationed; 2.the expression of ADRP and PKC in unstable plaques is higher than in stable plaques ;3.The high expression of ADRP、PKC and CD36 in plaques may be associated with instability of plaque.
For many years, modification and accumulation of lipid in arterial endothelium has been considered the beginning of atherosclerosis. Recent studies have found that adipose differentiation-related protein (ADRP, adipophilin) is an important role in the pathogenesis of atherosclerotic disease. ADRP is a lipid droplets around the main protein, which distributed a large number of lipid droplets in the presence of cells. It is a specific marker of lipid accumulation.In early differentiation in the fat cells, it has high expression, but in mature fat cells its expression was decreased. ADRP involved in adipocyte lipid metabolism, lipid droplets and liver triglyceride (TG) synthesis and metabolism. Also promote macrophage, smooth muscle cell foam, and promote the long-chain fatty acid intake, milk secretion. ADRP expression was associated with the pathological processes of atherosclerosis, insulin resistance and tumor closely. Studies have confirmed that ADRP expression in animal models of atherosclerosis increased, but the mechanism in the formation of atherosclerosis is not fully understood. Recent years` study found, individe the plaque into stable plaque and unstable plaque based on anatomical characteristics atherosclerotic is more reasonable. Because of its tendency to thrombosis and rapid development of acute vascular events can lead to "criminal patch", how to effectively identify unstable plaques and inhibit plaque formation and development becomes more important. At present, the research of ADRP expression in unstable plaques is relatively less. This experiment has done research on this, and to explore the expression of protein kinase C in unstable atherosclerotic plaques, which may participate the pathogenesis of atherosclerosis.
This study based on deriving atheroscleros sample from lower limb artery. Limited in clinical conditions the control group failed to obtain lower limb artery, take the mesenteric artery as control group, they are all medium-sized artery. By HE staining the plaque lesion group are divided into two subgroups :stable and unstable plaque group. Observe the ADRP expression in atherosclerotic plaques by immunohistochemistry and reverse transcription polymerase chain reaction, and observe the expression of both PKC and CD36 in atherosclerotic plaques with different stability by immunohistochemistry experiments for they may have association with the Regulation of ADRP`s expression.
Objective: To evaluate the expressions of adipophilin, PKC and CD36 in atherosclerotic lesion plaque, and to further explore the relationship between plaque stability and their expression.
Methods: Take the popliteal artery in 18 patients of arteriosclerosis obliterans who take treatment of amputation or prosthetic vessel replacement as the experimental group, Individe the samples at prominent plaque place into two part, one fixed with 10% formaldehyde solution for HE staining and immunohistochemical analysis, the other freese in -80℃refrigerator for PCR. Take mesenteric artery from pations ofgastrectomy and without risk factors of atherosclerosis as normal control group, samples were also divided into two parts for formaldehyde fixed and frozen in -80℃refrigerator. Take all of formaldehyde fixed paraffin embedded samples, sections, first HE staining. Select the HE stained specimens with clear structure for the study. Then divided the tissues into stable plaque and unstable plaque group in accordance with the reference criteria Currently widely used for the classification of plaque instability(lipid core plaque area of more than 40%). Frozen samples of atherosclerosis group were divided into a stable plate group and an unstable plaque group according to the corresponding mark. Specimens of each group take ADRP、PKC and CD36 immunohistochemistry respectively. Select five clear stain specimens from stable plaque group and unstable plaque group respectively ,observe each specimen under a 200 optical microscope of consecutive detection of 4-5 fields of vision. Images collected under the same conditions and analyzed the relative average gray of the observation site. Frozen samples of atherosclerosis group were divided into a stable plate group and an unstable plaque group according to the corresponding mark. Randomly selected six samples from unstable plaque group and control group respectively.Take all the samples in the stablegroup, determination for PT-PCR detection of ADRP expression. Analyze the PCR products by agarose gel electrophoresis, and analyze the optical density of electrophoresis strip by gel electrophoresis image ,determine their content with the ratio of the target gene and internal reference GAPDH . The experimental data were expressed with mean±standard deviation ( x±S) ,comparisons between groups were analyzed using analysis of variance and t test, completed by the software SPSS11.0, determined significant difference by P<0.05.
Results: 1.HE pathological staining: intima of vascular wall is smooth in normal control group, luminal side of endothelial cells stained blue, arranged in neat rows ,medial smooth muscle cells were spindle, no hyperplasia. Experimental group are of severely damaged vessel wall and typical of formation plaque, intimal thickening, fibrosis, vascular intimal the lumen prominent, visible plaque lipid deposition and foam cells can be seen. There are large number of inflammatory cells and intimal proliferation of smooth muscle cells in endomembrane, endocardium and calcium deposition in necrotic material, significant proliferation of medial smooth muscle cells and calcification were spotty or flake.2. Semi-quantitative immunohistochemical analysis showed that: ADRP、PKC and CD36 expressed high in the experimental group , they are slightly higher in unstable plaques than in stable plaques. 3.RT-PCR test results: ADRP expression increase in atherosclerotic plaques, it is higher in unstable plaques than in stable plaques.
Conclusion: 1. ADRP、PKC and CD36 in the atherosclerotic plaque are upregulationed; 2.the expression of ADRP and PKC in unstable plaques is higher than in stable plaques ;3.The high expression of ADRP、PKC and CD36 in plaques may be associated with instability of plaque.
引文
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[2]史旭波,胡大一.动脉粥样硬化发病机制的新认识. [J]临床荟萃, 2006,21(24):1751-1753.
[3] Liu PS, Ying YS, Zhao YM, et al Chinese hamsterovary K2 cell lipid droplets appear to metabolic organelles involved in membrane traffic[J]. Biol Chem, 2004,279(5):3787-3792.
[4] Kumi TS, Shintaro O, Toshiaki H, et al. The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition. [J]. J BiolChem, 2002,277(46):44507-44512.
[5] Larigauderie G, Fuman C, Jaye M, et al. ADRP enhances lipid accumulation and lipid efflux from macrophages potential role in atherogenesis[J]. Arterioscler Thromb Vasc Biol, 2004,24(3):504- 510.
[6] Targett Adams P, Chambers D, Gledhill S, et al. Live cell analysis and targeting of lipid droplet binding adipocyte differentiation-related protein[J]. Biol Chem, 2003,278(18):15998-16007.
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