泛素蛋白连接酶1在大鼠颈动脉球囊损伤后新生内膜中的表达及意义

doi:10.3969/j.issn.1674-5817.2016.03.001

实验动物与比较医学 ›› 2016, Vol. 36 ›› Issue (3): 161-167.DOI: 10.3969/j.issn.1674-5817.2016.03.001

• 论著 • 下一篇

泛素蛋白连接酶1在大鼠颈动脉球囊损伤后新生内膜中的表达及意义

杨波¹, 林新铎¹, 王星人², 唐俊明², 杨建业², 张蕾², 曹腾¹, 姜峰波³, 王家宁²

1.武汉大学人民医院心血管内科, 武汉430060;
2.湖北医药学院附属人民医院临床医学研究所, 十堰442000;
3.湖北医药学院附属人民医院神经内科, 十堰442000

收稿日期:2016-03-01 出版日期:2016-06-25 发布日期:2016-06-25
作者简介:杨波(1963-), 男, 医学博士, 主任医师, 教授, 硕士生、博士生导师, 主要研究方向: 抑郁症与心律失常。E-mail: yybb112@whu.edu.cn
基金资助:
国家自然科学基金资助项目(81270221)

Expression and Role of KIP1 Ubiquitylation-promoting Complex 1 in Neointimal after Carotid Arteries Balloon Injury in Rats

YANG Bo¹, LIN Xin-duo¹, WANG Xing-ren², TANG Jun-ming², YANG Jian-ye², ZHANG Lei², CAO Teng¹, JIANG Feng-bo³, WANG Jia-ning²

1. Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, China;
2. Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan 442000, China;
3. Department of Neurology, Renmin Hospital, Hubei University of Medicine, Shiyan 442000, China

Received:2016-03-01 Online:2016-06-25 Published:2016-06-25

摘要/Abstract

摘要： 目的研究胞质泛素蛋白连接酶1(KPC1）在大鼠颈动脉球囊损伤后的蛋白表达情况,探讨其在新生内膜形成过程中的生物学功能。方法将成年雄性SD大鼠(450~500 g)随机分为假手术组和颈动脉球囊损伤模型组,建模后以左侧损伤动脉为实验组,同时以假手术组的左侧正常颈动脉为对照组。实验组大鼠于损伤后1 d、3 d、7 d、14 d、28 d分别处死并取材,采用 Western blot 检测KPC1蛋白、内膜增殖细胞核抗原(PCNA)表达情况,以qRT-PCR检测KPC1的mRNA表达,苏木精-伊红(HE)染色后比较内膜增生程度; 采用体外培养血管平滑肌细胞(VSMC)建立增殖模型,运用Western blot及qRT-PCR检测其KPC1蛋白、KPC1 mRNA表达特征。结果 Western blot 结果显示,颈动脉球囊损伤后实验组KPC1蛋白表达水平明显低于正常对照组(P<0.05),而PCNA蛋白表达量明显高于正常对照组(P<0.05); qRT-PCR检测结果显示,实验组KPC1的mRNA水平明显低于对照组(P<0.05); HE染色结果示颈动脉球囊损伤后1 d、3 d时光镜下血管内膜增生不明显; 7 d时光镜下可见新生内膜,增生的VSMC向内膜下迁移,管腔开始狭窄; 14 d时,可见明显新生内膜,VSMC排列紊乱,内弹力膜断裂,管腔明显狭窄,内膜厚度超过中膜; 28 d时,血管内膜继续不规则增生,血管腔狭窄程度继续加重,狭窄超过50%。体外培养并模拟VSMC增殖,提取细胞进行 Western blot和qRT-PCR检测显示,KPC1蛋白、KPC1 mRNA表达在血小板源性生长因子-BB刺激增殖后开始逐渐减低。结论抑制KPC1基因表达可促进损伤内膜增生,其作用机制可能与促进VSMC增殖有关。

关键词: 胞质泛素蛋白连接酶1 (KPC1), 新生内膜, 血管平滑肌细胞(VSMC), 增生

Abstract: Objective To study the expression of Kip1 ubiquitylation-promoting complex 1(KPC1) protein after carotid arteries balloon injury in SD rats and to explore its biological function in neointimal formation process. Methods The adult male SD rats (450-500 g) were randomly divided into sham group and carotid artery injury model group. The left damaged carotid arteries were considered as the experimental group after modelling, with the left normal carotid arteries of sham group being as control group. After 1, 3, 7 ,14 and 28 days of balloon injury, SD rats of the experimental group were sacrificed and harvested for histomorphology and molecular markers assay. Hematoxylin-eosin (HE)staining was used to assess the changes of neointimal formation in the carotid artery while Western blot and qRT-PCR were used to evaluate the expression of KPC1. What’s more, Western blot was also used to assess the levels of proliferating cell nuclear antigen (PCNA). The proliferation model was established by culturing vascular smooth muscle cells (VSMCs) in vitro, then detected the KPC1 protein, KPC1 mRNA expression by Western blot and qRT-PCR. Results Western blot and qRT-PCR showed that the KPC1 expression levels of the experimental group were significantly lower than that of control group (P<0.05), while the PCNA expression was significantly higher than that of control group (P<0.05). Compared to 1 day and 3 days after carotid arteries injury, for VSMC hyperplasia, neointimal generation and luminal stenosis were observed at 7 days by HE staining. With VSMCs hyperplasia, neointimal and luminal stenosis were getting more and more serious, the thickness of intimal exceeded medial at 14 days. At 28 days, the irregular intimal hyperplasia was continued and the degree of vascular stenosis was more than 50%. After stimulation of VSMCs proliferation by platelet-derived growth factor BB (PDGF-BB) in vitro, then Western blot and qRT-PCR data showed a gradual decreasing trend of KPC1 at protein and mRNA levels. Conclusion Inhibiting KPC1 expression can significantly decrease intima hyperplasia in this model, and its mechanism may be related to the promotion of VSMCs proliferation.

Key words: Kip1 ubiquitylation-promoting complex 1(KPC1), Neointima, Vascular smooth muscle cells (VSMCs), Hyperplasia

中图分类号:

Q95-33

杨波, 林新铎, 王星人, 唐俊明, 杨建业, 张蕾, 曹腾, 姜峰波, 王家宁. 泛素蛋白连接酶1在大鼠颈动脉球囊损伤后新生内膜中的表达及意义[J]. 实验动物与比较医学, 2016, 36(3): 161-167.

YANG Bo, LIN Xin-duo, WANG Xing-ren, TANG Jun-ming, YANG Jian-ye, ZHANG Lei, CAO Teng, JIANG Feng-bo, WANG Jia-ning. Expression and Role of KIP1 Ubiquitylation-promoting Complex 1 in Neointimal after Carotid Arteries Balloon Injury in Rats[J]. Laboratory Animal and Comparative Medicine, 2016, 36(3): 161-167.

参考文献

[1] Libby P, Braunwald E.Braunwald’s heart disease : a textbook of cardiovascular medicine[M]. Philadelphia:Saunders/Elsevier, 2008.
[2] Joner M, Finn AV, Farb A, et al.Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk[J]. J Am Coll Cardiol, 2006, 48(1):193-202.
[3] Chatterjee S, Pandey A.Drug eluting stents: friend or foe? A review of cellular mechanisms behind the effects of Paclitaxel and sirolimus eluting stents[J]. Curr Drug Metab, 2008, 9(6):554-566.
[4] Polimeni A, De Rosa S, Indolfi C.Vascular miRNAs after balloon angioplasty[J]. Trends Cardiovasc Med, 2013, 23:9-14.
[5] Curcio A, Torella D, Indolfi C.Mechanisms of smooth muscle cell proliferation and endothelial regeneration after vascular injury and stenting: approach to therapy[J]. Circ J, 2011, 75(6):1287-1296.
[6] Lowe HC, Oesterle SN, Khachigian LM.Coronary in-stent restenosis: current status and future strategies[J]. J Am Coll Cardiol, 2002, 39(2):183-193.
[7] Kamura T, Hara T, Matsumoto M, et al.Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase[J]. Nat Cell Biol, 2004, 6(12):1229-1235.
[8] Kravtsova-Ivantsiv Y, Shomer I, Cohen-Kaplan V, et al.KPC1-mediated ubiquitination and proteasomal processing of NF-kappaB1 p105 to p50 restricts tumor growth[J]. Cell,2015, 161(2):333-347.
[9] Wang JN, Shi N, Xie WB, et al.Response gene to complement 32 promotes vascular lesion formation through stimulation of smooth muscle cell proliferation and migration[J]. Arterioscler Thromb Vasc Biol, 2011, 31(8):e19-e26.
[10] 杨波, 吴冰, 唐俊明, 等. 大鼠颈总动脉球囊拉伤所致血管狭窄模型的建立[J]. 实验动物与比较医学, 2015, 35(2):87-91.
[11] Gruntzig A.Transluminal dilatation of coronary-artery stenosis[J]. Lancet, 1978, 1(8058):263.
[12] Muzina A, Wei L.Use of intravascular ultrasound vs. optical coherence tomography for mechanism and patterns of in-stent restenosis among bare metal stents and drug eluting stents[J]. J Thorac Dis, 2016, 8(1):E104-E108.
[13] Forrester JS, Fishbein M, Helfant R, et al.A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies[J]. J Am Coll Cardiol, 1991, 17(3):758-769.
[14] Costa MA, Simon DI.Molecular basis of restenosis and drug-eluting stents[J]. Circulation, 2005, 111(17):2257-2273.
[15] Wang JN, Shi N, Chen SY.Manganese superoxide dismutase inhibits neointima formation through attenuation of migration and proliferation of vascular smooth muscle cells[J]. Free Radic Biol Med, 2012, 52(1):173-181.
[16] Teyssier J R, Rey R, Ragot S, et al.Correlative gene expression pattern linking RNF123 to cellular stress-senescence genes in patients with depressive disorder: implication of DRD1 in the cerebral cortex[J]. J Affect Disord, 2013, 151(2):432-438.
[17] Lu C, Huang X, Zhang X, et al.miR-221 and miR-155 regulate human dendritic cell development, apoptosis, and IL-12 production through targeting of p27kip1, KPC1, and SOCS-1[J]. Blood, 2011, 117(16):4293-4303.
[18] Hristova NR, Tagscherer KE, Fassl A, et al.Notch1-dependent regulation of p27 determines cell fate in colorectal cancer[J]. Int J Oncol, 2013, 43(6):1967-1975.
[19] 景彩萍. NF-κB抑制剂对乳腺癌细胞增殖作用的影响及P65、MMP-9蛋白表达研究[D]. 延安: 延安大学, 2015.
[20] Siggers T, Chang AB, Teixeira A, et al.Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-kappaB family DNA binding[J]. Nat Immunol, 2012, 13(1):95-102.
[21] 杨春妮,杨一兵. NF-κB及其信号传导通路与肿瘤放射耐受的研究进展[J]. 中国现代医药杂志, 2015(12):106-109.

泛素蛋白连接酶1在大鼠颈动脉球囊损伤后新生内膜中的表达及意义

Expression and Role of KIP1 Ubiquitylation-promoting Complex 1 in Neointimal after Carotid Arteries Balloon Injury in Rats

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