趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究 <sup>*</sup>

doi:10.13523/j.cb.20190802

中国生物工程杂志

2019, Vol. 39

Issue (8): 7-16 DOI: 10.13523/j.cb.20190802

研究报告

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究 ^*

朱颖,范梦恬,李具琼,陈彬,张盟浩,吴静红,施琼(

)

重庆医科大学检验医学院临床诊断教育部重点实验室重庆 400016

Effect of Chemokine Receptor CX3CR1 on Osteogenic Differentiation of Human Aortic Valve Interstitial Cells

ZHU Ying,FAN Meng-tian,LI Ju-qiong,CHEN Bin,ZHANG Meng-hao,WU Jing-hong,SHI Qiong(

)

Key Laboratory of Clinical Laboratory Diagnostics, Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

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摘要：

目的: 探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。 方法: 取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。 结果: 临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P<0.05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P<0.05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,siCX3CR1+OM组Runx2、OPN和p-AKT表达下调(P<0.05),且茜素红S染色可见钙结节减少。 结论: 趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。

关键词： 钙化性主动脉瓣膜疾病; 人主动脉瓣膜间质细胞; 趋化因子受体CX3CR1; 成骨分化

Abstract:

Objective: To investigate the role and mechanism of chemokine receptor CX3CR1 in the regulation of osteogenic differentiation of human aortic valve interstitial cells, and to provide new ideas for early intervention and treatment of calcific aortic valve disease.Methods: Three non-calcified aortic valves and five calcified aortic valveswere taken.Immunohistochemical staining was used to detected the expression of osteogenic-related transcription factors Runx2,osteopontin (OPN) andosteocalcin (OCN).Three non-calcified aortic valves were taken to culture human aortic valve interstitial cells were isolated by collagenase digestion.Cell morphology and growth status were observed,and phenotypic identification was performed by immunofluorescence staining.The overexpression plasmid CX3CR1 were added into the osteoblast-induced culture of human aortic valve interstitial cells (CX3CR1+OM),the normal complete medium culture (CM) group,the osteogenic induction medium culture (OM) group, and the negative control plasmid was added into the osteogenic induction medium (negative control+OM) group were arranged in parallel.the expression of Runx2,OPN and OCN was detected by qPCR and Western blot, the expression of AKT and p-AKT was detected by Western blot.Alizarin red S staining was used to evaluate the formation of advanced calcium nodules.The interference plasmid siCX3CR1 were added into the osteoblast-induced culture of human aortic valve interstitial cells(siCX3CR1+OM), the normal complete medium culture (CM) group, the osteogenic induction medium culture (OM) group, andthe negative control siRNA was added into the osteogenic induction medium (negative siRNA control+OM) group were arranged in parallel. The expression of Runx2, OPN and OCNwere detected by qPCR and Western blot,the expression of AKT and p-AKT were detected by Western blot.Alizarin red S staining was used to evaluate the formation of advanced calcium nodules.Results: Clinical specimens showed that calcified valves had higher expression of CX3CR1 than non-calcified valves (P<0.05). Successful isolation of human aortic valve interstitial cells, stromal cell-specific marker protein smooth muscle actin α-SMA and Vimentin positive, endothelial cell specific marker protein vWF is negative. Compared with the CM, OM,and negative control group, CX3CR1+OM group have the expression of Runx2, OPN and p-AKT (P<0.05), and alizarin red S staining shows obvious calcium nodules. Compared with the CM,OM,negative siRNA control+OM group, siCX3CR1+OM shows a down-regulated expression of Runx2,OPN and p-AKT (P<0.05), Alizarin red S staining shows a decrease in calcium nodules.Conclusion: The chemokine receptor CX3CR1 promotes osteogenic differentiation of human aortic valve interstitial cells through the AKT signaling pathway.

Key words: Calcific aortic valve disease Human aortic valve interstitial cells Chemokine receptor CX3CR1 Osteogenic differentiation

收稿日期: 2019-01-27 出版日期: 2019-09-18

ZTFLH:

R44

基金资助: *国家自然科学基金(81672103)

通讯作者: 施琼 E-mail: shiqiong@cqmu.edu.cn

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引用本文:

朱颖,范梦恬,李具琼,陈彬,张盟浩,吴静红,施琼. 趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究 ^*[J]. 中国生物工程杂志, 2019, 39(8): 7-16.

ZHU Ying,FAN Meng-tian,LI Ju-qiong,CHEN Bin,ZHANG Meng-hao,WU Jing-hong,SHI Qiong. Effect of Chemokine Receptor CX3CR1 on Osteogenic Differentiation of Human Aortic Valve Interstitial Cells. China Biotechnology, 2019, 39(8): 7-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190802 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I8/7

表1 qPCR引物序列

图1 临床标本CX3CR1 mRNA检测(a)和免疫组织化学检测CX3CR1、Runx2、OPN(b)

图2 临床标本CX3CR1、α-SMA、Runx2和OPN的表达

图3 人主动脉瓣膜间质细胞生长状态(a)和表型鉴定(b)

图4 过表达CX3CR1对人主动脉瓣膜间质细胞的影响

图5 干扰CX3CR1对人主动脉瓣膜间质细胞的影响

图6 Western blot检测过表达和干扰CX3CR1后AKT和p-AKT的表达水平

[1]	Yutzey K E, Demer L L, Body S C , et al. Calcific aortic valve disease: a consensus summary from the alliance of investigators on calcific aortic valve disease. Arteriosclerosis, Thrombosis, and Vascular Biology, 2014,34(11):2387-2393.
[2]	Miller J D, Weiss R M, Heistad D D . Calcific aortic valve stenosis: methods,models, and mechanisms. Circulation Research, 2011,108(11):1392-1412. doi: 10.1161/CIRCRESAHA.110.234138
[3]	Dweck M R, Boon N A, Newby D E . Calcific aortic stenosis:a disease of the valve and the myocardium. Journal of the American College of Cardiology, 2012,60(19):1854-1863. doi: 10.1016/j.jacc.2012.02.093
[4]	Lee S H, Choi J H . Involvement of inflammatory responses in the early development of calcific aortic valve disease:lessons from statin therapy. Animal Cells and Systems, 2018,22(6):390-399.
[5]	Bouchareb R, Mahmut A, Nsaibia M J , et al. Autotaxin derived from lipoprotein (a) and valve interstitial cells promotes inflammation and mineralization of the aorticvalve. Circulation, 2015,132(8):677-690.
[6]	Otto C M, Prendergast B . Aortic-valve stenosis - from patients at risk to severe valve obstruction. New England Journal of Medicine, 2014,371(8):744-756. doi: 10.1056/NEJMra1313875
[7]	Kufareva, Irina . Chemokines and their receptors:insights from molecular modeling and crystallography. Current Opinion in Pharmacology, 2016,30:27-37.
[8]	Wong B W C, Wong D, Mcmanus B M . Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis,diabetes mellitus,and transplant vascular disease. Cardiovascular Pathology, 2002,11(6):332-338. doi: 10.1016/S1054-8807(02)00111-4
[9]	Greaves D R, H?kkinen T, Lucas A D , et al. Linked chromosome 16q13 chemokines, macrophage-derived chemokine,fractalkine,and thymus- and activation-regulated chemokine,are expressed in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol, 2001,21(6):923-929.
[10]	Ali M T, Martin K, Kumar A H , et al. A novel CX3CR1 antagonist eluting stent reduces stenosis by targeting inflammation. Biomaterials, 2015,69:22-29.
[11]	White G E, Tan T C C, John A E , et al. Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling. Cardiovascular Research, 2009,85(4):825.
[12]	Liu H, Jiang D, Zhang S , et al. Aspirin inhibits fractalkine expression in atherosclerotic plaques and reduces atherosclerosis in apoe gene knockout mice. Cardiovascular Drugs & Therapy, 2010,24(1):17-24.
[13]	Lee S H, Choi J H . Involvement of immune cell network in aortic valve stenosis: communication between valvular interstitial cells and immune cells. Immune Network, 2016,16(1):26-32.
[14]	Rajamannan N M, Subramaniam M, Rickard D , et al. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation, 2003,107(17):2181-2184.
[15]	Patrick M, Rihab B, Boulanger M C . Innate and adaptive immunity in calcific aortic valve disease. Journal of Immunology Research, 2015,2015:1-11.
[16]	Zhan Q, Song R, Zeng Q , et al. Activation of TLR3 induces osteogenic responses in human aortic valve interstitial cells through the nf-κb and erk1/2 pathways. International Journal of Biological Sciences, 2015,11(4):482-493.
[17]	Quintar A, Mcardle S, Wolf D , et al. Endothelial protective monocyte patrolling in large arteries intensified by western diet and atherosclerosisnovelty and significance. Circulation Research, 2017,120(11):1789-1799.
[18]	Thanassoulis G, Campbell C Y, Owens D S , et al. Genetic associations with valvular calcification and aortic stenosis. New England Journal o Medicine, 2013,368(6):503-512.
[19]	Fu L, Jamieson D G, Usher D C , et al. Gene expression of apolipoprotein(a) within the wall of human aorta and carotid arteries. Atherosclerosis, 2001,158(2):303-311.
[20]	Hutcheson J D, Aikawa E, Merryman W D . Potential drug targets for calcific aortic valve disease. Nature Reviews Cardiology, 2014,11(4):218-231. doi: 10.1038/nrcardio.2014.1
[21]	Leopold J A . Cellular mechanisms of aortic valve calcification. Circulation: Cardiovascular Interventions, 2012,5(4):605-614.
[22]	Lis G J, Czapla-Masztafiak J, Kwiatek W M , et al. Distribution of selected elements in calcific human aortic valves studied by microscopy combined with SR-μXRF: Influence of lipids on progression of calcification. Micron, 2014,67:141-148.
[23]	Nagy E, Andersson D C, Hansson G K , et al. 375 leukotiene-induced calcification and osteogenic differentiation of human aortic valve interstitial cells. Atherosclerosis Supplements, 2011,12(1):80-80.
[24]	Li C, Xu S, Gotlieb A I . The progression of calcific aortic valve disease through injury,cell dysfunction,and disruptive biologic and physical force feedback loops. Cardiovascular Pathology, 2013,22(1):1-8. doi: 10.1016/j.carpath.2012.06.005
[25]	Steiner I, Lukas K, Tomas R , et al. Calcific aortic valve stenosis:Immunohistochemical analysis of inflammatory infiltrate. Pathology - Research and Practice, 2012,208(4):231-234.
[26]	Weiss R M, Miller J D, Heistad D D . Fibrocalcific aortic valve disease:opportunity to understand disease mechanisms using mouse models. Circulation Research, 2013,113(2):209-222. doi: 10.1161/CIRCRESAHA.113.300153
[27]	Jansen F, Xiang X, Werner N . Role and function of extracellular vesicles in calcific aortic valve disease. European Heart Journal, 2017,38(36):2714-2716.

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