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

doi:10.13523/j.cb.20190802

China Biotechnology

2019, Vol. 39

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

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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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

Received: 27 January 2019 Published: 18 September 2019

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Corresponding Authors: Qiong SHI E-mail: shiqiong@cqmu.edu.cn

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

Cite this article:

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.

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

Table 1 Primer sequence used for qPCR

Fig.1 The expression of CX3CR1 mRNA at clinical specimen (a) and the expression of CX3CR1, Runx2 and OPN were used by immunohistochemistry(b)

χ ?

±S,**:P<0.01(non-calcified aortic valves vs calcified aortic valves); **:P<0.01(non-calcified bicuspid valves vs calcified bicuspid valves)

Fig.2 Expression of CX3CR1, α-SMA, Runx2 and OPN in clinical specimens (a) The expression of CX3CR1, α-SMA,Runx2 and OPN were examined by Western blot analysis (b) Bar graphs exhibited the relative protein lever of CX3CR1, α-SMA ,Runx2 and OPN by Western blot analysis

χ ?

±S,*:P<0.05(non-calcified aortic valves vs calcified aortic valves);*:P<0.05(non-calcified bicuspid valves vs calcified bicuspid valves)

Fig.3 Growth status (a) and phenotype identification (b) of human aortic valve interstitial cells

Fig.4 Effect of overexpression of CX3CR1 on human aortic valve interstitial cells

χ ?

±S,*:P<0.05(CX3CR1+OM vs negative control+OM); ***:P<0.01(CX3CR1+OM vs negative control+OM)

Fig.5 Effect of silence of CX3CR1 on human aortic valve interstitial cells

χ ˉ

±S,*:P<0.05(siCX3CR1+OM vs negative siRNA control+OM)

Fig.6 The level of AKT and p-AKT in the humanaortic valve interstitial cells after transfection *:P<0.05(CX3CR1+OM vs negative control+OM); *:P<0.05(siCX3CR1+OM vs negative siRNA control+OM)


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