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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (5): 63-71    DOI: 10.13523/j.cb.20190507
研究报告     
BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化 *
程瑜,施琼,安利钦,范梦恬,皇改改,翁亚光()
重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016
BMP7 Gene Silencing Inhibits Osteogenic Differentiation of Porcine Arotic Valve Interstitial Cells Induced by Osteogenic Induction Medium
Yu CHENG,Qiong SHI,Li-qin AN,Meng-tian FAN,Gai-gai HUANG,Ya-guang WENG()
Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine,Chongqing Medicine University, Chongqing 400016, China
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摘要：

目的:探究小干扰RNA(small interference RNA,siRNA)介导的骨形态发生蛋白7(bone morphogenetic protein7,BMP7)基因沉默对钙盐诱导猪主动脉瓣膜间质细胞成骨分化的影响及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的干预及治疗提供理论依据。方法:非CAVD瓣膜组织(non-CAVD组)取自手术治疗的主动脉夹层患者,CAVD瓣膜组织(CAVD组)取自因钙化性主动脉瓣狭窄而进行主动脉瓣膜置换术的患者,采用免疫组化和Western blot法检测non-CAVD组和CAVD组中BMP7、Runt相关转录因子2(Runx2)的蛋白质表达水平。选取健康家猪处死后即刻于无菌条件下取主动脉瓣叶,采用胶原酶连续消化法分离主动脉瓣膜间质细胞,观察其形态特征,并用免疫荧光染色行表型鉴定。采用脂质体转染法将BMP7-siRNA转染猪主动脉瓣膜间质细胞,采用qPCR和Western blot法验证BMP7表达的变化;利用钙盐培养基诱导细胞成骨分化,建立体外主动脉瓣膜间质细胞钙化模型后,采用ALP染色和茜素红S染色实验分别检测细胞早期及晚期成骨分化能力;采用qPCR和Western blot法分别检测细胞成骨相关基因及蛋白质Runx2、OCN和OPN的表达情况。并用Western blot法检测BMP7下游信号通路中Smad1/5/8的磷酸化水平。结果:BMP7和Runx2蛋白在CAVD组中表达明显高于non-CAVD组。成功分离出原代猪主动脉瓣膜间质细胞,α-平滑肌肌动蛋白(α-SMA)及波形蛋白(vimentin)染色阳性,血管性血友病因子(von willebrand factor,vWF)染色阴性。转染BMP7-siRNA后猪主动脉瓣膜间质细胞中BMP7的mRNA和蛋白质水平均明显下调,早期及晚期成骨分化能力均明显降低。沉默BMP7基因的表达,可下调Runx2、OCN和OPN的基因及蛋白质表达,且磷酸化的Smad1/5/8(p-Smad1/5/8)蛋白水平明显降低。结论:BMP7基因沉默抑制钙盐诱导的主动脉瓣膜间质细胞的成骨分化能力,BMP7/Smads信号通路可能在该过程中发挥重要作用。
关键词： 钙化性主动脉瓣膜病;  瓣膜间质细胞;  骨形态发生蛋白7;  成骨分化;  RNA干扰    
Abstract:

Objective: To investigate the effect of small interfering RNA (siRNA) -mediated bone morphogenetic protein7 (BMP7) gene silencing on osteogenic differentiation of porcine aortic valve interstitial cells induced by osteogenic induction medium,and to provide a theoretical basis for the intervention and treatment of calcific aortic valve disease.Methods:The non-CAVD valvular tissues (non-CAVD group) were taken from patients with surgical treatment of aortic dissection,the CAVD valvular tissues (CAVD group) were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis.The expression levels of BMP7 and Runx2 in the non-CAVD group and CAVD group were tested by immunohistochemistry and Western blot. Healthy domestic pigs were sacrificed and the aortic valve leaflets were aseptically removed immediately.The aortic valve interstitial cells(VICs) were isolated by continuous collagenase digestion,its morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining. VICs were transfected with BMP7-siRNA by liposome method.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was detected by qPCR and Western blot,respectively.The conditioned medium induced osteogenic differentiation of VICs for the establishment of calcification model of aortic valve interstitial cells in vitro,then Alkaline phosphatase (ALP)staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The mRNA and protein levels of Runx2,OCN and OPN were determined by qPCR and Western blot,respectively.The protein levels of p-drosophila mothers against de-capentaplegic 1/5/8 (p-Smad1/5/8) was also determined by Western blot.Results:The expression of BMP7 and Runx2 in CAVD group was significantly higher than that in non-CAVD group.The primary porcine aortic valve interstitial cells were successfully isolated and the staining of α-smooth muscle actin (α-SMA) and Vimentin were positive,the staining of von Willebrand factor (vWF) was negative.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was significantly decreased,and the cell early and late osteogenic differentiation abilities were significantly decreased.The mRNA and protein levels of Runx2,OCN and OPN were significantly reduced.Meanwhile,the protein levels of p-Smad1/5/8 were down-regulated.Conclusion:BMP7 gene silencing obviously inhibits the osteogenic differentiation of aortic valve interstitial cells induced by osteogenic induction medium.The BMP7/Smads signaling pathways may play an important role in these processes.
Key words: Calcific aortic valve disease    Valve interstitial cell    BMP7    Osteogenic differentiation    RNA interference
收稿日期: 2018-10-24 出版日期: 2019-06-04
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(NSFC81672103);重庆市技术创新与应用示范项目资助项目(cstc2018jscx-msybX0113)
通讯作者: 翁亚光     E-mail: yaguangweng@126.com
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程瑜
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引用本文:

程瑜,施琼,安利钦,范梦恬,皇改改,翁亚光. BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化 *[J]. 中国生物工程杂志, 2019, 39(5): 63-71.

Yu CHENG,Qiong SHI,Li-qin AN,Meng-tian FAN,Gai-gai HUANG,Ya-guang WENG. BMP7 Gene Silencing Inhibits Osteogenic Differentiation of Porcine Arotic Valve Interstitial Cells Induced by Osteogenic Induction Medium. China Biotechnology, 2019, 39(5): 63-71.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190507    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I5/63

Gene Forward primer(5 'to 3') Reverse primer(5' to 3')
BMP7 GACTTCAGCCTGGACAACGA AGGTCCAGCATGAACATGGG
Runx2 GCACTACCCAGCCACCTTTA TATGGAGTGCTGCTGGTCTG
OCN TCACACTGCTTGCCCTACTG TGCCATAGAAGCGCCGATAG
OPN GAGCAAACAGACGATGTGGA GACCAGCTCATCGGATTCAT
GAPDH GGTGAAGGTCGGAGTGAACG CGTGGGTGGAATCATACTGGA
表1  引物序列(猪)
图1  BMP7和Runx2蛋白在CAVD组中的表达明显高于non-CAVD组
图2  猪主动脉瓣膜间质细胞形态及表型鉴定
图3  VICs细胞转染BMP7-siRNA后BMP7的mRNA和蛋白质表达水平
图4  BMP7表达下调抑制VICs细胞的早期及晚期成骨分化能力
图5  BMP7表达下调抑制VICs细胞成骨相关基因及蛋白质的表达
图6  BMP7表达下调对BMP-Smad1/5/8信号通路的影响
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