Effect of calcitriol on osteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide

Xianfeng XIE; Wenping HU; Qiong SHI; Yingjiu JIANG

doi:10.16016/j.1000-5404.201804038

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Effect of calcitriol on osteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide

Basic Medicine | 更新时间：2026-01-09

- Effect of calcitriol on osteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide
- Journal of Third Military Medical University Vol. 40, Issue 19, Pages: 1770-1776(2018)
- 作者机构：
  
  1.400016 重庆，重庆医科大学附属第一医院胸心外科
  2.400016 重庆，重庆医科大学检验医学院
- 作者简介：
- 基金信息：
- DOI：10.16016/j.1000-5404.201804038
  CLC： R363.1+3;R542.5+2
- Received：18 April 2018，
  
  Revised：2018-06-18，
  
  Published：15 October 2018
- 稿件说明：
移动端阅览
Xianfeng XIE, Wenping HU, Qiong SHI, et al. Effect of calcitriol on osteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide[J]. Journal of Third Military Medical University, 2018, 40(19): 1770-1776.
DOI：

Xianfeng XIE, Wenping HU, Qiong SHI, et al. Effect of calcitriol on osteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide[J]. Journal of Third Military Medical University, 2018, 40(19): 1770-1776. DOI： 10.16016/j.1000-5404.201804038.

摘要

目的

探究骨化三醇[1α

25-dihydroxycholecalciferol，1

25(OH)

]

对脂多糖(Lipopolysaccharide，LPS)诱导猪主动脉瓣膜间质细胞成骨分化的影响，为钙化性主动脉瓣膜病的预防提供理论依据。

方法

6只雄性健康家猪(8~10月龄，体质量100~120 kg)，处死后即刻于无菌条件下取主动脉瓣叶。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。利用条件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，并予加入不同浓度骨化三醇处理72 h，并设立空白对照组和DMSO载体对照组。LPS诱导细胞成骨分化，筛选最适LPS效应浓度；实验分：空白对照组，骨化三醇组，LPS组，骨化三醇+LPS组。1 nmol/L骨化三醇与100 ng/mL LPS先后共同处理细胞48 h。分别用实时荧光定量PCR和Western blot检测细胞成骨分化标志物骨形态发生蛋白2(bone morphogenetic protein 2，BMP-2)、骨钙蛋白(osteocalcin，OC)的mRNA和蛋白水平表达；茜素红染色评估晚期钙结节形成情况。

结果

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(vWF)染色阴性。骨化三醇抑制条件培养基诱导的细胞成骨分化，BMP-2和OC的表达较空白对照组和DMSO载体对照组均显著降低(

0.05)。LPS处理后可促进细胞成骨分化，与其他浓度LPS组比较，其中100 ng/mL LPS组，BMP-2和OC的表达量最多(

0.05)；加入骨化三醇预处理后，可减弱LPS诱导的细胞成骨分化标志物表达，骨化三醇+LPS组BMP-2(

=3，1.94±0.27、0.41±0.03)和OC(

=3，0.21±0.04、0.44±0.03)的表达较LPS组(2.54±0.36、0.88±0.02；2.49±0.53、0.86±0.02)明显降低(

0.01)，并且晚期钙结节形成减少。

结论

骨化三醇可抑制条件培养基及LPS诱导的主动脉瓣膜间质细胞成骨分化，可能对主动脉瓣膜钙化具有一定的保护作用。

Abstract

Objective

To investigate the effect of calcitriol [1α

25-dihydroxycholecalciferol

25(OH)

]

on osteogenic differentiation of porcine aortic valve interstitial cells (VICs) induced by lipopolysaccharide (LPS) in order to provide a theoretical basis for the prevention of calcific aortic valve disease.

Methods

After 6 healthy male domestic pigs (8~10 months old

body weight 100~120 kg) were sacrificed

the aortic valve leaflets were removed immediately under aseptic condition. The aortic VICs were isolated by continuous collagenase digestion

and their morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining. The conditioned medium was used to induce osteogenic differentiation in VICs to establish a calcification model of aortic VICs. Then the differentiated cells were treated with different concentrations of calcitriol for 72 h

and untreated and DMSO treated cells served as blank control and vehicle control. Optimal concentration of LPS were screened

LPS induced osteogenic differentiation of VICs

to select the best effect; Experimental groups were blank control group

calcitriol group

LPS group

and calcitriol+LPS group. VICs were treated with 1 nmol/L calcitriol and 100 ng/mL LPS for 48 h. The expression of bone morphogenetic protein 2 (BMP-2) and osteocalcin (OC) at mRNA and protein levels were detected by real-time fluorescence quantitative PCR and Western blotting

respectively

and alizarin red staining was used to evaluate the formation of late calcium nodules.

Results

The primary porcine aortic VICs were successfully isolated

with positive staining of α-smooth muscle actin (α-SMA) and vimentin

and negative staining of von Willebrand factor (vWF). Calcitriol inhibited osteogenic differentiation of VICs induced by conditioned medium

and resulted in significantly lower

expression levels of BMP-2 and OC when compared with those of blank control group and vehicle control group (

0.05). LPS treatment promoted osteogenic differentiation of VICs

and 100 ng/mL LPS induced highest expression levels of BMP-2 and OC than LPS of other concentrations (

0.05). Calcitriol pretreatment reduced the expression levels of the markers of osteogenic differentiation induced by LPS. The expression levels of BMP-2 (

1.94±0.27

0.41±0.03) and OC (

0.21±0.04

0.44±0.03) in the calcitriol+LPS group were obviously lower than those of LPS group (2.54±0.36

0.88±0.02; 2.49±0.53

0.86±0.02

0.01)

and the formation of late calcium nodules was also decreased.

Conclusion

Calcitriol inhibits the osteogenic differentiation of aortic VICs induced by conditioned medium and LPS

which may have a protective effect on aortic valve calcification.

关键词

Keywords

references

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