维生素D调节血管内皮生长因子在支气管肺发育不良新生大鼠中的作用及机制研究

The Role and Mechanism of Vitamin D in Regulating Vascular Endothelial Growth Factor in Neonatal Rats with Broncho Pulmonary Dysplasia

目的:研究维生素D(VD)及其受体(VDR)通过调节VEGF对新生大鼠支气管肺发育不良(BPD)的调节作用和机制。方法:构建BDP模型新生大鼠,分为对照组和BDP组。培养大鼠肺上皮Ⅱ型细胞(RLE-6TN),RLE-6TN细胞分为VDR过表达(Le-VDR慢病毒感染)、VDR过表达对照组(Le-Ctrl)、VDR敲低(Sh-VDR感染)、VDR敲低对照组(Sh-Ctrl),VD单独处理,VEGF-A沉默组(siVEGF-A);Le-VDR与siVEGF-A共处理(Le-VDR+siVEGF-A);通过CCK-8检测、流式细胞术、伤口愈合检测和体外粘附检测明确VD和VDR对肺泡上皮细胞的调节作用。RTqPCR检测VDR和VEGF-A mRNA水平,Western blot检测VDR和VEGF-A的蛋白水平。结果:与对照相比,BPD新生大鼠的VD水平和VDR表达降低(P<0.01);在RLE-6TN细胞中过表达VDR,细胞增殖、迁移、粘附能力增加(均P<0.01),凋亡率减少(P<0.01);细胞中敲低VDR效果相反,VD刺激后细胞凋亡被抑制,细胞增殖、迁移和粘附能力增加(P<0.05);VDR过表达正向调节血管内皮生长因子(VEGF)-A(P<0.05),而且VEGF-A和VDR共定位于BPD大鼠肺组织和细胞中;且BPD新生大鼠肺组织中VEGF-A的表达水平降低(P<0.05)。结论:VD通过上调VEGF-A表达抑制BPD的发展。

Objective:To study the effect of VEGF regulated by vitamin D(VD)and/or VD receptor(VDR)on bronchopulmonary dysplasia(BPD)in neonatal rats.Methods:BDP neonatal rats were constructed and divided neonatal rats into control group and BDP group.Rat lung epithelial typeⅡcells(RLE-6TN)were cultured,and divided into VDR overexpression group(Le-VDR lentivirus infection group),VDR overexpression control group(Le-Ctrl group),VDR knockdown group(Sh-VDR infection group),VDR knockdown control group(Sh-Ctrl group),VD treatment alone group,VEGF-A silencing group(siVEGF-A group),and Le-VDR and siVEGF-A co-treatment group(Le-VDR+siVEGF-A group).The regulated effect of VD and VDR on lung cells was confirmed by CCK-8 detection,flow cytometry,wound healing detection and in vitro adhesion detection.VDR and VEGF-A mRNA were detected by RTqPCR,and VDR and VEGF-A protein were detected by Western blot.Results:Compared with the control group,the VD level and VDR expression in BPD neonatal rats were downregulated(P<0.01);overexpression of VDR in RLE-6TN cells increased the cell proliferation,migration,and adhesion ability(all P<0.01),and the apoptosis rate were decreased(P<0.01);however,the effect of knocking down VDR in cells was opposite.VD stimulation inhibited cell apoptosis and increased cell proliferation,migration and adhesion(P<0.05);VDR overexpression positively regulated vascular endothelial growth factor(VEGF)-A(P<0.05);Moreover,VEGF-A and VDR were co-localized in the lung tissue and cells of BPD rats;furthermore,the expression levels of VEGF-A in lung tissue of BPD neonatal rats were decreased(P<0.05).Conclusion:VD inhibits the development of BPD by up-regulating the expression of VEGF-A.