1,25(OH)_(2)D_(3)及VDR敲除对山羊附睾头上皮细胞β防御素家族表达的影响

Effect of 1,25(OH)_(2)D_(3)and VDR Knockout on the Expression ofβ-defensins Family in the Caprine Epididymal Caput Epithelial Cells

旨在研究1,25(OH)_(2)D_(3)是否通过VDR途径调节山羊附睾头上皮细胞β防御素基因表达。本试验选取3只6月龄太行黑山羊,分别采集附睾头组织。采用差速贴壁法分离山羊附睾头上皮细胞,用细胞免疫荧光鉴定上皮细胞纯度。添加100 nmol·L^(-1)1,25(OH)_(2)D_(3)处理附睾头上皮细胞以及筛选出敲除效率最高的pCas9/gRNA1质粒载体进行细胞转染,同时设置阴性对照组和空白对照组,每组3个重复孔。附睾头上皮细胞经1,25(OH)_(2)D_(3)处理以及VDR基因敲除后,分别用qRT-PCR检测VDR和17种β防御素基因的表达,用Western blot检测VDR蛋白和3种β防御素蛋白的表达。结果表明,1,25(OH)_(2)D_(3)能极显著提高VDR、gBD124、gBD126和gBD104a的mRNA和蛋白表达(P<0.01),同时极显著提高gBD104、gBD 109tr1、gBD 109tr2、gBD113、gBD116、gBD120、gBD 121以及gBD 123基因的表达(P<0.01),显著提高gBD106、gBD127、gBD 129以及gBD 134基因的表达(P<0.05),而对gBD 110like和gBD 128基因没有显著影响(P>0.05);3个基因敲除载体进行细胞转染后,pCas9-VDR-V1组VDR蛋白表达极显著降低(P<0.01)。VDR基因敲除极显著降低gBD124的mRNA和蛋白表达(P<0.01),显著降低gBD126和gBD104a的mRNA和蛋白表达(P<0.05),同时VDR基因敲除组gBD 109tr1、gBD 109tr2、gBD116、gBD123、gBD127、gBD 128以及gBD 134基因的表达极显著低于其他组(P<0.01),VDR基因敲除组gBD104、gBD106、gBD 120以及gBD 129基因的表达显著低于其他组(P<0.05),而对gBD121、gBD 110like以及gBD 113的相对表达则无显著影响(P>0.05)。综上所述,1,25(OH)_(2)D_(3)可以上调VDR和部分β防御素表达;VDR基因敲除后降低部分β防御素表达,结果表明1,25(OH)_(2)D_(3)通过上调VD/VDR信号通路关键基因VDR的表达调控山羊附睾头上皮细胞部分β防御素表达。

The aim of this study was to explore whether 1,25(OH)_(2)D_(3)regulates beta-defensin gene expression in the caprine epididymal caput epithelial cells by the VDR signaling pathway.Three 6-month-old Taihang black goats were selected,and the epididymal caput tissues were collected.The caprine epididymal caput epithelial cells were isolated by differential adherence time,and the purity of epithelial cells was identified by cellular immunofluorescence;The epididymal caput epithelial cells were treated with 100 nmol·L^(-1)1,25(OH)_(2)D_(3);And the pCas9/gRNA1 plasmid vector with the highest knockout efficiency was selected for cell transfection.And negative control group and blank control group were set up,with three replicate in each group.After the cells were treated with 1,25(OH)_(2)D_(3)or VDR gene knockout,the expressions of VDR and 17β-defensin genes were detected by qRT-PCR,and the expression of VDR protein and 3β-defensin proteins were detected by Western blot.The results showed that 1,25(OH)_(2)D_(3)could significantly increase mRNA and protein expressions of VDR,gBD124,gBD126 and gBD104a(P<0.01),and markedly increased the expression of gBD104,gBD 109tr1,gBD 109tr2,gBD113,gBD116,gBD120,gBD 121 and gBD 123 genes(P<0.01),and significantly increased the expression of gBD106,gBD127,gBD 129 and gBD 134 genes(P<0.05),but had no significant effect on gBD 110like and gBD128 expression(P<0.05);After the three gene knockout vectors were transfected into cells,the expression of VDR protein in pCas9-VDR-V1 group was significantly decreased(P<0.01).VDR gene knockout significantly increased gBD124 mRNA and protein expression(P<0.01),and significantly reduced the mRNA and protein expression of gBD126 and gBD104a(P<0.05),the mRNA level of gBD 109tr1,gBD 109tr2,gBD116,gBD123,gBD127,gBD 128 and gBD 134 genes in VDR gene knockout group were significantly lower than those in the other groups(P<0.01),the mRNA level of gBD104,gBD106,gBD 120 and gBD 129 genes in VDR gene knockout group were significantly lower than those in the other groups(P<0.05),but had no significant effect on gBD121,gBD 110like and gBD113(P<0.05).In conclusion,1,25(OH)_(2)D_(3)could up-regulate the expression of VDR and some beta-defensins,VDR gene knockout increased the expression of some beta-defensins.The results showed that 1,25(OH)_(2)D_(3)could increase the expression of part beta-defensins in caprine epididymal caput epithelial cells by up-regulating the expression of VDR and activating the VD/VDR signaling pathway.