鸡软骨细胞体外分离培养鉴定以及过表达miR-15a对软骨细胞的影响

Isolation,Culture and Identification of Chicken Chondrocytes and the Effect of Overexpression of miR-15a on Chondrocytes

为分析miR-15a在肉鸡不同组织中的表达情况,并探究过表达miR-15a对体外培养鸡软骨细胞的影响。本研究首先通过倒置显微镜观察、PCR、凝胶电泳和甲苯胺蓝染色对体外分离培养的软骨细胞进行鉴定。通过实时荧光定量PCR检测miR-15a在肢体内外翻畸形(valgus-varus deformity,VVD)组和健康组肉鸡中(各3只)各组织的表达量。之后通过CCK8和EDU方法分析软骨细胞过表达miR-15a后细胞增殖情况。软骨细胞转染miR-15a mimics后,通过qPCR检测软骨细胞的标志基因Collagen-2、Aggrecan、Collagen-10,成熟分化基因Runx2、Sox9、VEGF、MMP9,炎性因子IL-1β、IL-6、IL-8、IL-10、TNF-α、TGF-β3以及凋亡基因Fas、FasL、Bcl-2的表达量。并构建FKBP53′UTR的野生型载体和突变型载体,通过双荧光素酶检测报告检测miR-15a与FKBP5的靶向关系。结果表明,本研究所用的胰蛋白酶、Ⅱ型胶原酶和透明质酸酶联合消化法成功分离得到状态良好的软骨细胞。荧光定量结果显示,miR-15a在各组织中均有表达,与健康组相比,miR-15a在VVD组的肝(P<0.01)、脾(P<0.05)、胸腺(P<0.01)中的表达量显著升高,在心和胸肌组织中的表达量显著降低(P<0.01)。CCK8与EDU分析结果显示,与NC组相比,过表达miR-15a组软骨细胞增殖速度显著降低(P<0.01),增殖细胞数量显著减少(P<0.01)。qPCR结果显示,与mimics NC组相比,miR-15a mimics组的软骨细胞标志基因Aggrecan、成熟分化基因Sox9、Runx2表达量显著降低(P<0.05),Fas基因表达量极显著上升(P<0.01),FasL基因和抗凋亡基因Bcl-2极显著下降(P<0.01)。成功构建了FKBP53′UTR野生型和突变型载体,双荧光素酶检测报告结果显示预测的靶基因FKBP5与miR-15a没有靶向关系。本研究成功分离并鉴定了鸡软骨细胞,过表达miR-15a抑制鸡软骨细胞增殖、成熟和分化并促进细胞凋亡。

The study aimed to investigate the expression of miR-15a in different tissues of broilers and explore the effect of overexpression of miR-15a on chondrocytes.In this study,the chondrocytes were isolated and culture,and it was identified by inverted microscope observation,PCR,gel electrophoresis and toluidine blue staining.Real-time fluorescence quantitative PCR was used to detect the expression level of miR-15a in each tissue of broilers in the valgus-varus deformity(VVD)group and the healthy group(3 each).After that,the cell proliferation of chondrocytes in miR-15a overexpression group was detected by CCK8 and EDU assay.After chondrocytes were transfected with miR-15a mimics,the expression of marker genes of chondrocytes Collagen-2,Aggrecan,Collagen-10,mature differentiation genes Runx2,Sox9,VEGF,MMP9,inflammatory factors IL-1β,IL-6,IL-8,IL-10,TNF-α,TGF-β3 and the apoptosis genes Fas,FasL and Bcl-2 were detected by qPCR.The wild-type vector and mutant vector of FKBP53′UTR were constructed,and the targeting relationship between miR-15a and FKBP5 was detected by dual-luciferase detection reporter.The results showed that the trypsin,collagenase II and hyaluronidase combined digestion method used in this study could successfully isolate chondrocytes.The qPCR results showed that miR-15a was expressed in all detected tissues.The expression of miR-15a in the liver(P<0.01),spleen(P<0.05)and thymus(P<0.01)in the VVD group were significantly higher compared with the healthy group.And the expression level in the heart and pectoral muscle tissues was significantly lower in the VVD group(P<0.01).The results of CCK8 and EDU showed that,compared with the NC group,the proliferation rate of chondrocytes in the miR-15a overexpression group was significantly decreased(P<0.01),and the number of proliferating cells was significantly decreased(P<0.01).The qPCR results showed that,the expression of Aggrecan,Sox9 and Runx2 genes in the miR-15a mimics group were significantly decreased(P<0.05),and the expression of Fas gene was extremely significantly increased(P<0.01),the expression of FasL gene and anti-apoptotic gene Bcl-2 were extremely significantly decreased(P<0.01)compared with the NC group.The FKBP53′UTR wild-type and mutant vectors were successfully constructed,and the dual-luciferase detection reporter showed that the predicted target gene FKBP5 had no targeting relationship with miR-15a.In this study,chicken chondrocytes were successfully isolated and identified,and the results suggest that the overexpression of miR-15a can inhibit the proliferation,maturation,differentiation and promote apoptosis of the chicken chondrocytes.