体外转染Klotho基因对原代成骨细胞活性的影响

The effect of klotho gene transfection on the vitality of primary osteoblasts

目的探讨转染克老素(KL)基因对成骨细胞活性的影响。方法提取原代骨髓间充质干细胞(BMSCs)并培养,用倒置显微镜观察BMSCs形态。BMSCs培养至第三代时做流式鉴定,之后换为诱导培养基培养14~21 d,诱导为成骨细胞,用茜素红染色鉴定是否诱导成功。通过重组腺相关病毒(r AAV)介导小鼠KL基因转染成骨细胞。实验分为五组:空白对照组(control组),KL转染组(KL组),klotho转染+FGFR1抑制剂(BGJ398)组(KL+BGJ398组),空载体组(r AAV组),空载体+FGFR1抑制剂(BGJ398)组(r AAV+BGJ398组)。分组处理72 h后,荧光倒置显微镜观察成骨细胞腺病毒表达情况,RT-PCR检测成骨细胞的KL mRNA的表达,ELISA检测上清液KL蛋白的含量;RT-PCR检测成骨细胞分泌物:骨钙素(OCN)、骨桥蛋白(OPN)的mRNA表达。结果成功提取原代骨髓间充质干细胞,并且成功向成骨方向诱导。通过r AAV转染KL后,成骨细胞高表达外源性KL蛋白(P<0.01),并在KL-FGF23-FGFR1通路正常的情况下可显著促进成骨细胞分泌OCN(P<0.05),显著抑制成骨细胞分泌OPN(P<0.05)。结论 KL可以促进成骨细胞的活力,可能是通过KL-FGF23-FGFR1通路发挥作用。

Objective To investigate the effect of klotho( KL) gene transfection on the vitality of primary osteoblasts,and to reveal the effect of KL via fibroblast growth factor( FGF)-23 signal path on bone metabolism. Methods The primary mesenchymal stem cells( BMSCs) were abstracted from male rats. The morphologies of BMSCs was observed by inverted microscope. After the third generation culture,the specific surface markers of them were identified by flow cytometer. Then BMSCs were cultured by conditioned medium particularly for inducing into osteoblasts for 14 ~ 21 days. Alizarin red staining was used to test whether the induction was successful. After successfully inducted,the primary osteoblasts were transfected by adeno associated virus mouse KL gene( r AAV / k L). The experiment was divided into control,KL,KL with BGJ398( FGFR1 inhibitor),adenovirus empty vector,adenovirus empty vector with BGJ398( FGFR1 inhibitor) groups. After treatments,the expression of KL in osteoblasts was detected by RT-PCR,inversed fluorescent microscope and ELISA. The expression of osteoblast discharges: osteopontin( OPN),osteocalcin( OCN) were detected by RT-PCR. Results BMSCs were successfully abstracted from rats,and also primary osteoblasts were inducted. After transfected,the exogenous KL-derived genes were highly expressed( P < 0. 01). KL improved discharge of OCN( P < 0. 05) and inhibited OPN( P < 0. 05) in osteoblasts when KL-FGF23-FGFR1 pathway was normal. Conclusions KL could promote the vitality of primary osteoblast probably through KL-FGF23-FGFR1 pathway.