脂联素受体激动剂(AdipoRon)对鸡成骨细胞增殖和凋亡的影响

Effect of AdipoRon on proliferation and apoptosis of chicken osteoblasts

脂联素受体激动剂(AdipoRon)是人工合成的与脂联素(ADPN)有相似作用的口服活性小分子,研究显示ADPN可调节成骨细胞生长和分化,但AdipoRon是否具有类似的功能目前鲜见报道。本试验主要研究AdipoRon对鸡成骨细胞的影响。从14日龄鸡胚额骨中分离获得成骨细胞,细胞培养第4日,分别添加100,200 mg/L AdipoRon处理成骨细胞,处理72 h后,MTT法检测细胞增殖活性,并进行碱性磷酸酶(ALP)染色。Real-time PCR检测脂联素受体1(AdipoR1)、脂联素受体2(AdipoR2)、成骨细胞成熟标志基因骨钙素(osteocalcin,OC)、Ⅰ型胶原α2链(alpa2 of type I collegen,COL1A2)、细胞凋亡相关基因Caspase-3、Bcl-2及Bax的基因表达量,计算Bcl-2与Bax基因表达量的比值。结果显示,100,200 mg/L AdipoRon处理后的成骨细胞正常形态消失,细胞数量减少,体积变小,细胞核明显固缩,细胞存活率极显著降低(P<0.01)。AdipoRon可增加成骨细胞中AdipoR1、AdipoR2、OC、Bcl-2、Bax和Caspase-3的表达量(P<0.05),并呈剂量依赖性,但对COL1A2和Bcl-2/Bax的表达无显著性影响。结果表明,100,200 mg/L AdipoRon均可促进鸡成骨细胞AdipoR1/2的表达,且能抑制成骨细胞的增殖,促进成骨细胞的成熟及凋亡。

AdipoR agonist(AdipoRon) is artificial synthesis oral active small molecule which has similar functions with adiponectin(ADPN).Some researchers have showed the ADPN stimulates osteoblasts proliferation and differentiation.However,the effect of AdipoRon on bone metabolism is not known.Therefore,the experiment is to study the effect of AdipoRon on the primary chicken embryo osteoblasts.Osteoblasts were isolated from the frontal bone of 14-day-old chicken embryos by enzyme digestion.On the 4 day of cell culture,osteoblasts were treated with 100 or 200 mg/L AdipoRon,respectively.After 72 hours of AdipoRon treatment,MTT assay was used to detect the proliferation activity of the cells,and alkaline phosphatase(ALP) staining of these cells was performed.The gene expression levels of adiponectin receptor 1(AdipoR1),adiponectin receptor 2(AdipoR2),the marker gene for osteoblast maturation including osteocalcin(OC) and type Ⅰ collagen α2 chain(COL1 A2),apoptosis-related genes including Caspase-3,Bcl-2 and Bax were analyzed by real-time PCR and the radio of Bcl-2 to Bax were calculated.The normal morphology of osteoblasts disappeared,the number of cells decreased,the volume became smaller,the nucleus became obviously pyknotic,and the cell survival rate was significantly reduced(P<0.01) by 100 or 200 mg/L AdipoRon.Real-time PCR showed that 100 or 200 mg/L AdipoRon increased the gene expression of AdipoR1,AdipoR2,OC,Bcl-2,Bax and Caspase-3(P<0.05)in a does dependent manner.However,the gene expression of COL1 A2 and the radio of Bcl-2 to Bax were not affected by 100 or 200 mg/L AdipoRon treatment.This study indicated that both 100 and 200 mg/L AdipoRon promoted the gene expression of AdipoR1/2,and AdipoRon inhibited the proliferation of osteoblasts and accelerated the maturation and apoptosis of osteoblasts.