阿托伐他汀钙对原代成骨细胞OPG、RANKL、M-CSF基因表达的影响

Effects of atorvastatin calcium on OPG,RANKL,M-CSF gene expression in osteoblast

目的观察不同浓度阿托伐他汀钙对体外培养原代成骨细胞增生和对OPG、RANKL、M-CSF基因表达的影响,探讨阿托伐他汀钙抗骨质疏松的分子机制,为临床用药提供实验依据。方法采用胰酶和Ⅰ型胶原酶混合酶消化法分离获得原代成骨细胞;采用差速贴壁法进行纯化。光镜下观察细胞的形态结构和生长状况,通过碱性磷酸酶染色和Ⅰ型胶原免疫组化SABC法对细胞进行鉴定。用不同浓度的阿托伐他汀钙(10^(-9)、10^(-8)、10^(-7)、10^(-6)和10^(-5) mol/L)干预成骨细胞,在24、48和72 h后,用实时荧光定量方法检测OPGmRNA、RANKL mRNA、M-CSF mRNA的表达水平。结果镜下观察显示细胞具有成骨细胞的典型特征,细胞多为单核,呈三角、多边、长梭等形态,并伸有粗大伪足;碱性磷酸酶染色及Ⅰ型胶原免疫组化染色呈阳性。实时荧光定量PCR结果显示:不同浓度的阿托伐他汀钙(10^(-9)、10^(-8)、10^(-7)、10^(-6)、10^(-5)mol/L)均可以促进OPG mRNA的表达,抑制RANKL mRNA的表达,对M-CSF mRNA基因的表达也具有促进作用,且与药物浓度及作用时间有关。结论采用胰酶和Ⅰ型胶原酶混合酶消化法体外分离成骨细胞,采用多次"差速贴壁法"可以获得数量多、纯度高且活性好的成骨细胞。阿托伐他汀钙可以促进成骨细胞OPG基因mRNA的表达,抑制RANKL基因mRNA的表达,对M-CSF基因的表达也有一定的促进作用。

Objective To research the effects of different concentrations atorvastatin calcium on cell proliferation and OPG, RANKL, M-CSF gene expression in osteoblast in vitro, to explore the molecular mechanism of atorvastatin calcium anti osteoporosis, and to provide the experiment basis for clinical drug use. Methods The rats crinial bones were digesting by mixed enzyme which contains enzyme and type I collagenase. The osteoblast were purified by selective plating technique. The osteoblasts were identified through morphology observation, alkaline phosphatase dyeing, collagen I immunohistochemical staining. With different concentrations of atorvastatin calcium （10-9, 10-s, 10-7, 10-6, 10-5 mol/L） intervention osteoblasts, the gene expression of OPG mRNA, RANKL mRNA, M-CSF mRNA was meas- ured by real-time fluorescence quantitative PCR. Results The cultured cells had the typical features of osteoblast. The cells are triangle, polygonal, long spindle shape, etc. And mostly are mononuclear cells, have large pseudopodia. Alka-line phosphatase dyeing and collagen I immunohistochemical staining were both positive. The result of real-time fluorescence quantitative polymerase chain reaction （PCR） showed that atorvastatin calcium （10-9, 10-8, 10-7, 10-6, 10-s mol/L） could promote OPG mRNA and M-CSF mRNA expression, restrain RANKL mRNA expression, and this effect may be concentration and time denpendent. Conclusion A large quantity and activity of cells were obtained by mixed enzyme digestion method. High purity osteoblasts were obtained by selective plating technique that can remove impurities such as fibroblast cells. Atorvastatin calcium could promote OPG and M-CSF gene＇s expression of osteoblast, inhibit RANKL gene＇s expression of osteoblast.