生理和药理浓度下1,25(OH)2D3对软骨细胞增殖、成熟和钙化的影响研究

Effects of 1,25(OH)2D3 on proliferation,maturation and calcification of chondrocytes at physiological and pharmacological concentrations

目的体外观察生理和不同药理浓度的1,25(OH)2D3对原发性股骨头软骨细胞(femoral chondrocytes,FCs)和继发性髁突软骨细胞(condylar chondrocytes,CCs)增殖、成熟和钙化的影响。方法通过分离培养1周龄雌性SD仔鼠的FCs和CCs,体外分为6组:(1)空白对照组(不做处理);(2)溶剂组(0.01%酒精处理);(3)^(6)分别为0.1、1、10、100 nmol/L 1,25(OH)2D3处理组。各组按相应处理因素处理24 h后通过CCK8检测对2种软骨细胞增殖能力的影响,并对增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、Ⅱ型胶原(collagenⅡ,COLⅡ)以及X型胶原(collagen X,COL X)进行q RT-PCR和Western blot检测。结果 (1)CCK8检测结果显示,1,25(OH)2D3呈浓度依赖性抑制2种软骨细胞的增殖,且CCs的抑制率较FCs明显;PCNA的qRT-PCR和Western blot检测结果与之基本一致。(2)相对于空白对照组,体外生理浓度(0.1 nmol/L)的1,25(OH)2D3对2种软骨细胞COLⅡ的表达影响差异无统计学意义(P>0.05),药理浓度(1、10、100 nmol/L)的1,25(OH)2D3可上调2种软骨细胞COLⅡ的表达,其中FCs最佳浓度在10 nmol/L,而CCs呈浓度依赖性上升。(3)相对于空白对照组,1,25(OH)2D3对2种软骨细胞COL X的m RNA和蛋白水平表达均无统计学意义(P>0.05)。结论体外1,25(OH)2D3呈浓度依赖性抑制软骨细胞增殖,药理浓度的1,25(OH)2D3可促进软骨细胞成熟,且其对CCs的影响较FCs显著;而其对2种软骨细胞的钙化无影响。

Objective To investigate the effects of 1,25(OH)2 D3 on the proliferation,maturation and calcification for primary femoral chondrocytes(FCs)and secondary condylar chondrocytes(CCs)at physiological and different pharmacological concentrations in vitro. Methods Isolate and culture FCs and CCs in 1-week-old female SD rats,which were divided in vitro into six groups:(1)blank control group(without treatment);(2)vehicle group(treated with 0.01% alcohol);group 3,4,5,and 6 were treated with 1,25(OH)2 D3 at the concentration of 0.1,1,10,and 100 nmol/L. After 24 h,CCK8 assay was used to measure its effect on the proliferation ability of two kinds of chondrocytes. Proliferating cell nuclear antigen(PCNA),collagen Ⅱ(COLⅡ)and collagen X(COL X)were determined by qRT-PCR and Western blot.Results(1)CCK8 results showed that 1,25(OH)2 D3 inhibited the proliferation of two kinds of chondrocytes in a concentration-dependent manner,and the inhibition rate on CCs was more obvious than that on FCs. The results of PCNA were basically consistent with them.(2)Compared with the control group,at the physiological concentration of 0.1 nmol/L,the effect of 1,25(OH)2 D3 had no statistically significant difference on the expression of COLⅡ in the two types of chondrocytes(P>0.05). At pharmacological concentration of 1,10,and 100 nmol/L,1,25(OH)2 D3 could up-regulate the expression of COL Ⅱ in the two types of chondrocytes,with the optimal concentration of FCs at 10 nmol/L,while CCs showed a concentration-dependent increase.(3)Compared with the control group,the effect of 1,25(OH)2 D3 had no statistically significant difference on the expression of COLX m RNA and protein levels in two chondrocytes(P>0.05). Conclusion The 1,25(OH)2 D3 inhibits chondrocyte proliferation in a concentrationdependent manner. At pharmacological concentration,1,25(OH)2 D3 can promote chondrocyte maturation,and it has a greater effect on CCs than on FCs. It has no effect on calcification of two types of chondrocytes.