羊骨髓肽及其钙螯合物对成骨细胞增殖和活性的影响

Function of Bone Marrow Peptide and Its Calcium-Chelated Form in the Proliferation and the Osteogenic Activity of Cultured Osteoblasts

成骨细胞在骨代谢过程中发挥成骨作用,笔者制备羊骨髓肽钙螯合物(MP-Ca),探究其对体外培养成骨细胞的增殖及活性的影响,并将其效果同羊骨髓肽(MP)进行比较。制备的MP-Ca经傅里叶红外光谱仪进行扫描鉴定;从新生SD大鼠颅骨中分离纯化的成骨细胞经碱性磷酸酶(ALP)和茜素红染色鉴定;在培养液中添加不同浓度的受试物,CCK-8法确定最佳作用浓度。以17-β雌二醇(10~2μg·mL^(-1))为阳性对照,通过测定各组细胞OD_(450 nm)和ALP、骨钙素(BGP)的含量,考察MP、MP-Ca在最适作用浓度(10~3μg·mL^(-1))下对成骨细胞增殖和活性的影响。结果表明,MP和MP-Ca的红外光谱图未完全重叠,吸收峰发生了明显的位移变化,说明MP与CaCl_2反应形成了MP-Ca。分离的成骨细胞经ALP染色呈阳性,胞内可见大量黑褐色颗粒;茜素红染色后细胞呈深红色,并形成钙化结节。MP-Ca处理组的OD_(450 nm)和ALP、BGP水平均显著(P <0. 05或P <0. 01)高于MP处理组,但均显著(P <0.05或P <0. 01)低于雌激素组。综上,尽管效果不及雌激素,MP-Ca和MP均可促进体外培养成骨细胞的增殖并增强其成骨活性,效果MP-Ca优于MP。

Osteoblasts play bone-forming roles in the process of bone metabolism.Calcium-chelated bone marrow peptides(MP-Ca)was prepared and its effects on the proliferation and osteogenic activity of osteobalst(OB)were investigated and compared with calcium-free peptides of sheep bone marrow(MP).Prepared MP-Ca was identified via Fourier transform infrared spectrometer.The osteoblasts isolated from the skull of newborn SD rats were identified through alkaline phosphatase(ALP)and alizarin red staining.Different concentrations of test substance,including the positive control17β-estradiol,were added to the medium and the CCK-8method was used to determine the optimal concentration for the subsequent study.The proliferation and the osteogenic activity of OB were expressed as OD450nm and the content of ALP and osteocalcin(BGP)in cell lysate prepared from MP-Ca,MP,estrogen treated OB cells at optimal concentrations.MP and MP-Ca showed no completely overlapped infrared spectra and apparent shifts of absorption peaks were observed,which indicated that MP-Ca was different form MP and it was prepared successfully.After ALP staining the isolated and purified OB cells appeared positive reaction.The results showed that OD450nm,ALP and BGP levels of the MP-Ca treated cells were significantly higher than those of the MP treatment(P<0.05or P<0.01),but they were significantly lower than those of17β-estradiol treated group(P<0.05or P<0.01).It can be concluded that although inferior to17β-estradiol,MP and MP-Ca were effective in promoting the proliferation of osteoblasts in vitro and enhance their bone formation activity,and MP-Ca was superior to MP.