低分子牛骨多肽制备及对大鼠成骨细胞的影响

目的从牛骨制备低分子多肽,并观察其对体外培养新生大鼠的成骨细胞增殖、碱性磷酸酶(ALP)活性和Ⅰ型胶原mRNA表达的影响。方法采用冷冻离心、等电点沉淀方法去除骨胶原,超滤法得到相对分子质量(Mr)10000以下的牛骨多肽,经Sephadex G-10脱盐,浓缩后用Tricine-SDS-PAGE电泳对Mr进行检测。采用四甲基偶氮唑盐(MTT)比色法测定牛骨多肽对成骨细胞增殖的影响,ALP检测试剂盒测定细胞的ALP活性,RT-PCR法检测细胞中Ⅰ型胶原mRNA表达。结果建立了牛骨多肽的制备方法。低浓度牛骨多肽组与实验对照组相比对成骨细胞的增殖不显著(P>0.05),高浓度具有显著的增殖作用(P<0.05),并呈现出剂量依赖性;中、高浓度(50~400μg/mL)牛骨多肽组可提高成骨细胞ALP的活性(P<0.05),低浓度组(10μg/mL)提高ALP的活性不显著(P>0.05);牛骨多肽能提高Ⅰ型胶原mRNA表达。结论牛骨多肽的制备工艺简单,适合规模化生产;牛骨多肽能促进成骨细胞增殖、ALP活性和Ⅰ型胶原mRNA的表达。

Preparation of low molecular mass polypeptides from bovine bone and it''''s effects on osteoblasts of rat

Purpose To prepare low molecular mass polypeptides from bovine bone and to study the effects on the proliferation,alkaline phosphatase activity and expression mRNA of collagenⅠ in osteoblasts of rat. Methodswe removed degenerative collagen from bovine bone solution by freezing centrifugalization and isoelectric point precipitation,obtained polypeptides from bovine bone whose molecular mass was below 10 000 by ultrafiltration,and desalted by Sephadex G-10 chromatography.The polypeptides were detected by Tricine-SDS-PAGE.The proliferation of osteoblast was tested by MTT and the ALP activity of osteoblasts was tested by AKP REAGENT and the expression of mRNA of collagenⅠ of osteoblasts was tested by RT-PCR.ResultsThe preparation of low molecular mass polypeptides from bovine bone was established.The low concentration was not significant compared with blank(P>0.05),while high concentration was significant(P<0.05) and showed dependence with the polypeptides dose on proliferation.The low concentration was not significant compared with blank(P>0.05),while middle and high concentrations were significant(P<0.05).The polypeptides can enhance the expression of mRNA of collagenⅠ of osteoblasts.ConclusionThe preparation technique is simple and suitable for factory manufacture.The experimentation results show that the polypeptides can enhancement proliferation,the ALP activity,and the mRNA of expression ofⅠcollagen of osteoblasts.