摘要
目的:观察不同浓度重组胰岛素生长因子对成骨细胞的增殖和分化功能的影响。方法:实验于2004-01/06在广东医学院动物中心完成。无菌取出生24h内的Wistar大鼠颅盖骨,进行成骨细胞的分离、培养、增殖,取第5代鼠成骨细胞,在含体积分数0.1的胎牛血清DMEM培养基中,以配制的5种不同浓度重组胰岛素生长因子犤0(对照组),1,10,20,50μg/L犦中继续培养1,3,5,7d,反映不同浓度重组胰岛素生长因子对成骨细胞增殖的影响,采用四甲基偶氮噻唑盐法在酶标仪上检测吸光度值表示;反映成骨分化作用采用酶标仪检测碱性磷酸酶活性;反映不同浓度重组胰岛素生长因子对成骨细胞脂质代谢的影响采用分光光度计检测丙二醛。结果:①对细胞增殖的量效作用:四甲基偶氮噻唑盐法吸光度值反映细胞增殖的活性,胰岛素生长因子对成骨细胞有明显的刺激作用,能使细胞的增殖率提高。不同浓度重组胰岛素生长因子对成骨细胞均有较强的促进作用,1～20μg/L范围随着浓度的增加,吸光度值随之增高,存在剂量-效应关系,20μg/L重组胰岛素生长因子的刺激作用最强,随浓度增至50μg/L,吸光度值有所下降,重组胰岛素生长因子的刺激作用不再增加。②对细胞增殖的时效作用:重组胰岛素生长因子对鼠成骨细胞的促增殖作用还存在时间-效应的关系,随着培养作用时间的延长,吸光度值增高,作用5d后,吸光度值达到最高峰,促增殖作用最为明显,到第7天后,吸光度值下降。③早期成骨活性:不同浓度的胰岛素生长因子,能促进反映成骨细胞的成骨活性、可作为早期分化指标的骨细胞碱性磷酸酶的表达,1～20μg/L范围随浓度的增加,碱性磷酸酶的表达亦增高,20μg/L组促碱性磷酸酶的活性作用最明显,增至50μg/L,碱性磷酸酶的表达有所下降。④对丙二醛表达的影响:1～20μg/L范围随浓度的增加,丙二醛的表达随之降低,20μg/L组的作用最明显,增至50μg/L,丙二醛的表达略有升高,提示胰岛素生长因子可降低成骨细胞的脂质过氧化物水平。结论:重组胰岛素生长因子可促进成骨细胞的增殖,提高增殖率,并具有量效、时效关系。同时能促进成骨细胞进一步的分化,可促进反映早期分化指标的骨细胞碱性磷酸酶的表达,提高成骨能力,有助于骨坏死修复过程。还具有影响成骨细胞脂质代谢,降低成骨细胞的脂质过氧化物水平的作用。
AIM: To observe the effect of different-dosage recombinant insulin growth factor 1 (rhIGF-1) on the proliferation and differentiation of osteoblasts. METHODS:The experiment was completed in the Animal Center of Guangdong Medical College from January to June 2004. Under sterile condition, skullcaps of Wistar rats were extracted within 24 hours after birth for separation, culture, and proliferation. The fifth generation of osteoblasts was selected and cultured in DMEM medium containing fetal calf serum of 0.1 volume fraction to prepare five different-concentraion rhIGF-1(0,1,10,20, 50 μg/L).Then,the osteoblasts were continuously cultured in medium for 1,3,5,and 7 days.Absorbance(A value) detected with enzyme-labeled instrument after co-culture with MTT,alkaline phosphatase(ALP) activity measured with enzyme-labeled instrument and malondialdehyde(MDA) levels measured with spectrophotometer were used to reflect the effects of rhIGF on the proliferation, differentiation and lipid metabolism of osteoblasts, respectively. RESULTS: ①Dose-effect on cell proliferation: IGF exerted a remarkable role in stimulating osteoblasts, leading to an increase in the rate of cell proliferation, reflected by the A value detected with MTT. IGF with different dosage could all stimulate the osteoblasts obviously, especially IGF of 20 μg/L.The A value was positively increased when the concentration of IGF ranged from 1 to 20 μg/L,indicating that dose-effect existed, and then began to decrease until the concentration of IGF increased to 50 μg/L indicating there was no stimulation of IGF on osteoblasts. ②Time-effect on cell proliferation: The A value was also increased with the culture time. After 5 days, the A value reached the peak, indicating the effect on proliferation was the most obvious.The A value began to decrease until the 7th day.③Activity of osteoblasts in the early period: As the marker of differentiation,the activity of ALP was increased positively in relation to the concentration of IGF within 1 to 20 μg/L,especially 20 μg/L IGF,and began to decrease gradually until the concentration of IGF increased to 50 μg/L. ④Effect on the levels of MDA:The level of MDA was decreased with the increase of IGF concentration within 1 to 20 μg/L,and began to increase until IGF concentration reached to 50 μg/L,indicating that IGF could lower the level of Lipid peroxidation products in osteoblasts. CONCLUSION:Cell proliferation and differentiation in the cultured osteoblasts can be stimulated by rhIGF-1 in a dose-effect and time-effect manner, so as to promote the ability of bone formation. rhIGF can also influence the lipid metabolism of osteoblasts,and reduce the level of lipid peroxidation products in osteoblasts.
出处
《中国临床康复》
CSCD
北大核心
2005年第22期84-86,共3页
Chinese Journal of Clinical Rehabilitation
