摘要：
Objective To study the effects of differential strain on the proliferation of osteoblasts and the expression of inducible nitric oxide synthase mRNA,and to explore the molecular mechanism of bone lengthening.Methods The osteoblasts were collected from the skull of newborn SD rats and cultured,and then randomly divided into 4 groups (8 samples for each group):group A,group B,group C and group D.In groups A,B and C and D,the cells were treated with a tensile force of 1 000,2 000 and 3 000 and 0 μstrain respectively.The period of mechanical stimulation was 48 h in all the four groups.The proliferation ratio of osteoblasts was measured using methyl thiazol tetrazolium (MTT) reduction assay.The cells were collected and the total RNA was obtained.The expression of inducible nitric oxide synthase (iNOS) mRNA was detected by reverse transcription-polymerase chain reaction,and the amount of NO in the culture medium was examined by the method of nitric acid reduction enzyme.Results The proliferation ratio of osteoblasts in groups A,B,C and D was (28.23 ± 1.38) ％、(37.51 ± 4.08) ％、(21.59 ± 1.54) ％、(27.96 ± 2.22) ％ respectively.The expression of iNOS mRNA in groups A,B,C and D was 0.490 ±0.011,0.543 ±0.048,0.457 ±0.012,and 0.486 ±0.009 respectively.The level of NO in the culture medium of groups A,B,D and D was (14.47 ±0.39),(16.47 ±0.38),(12.28 ±0.41),and (14.32 ± 0.37) μmol/L respectively.As compard with group D,there were significant differences in the proliferation ratio of osteoblasts,the expression of iNOS mRNA,and level of NO in groups B and group C (P ＜ 0.05).There was no significant defference between group A and group D (P ＞ 0.05).Conclusion It demonstrates that tensile force of 2 000 μstrain increases significantly the proliferation of osteoblasts and the expression of iNOS mRNA.Tensile force of 3 000 μstrain declines significantly the proliferation of osteoblasts and the expression of iNOS mRNA.Under right strain,the high expression of iNOS mRNA is one of the molecular mechanisms of bone lengthening.%目的 观察不同的机械牵张应力对成骨细胞增殖和诱生型一氧化氮合酶(iNOS)基因表达的影响,探讨骨延长的分子生物学机制.方法 从新生SD大鼠颅盖骨分离培养成骨细胞,104个/cm2密度接种至细胞加力板上,随机分为4组,每组8份.A组采用1 000 μstrain的牵张应力(strain为细胞加力板变形量的数值,是四点弯曲细胞力学加载仪的计量单位);B组采用2000 μstrain的牵张应力;C组采用3 000 μstrain的牵张应力;D组作为空白对照,采用0μstrain的牵张应力.4组牵张应力的刺激时间均为48 h.噻唑蓝(MTT)法测定成骨细胞的增殖率;提取成骨细胞RNA,反转录-聚合酶链反应(RT-PCR)一步法分析成骨细胞iNOS mRNA表达,硝酸还原酶法测定细胞培养上清液中一氧化氮(NO)的含量.结果 A、B、C、D组细胞增殖率分别为(28.23±1.38)％、(37.51±4.08)％、(21.59±1.54)％、(27.96±2.22)％;成骨细胞iNOS mRNA基因表达比值分别为0.490 ±0.011、0.543±0.048、0.457 ±0.012、0.486±0.009;细胞培养上清液中NO含量分别为(14.47±0.39)、(16.47 ±0.38)、(12.28 ±0.41)、(14.32±0.37) μmol/L.B、C组与D组比较,成骨细胞增殖率、iNOS mRNA基因表达和细胞培养上清液中NO的含量,差异均有统计学意义(P＜0.05);A组与D组比较差异无统计学意义(P＞0.05).结论 2000 μstrain的牵张应力促进成骨细胞增殖和iNOS基因表达,3 000 μstrain则抑制成骨细胞增殖和iNOS基因表达.合适的牵张应力下,iNOS基因的高表达是骨延长的分子生物学机制之一.
