不同辐照时间低强度脉冲超声促进人成骨细胞生物学效应研究  

Effects of low intensity pulsed ultrasound on biological activity of osteoblasts at different exposure time

作　　者：祖海越 易雪婷 

机构地区：[1]大连大学附属中山医院骨科,辽宁大连116000 [2]大连大学附属中山医院超声碎石科,辽宁大连116000

出　　处：《生物医学工程与临床》2017年第4期356-362,共7页Biomedical Engineering and Clinical Medicine

摘　　要：目的研究低强度脉冲超声(LIPUS)对人成骨细胞FOB1.19生物学效应的影响并明确其最适辐照时间。方法选用频率为1.5 MHz、强度为30 m W/cm^2的LIPUS以不同时间(0、10、20、30、40、50 min)辐照人成骨细胞FOB1.19,以CCK-8法分别检测不同辐照时间并培养24、48、72 h时成骨细胞增殖情况,初步筛选出LIPUS作用于成骨细胞的相对最适辐照时间范围;并在此最适辐照时间范围内,通过流式细胞仪检测不同辐照时间(20、25、30 min)后培养72 h对细胞周期的影响;通过碱性磷酸酶染色检测不同辐照时间后培养24、72 h对细胞矿化能力的影响,通过实时定量聚合酶链反应(PCR)检测不同辐照时间对细胞内骨形成蛋白(BMP)-2的相对表达量的影响。结果经过LIPUS辐照后培养不同时间观察到成骨细胞增殖能力相比阴性对照组显著提高,LIPUS辐照时间在20~30 min内为相对最适辐照时间范围。细胞周期增殖指数(PI)在每天辐照25 min并培养72 h实验组高于阴性对照组[(0.57±0.01)%vs(0.45±0.02)%],差异有显著统计学意义(P<0.01);碱性磷酸酶阳性率在辐照25 min并培养细胞72 h最为明显,实验组高于阴性对照组[(67.05±3.46)%vs(28.37±4.12)%,P<0.01]。成骨细胞内BMP-2的相对表达量显著提高,其中以LIPUS辐照25 min、培养72 h时促进人成骨细胞生物学效应最为显著,培养72 h实验组BMP-2相对含量高于阴性对照组(4.16±0.13)倍,差异有显著统计学意义(P<0.01)。结论不同辐照时间LIPUS可促进成骨细胞生物学活性,但是其促进成骨细胞的效果不一,其中以辐照时间25 min/d的LIPUS辐照下促进成骨的效果最佳。Objective To study the effect and optimum exposure time of low intensity pulsed ultrasound（LIPUS） for enhancing biological effect of human FOB1.19 osteoblast. Methods LIPUS was selected at 1.5 MHz frequency, 30 m W/cm^2 intensity with different exposure time（0, 10, 20, 30, 40, 50 minutes） for the treatment of human osteoblast. CCK-8 method was used to detect the proliferation and incubation for 24, 48 and 72 hours of osteoblasts under different exposure time, and then the effect of LIPUS was screened to select relative optimum exposure range. Within the relative optimum exposure time range, the effects of different irradiation time（20, 25, 30 minutes） on osteoblast cell cycle were tested by flow cytometer. Alkaline phosphatase staining was used to detect the effect of 24, 72 hours incubation on cell mineralization after different irradiation time, and relative expression of bone morphogenetic protein（BMP）-2 was detected by real-time quantitative polymerase chain reaction（PCR）.Results Compared with negative control group, the osteoblast proliferation of experimental group was significantly increased after LIPUS treatment, and the relative optimum exposure range was 20-30 minutes. The cell cycle proliferation index（PI） of experimental group was significantly higher than that of negative control group[（0.57 ± 0.01） % vs（0.45 ± 0.02） %, P 〈 0.01],and the difference was statistically significant. The 25 minutes LIPUS exposure for 72 hours incubation was the most efficient,and the positive rate of alkaline phosphatase was significantly higher than that of negative control group[（67.05 ± 3.46） % vs（28.37 ± 4.12） %, P 〈 0.01], the relative expression of BMP-2 in osteoblast increased significantly, and 25 minutes LIPUS exposure for 72 hours was the most efficient, and the relative content of BMP-2 was significantly higher than that of negative control group[（4.16 ± 0.13） times], and the difference was statistically significant（P 〈 0.01）. Conclusion LIPUS can promo

关 键 词：低强度脉冲超声 骨组织工程 碱性磷酸酶 骨形成蛋白-2 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]