低强度脉冲超声波对兔骨髓基质细胞增殖和分化的影响

Effects of low intensity pulsed ultrasound on the proliferation and differentiation of rabbit bone marrow stromal cells

背景:研究发现低强度脉冲超声波对骨折愈合具有明显的促进作用,但其机制尚不明确。目的:观察低强度脉冲超声波对体外分离培养的兔骨髓基质细胞增殖和分化的影响。设计、时间及地点:随机分组设计,对比观察,于2008-06/2009-01在解放军第四军医大学西京医院完成。材料:4周龄新西兰白兔2只,体质量0.8～1.0kg,用于骨髓基质细胞的分离、培养。方法:将骨髓基质细胞以2×104个/孔的密度接种于6孔培养板中,采用辐射强度为30mW/cm2的脉冲超声波作用于体外培养的兔骨髓基质细胞,根据低强度脉冲超声波每天作用时间分为4组:5min/d组,10min/d组,20min/d组和空白对照组(无超声波处理)。主要观察指标:原代及传代细胞形态变化;四甲基偶氮唑盐法检测低强度脉冲超声波作用1,3,7d各组细胞吸光度的变化;作用3,7d后各组骨髓基质细胞碱性磷酸酶活性的变化;作用7d各组骨髓基质细胞分泌骨钙素水平的变化。结果:①原代骨髓基质细胞7d后融合成片,多数细胞呈梭形或多角形;第3代细胞经低强度脉冲超声波处理后,各组细胞形态较空白对照组无明显变化。②四甲基偶氮唑盐检测,各组细胞吸光度值均随时间延长而增加;且各实验组不同时间点吸光度值均明显高于空白对照组(P<0.05)。③与空白对照组相比,20min/d组低强度脉冲超声波作用3,7d后,单位质量骨髓基质细胞总蛋白碱性磷酸酶活性均显著升高(P<0.05),其他2组与空白对照组相比,差异无显著性意义(P>0.05)。④与空白对照组相比,20min/d组低强度脉冲超声波作用7d时,骨髓基质细胞分泌骨钙素显著增加(P<0.05),而其他2组骨钙素的分泌量与空白对照组相比,差异无显著性意义(P>0.05)。结论:低强度脉冲超声波能够显著促进兔骨髓基质细胞的增殖,其诱导分化作用与作用时间有关。

BACKGROUND： It was discovered that low intensity of pulsed ultrasound （LIPUS） could obviously improve the healing of bone fracture, but the related mechanism was unclear. OBJECTIVE： To investigate the biological effects of LIPUS on the proliferation and differentiation of rabbit bone marrow stromal cells （BMSCs） in vitro. DESIGN, TIME AND SETTING： A randomized study was performed from June 2008 to January 2009 at the Xijing Hospital of the Fourth Military Medical University of Chinese PLA. MATERIALS： Two New Zealand white rabbits, aged 4 weeks old, weighing 0.8 to 1.0 kg, for the isolation and culture of BMSCs. METHODS： BMSCs were incubated in a 6-well plate at 2×10^4 cells/well. LIPUS of 30 mW/cm2 was used to stimulate the cultured rabbit BMSCs. BMSCs were assigned to 4 groups, 5 min/d group, 10 min/d group, 20 min/d group and blank control group （no ultrasonic treatment）. MAIN OUTCOME MEASURES： Morphological changes in primary and subcultured cells were measured. Changes in absorbance in each group were detected at 1, 3 and 7 days following ultrasonic treatment using MTT assay. Alkaline phosphatase activities were determined at 3 and 7 days in each group. Osteocalcin levels secreted from BMSCs were detected in each group at 7 days following treatment. RESULTS： Following 7 days of culture, primary cultured BMSCs were confluent, and most cells were spindle or polygonal. Cells at passage 3 following LIPUS did not change in morphology in 5 min/d group, 10 min/d group, 20 min/d group compared with the blank control group. MTT assay indicated that absorbance was increased in each group over time. Absorbance was significantly greater in the 5 min/d group, 10 min/d group, 20 min/d group compared with the blank control group at various time points （P 〈 0.05）. Compared with the blank control group, alkaline phosphatase activities were significantly higher following 3 and 7 days of LIPUS treatment in the 20 min/d group （P 〈 0.05）. No significant difference in alkaline phosphatase activities was determined when comparing 5 min/d group, 10 min/d group to the blank control group （P 〉 0.05）. Compared with the blank control group, osteocalcin levels were significantly increased in the 20 min/d group following 7 days of LIPUS treatment （P 〈 0.05）. No significant difference in osteocalcin levels was determined when comparing 5 min/d group, 10 min/d group to the blank control group （P 〉 0.05）. CONCLUSION： The low intensity pulsed ultrasound could stimulate the proliferation of rabbit BMSCs. The cell differentiation-induced effect of LIPUS on BMSC depends on the exposure time.