不同声强低强度脉冲超声对SD大鼠骨髓间充质干细胞迁移的影响

目的探讨不同声强低强度脉冲超声(LIPUS)辐照对骨髓间充质干细胞(BMSCs)体外迁移的影响。方法将BMSCs分为空白对照组、30mW/cm~2组、60mW/cm~2组及90mW/cm~2组,对空白对照组仅行LIPUS假辐照操作,而对其余3组以相应声强进行辐照。以细胞划痕实验分析LIPUS对划痕愈合的促进作用,并通过MTT活性检测排除细胞增殖能力的干扰。采用transwell迁移实验评价各组BMSCs的迁移能力。通过FITC-鬼笔环肽染色检测F-肌动蛋白(Factin)表达结果 LIPUS辐照(空白对照组假辐照)后24h及48h,30mW/cm~2组、60mW/cm~2组、90mW/cm~2组及空白对照组间划痕面积差异均有统计学意义(F=26.559、106.110,P均0.001),且空白对照组划痕面积[辐照后24h:(0.93±0.26)mm~2,辐照后48h:(0.70±0.11)mm~2]最大,30mW/cm~2组[辐照后24h:(0.47±0.21)mm~2,辐照后48h:(0.19±0.10)mm~2]最小;而辐照后即刻各组间划痕面积差异无统计学意义(F=2.921,P=0.063)。LIPUS辐照(空白对照组假辐照)后即刻、24h及48h各组间吸光度差异均无统计学意义(F=1.616、0.720、1.408,P=0.196、0.544、0.378)。各组间穿过transwell小室上室的BMSCs细胞计数差异有统计学意义(F=43.145,P0.001),且30 mW/cm~2组细胞计数[(212.53±35.32)个]最大,空白对照组[(89.53±19.27)个]最小。F-actin染色显示,LIPUS辐照后BMSCs微丝增粗变长,数量增多。各组间相对荧光强度差异有统计学意义(F=64.350,P0.001),且30 mW/cm~2组相对荧光强度(125.43±17.43)最大,空白对照组(51.94±12.76)最小。结论 LIPUS可促进BMSCs体外迁移,声强为30mW/cm~2时促进效应最明显。

Impact on migration of SD rat bone marrow mesenchymal stem cells treated with different intensities of low intensity pulsed ultrasound

Objective To investigate the impact of low intensity pulsed ultrasound (LIPUS) with different intensities on the migration of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods BMSCs were divided into control group, 30 mW/cm² group, 60 mW/cm² group and 90 mW/cm² group. Control group was treated by sham LIPUS exposure, and the other three groups were treated by LIPUS with corresponding intensities. The impact of LIPUS on scratch healing was tested with scratch assay, and the interference of proliferation was eliminated with MTT assay. The migration of BMSCs were evaluated with transwell migration assay. The expression of F-actin was analyzed with fluorescein isothiocyanate (FITC) fluorescent coloration. Results 24 h and 48 h after LIPUS exposure, there were statistical differences of scratch area among groups (F=26.559, 106.110, both P<0.001),and the scratch area of control group was the largest ([0.93±0.26)mm² of 24 h after LIPUS exposure and[0.70±0.11] mm² of 48 h after LIPUS exposure), while that of 30 mW/cm² group was the smallest ([0.47±0.21] mm² of 24 h after LIPUS exposure and[0.19±0.10] mm² of 48 h after LIPUS exposure). There was no statistical difference of scratch area among the four groups immediately after LIPUS exposure (F=2.921, P=0.063). MTT assay results showed there was no statistical difference of absorbance among the four groups immediately, nor 24 h, 48 h after LIPUS exposure (F=1.616, 0.720, 1.408; P=0.196, 0.544, 0.378). Significant difference was found in the number of cells migrated through the transwell chamber among the four groups (F=43.145, P<0.001), and the cell number of 30 mW/cm² group was the largest (212.53±35.32), while that of the control group was the least (89.53±19.27). F-actin fluorescence staining results showed the morphology of F-actin was changed after LIPUS exposure. The cytoskeleton became narrow and elongated. Statistical difference of relative fluorescence intensity was found among the four groups (F=64.350, P<0.001).The relative fluorescence intensity of 30 mW/cm² group was the largest (125.43±17.43), while that of control group was the least (51.94±12.76). Conclusion LIPUS can promote the migration ability of BMSCs in vitro with the best intensity was 30 mW/cm².