低能量激光照射对人脂肪基质细胞增殖分化的影响

目的： 研究低能量激光照射（low level laser irradiation，LLLI）的照射剂量对人脂肪基质细胞（human adipose-derived stromal cells，hASCs）增殖与分化作用的影响，为LLLI进一步在骨组织工程中的应用奠定基础。方法： 将P4代hASCs接种于孔板中，24 h后利用半导体激光器（980 nm，100 mW~12 W）连续照射4 d。以增殖培养基为空白对照组(PM组)，实验组接受2、4、6、8 J/cm2的激光照射，连续7 d进行细胞计数试剂盒（cell counting kit-8，CCK-8）增殖检测。当细胞达到70%的融合后将一半细胞更换为成骨培养基（osteogenic medium，OM），根据培养基及累计接受的激光总能量密度分为6组：PM、OM、PM+LLLI2 J/cm2、OM+LLLI2 J/cm2、PM+LLLI4 J/cm2、OM+LLLI4 J/cm2。于第7天进行碱性磷酸酶（alkaline phosphatase，ALP）染色及定量检测，于第14、21天进行矿化沉积检测，于第7、14天进行实时定量PCR检测成骨相关基因的表达，探究低能量激光对hASCs成骨分化的影响,结果采用SPSS 19.0软件进行统计学分析。结果： PM+LLLI4 J/cm2组细胞增殖速率最快，OM各组的ALP染色均呈阳性表达，且染色深度随激光能量密度的增高而加重。第14天时，OM各组随激光照射能量升高，矿化结节体积明显增大、数目增多；第21天时，OM组间茜素红染色结果无明显差异。ALP及茜素红定量结果均与相应染色结果趋势一致。hASCs实时定量PCR结果提示，LLLI可促进ALP和Runx2的表达，且促进作用与照射剂量呈正相关。结论： LLLI具有促进hASCs增殖分化的作用，且在一定剂量范围内，这种促进作用会随照射剂量的增大而加强，当达到峰值后随照射剂量的增大而减弱。

Effect of low-level laser irradiation on proliferation and osteogenic differentiation of human adipose-derived stromal cells

Objective:To examine the in vitro effects of low-level laser irradiation (LLLI) on proliferation and differentiation of human adipose-derived stromal cells(hASCs).Methods: Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (980 nm;100 mW-12 W power output).The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated for four consecutive days with laser doses of 2,4,6 or 8 J/cm2,the cells without irradiation were used as controls.Half of the cells were changed to osteogenic medium (OM) when they had grown to 70％ confluence.The hASCs both with and without osteogenic supplements were divided into three groups,and each group was irradiated at doses of 0,2 and 4 J/cm2.In order to examine the in vitro effects of LLLI on osteogenic differentiation of hASCs,the alkaline phosphatase activity was assessed on day 7,and alizarin red staining (AR-S) and quantitative detection were assessed on days 14 and 21.The expression of osteoblast master genes (ALP and Runx2) were tested on days 7 and 14.Results: The proliferation medium(PM)+LLLI4 J/cm2 group had the highest multiplication rate.In the groups with osteogenic supplements,LLLI increased alkaline phosphatase activity and mineralized nodule formation,and stimulated the expression of ALP and Runx2.Furthermore,the effect became more obvious at high dose.Conclusion: Our data demonstrated that hASCs proliferation and osteogenic differentiation were enhanced by LLLI.With the increase of laser dose,the effect of LLLI would be enhanced at first,and then be decreased after reaching a peak.