红光LED照射促间充质干细胞增殖分化的量效关系研究

Study on Dose-Effect Relationship of 650nm LED Illumination for Mesenchymal Stem Cell Proliferation and Differentiation

目的探讨红光LED照射促进C3H10T1/2细胞增殖的量效关系以及对TNF-α抑制的成骨分化的影响。方法(1)细胞增殖生物学研究。将C3H10T1/2细胞传代培养后,随机分为对照组(A组)、TNF-α干预组(B组)和TNF-α+LED光照组(L组),其中L组再根据不同光照剂量分为LA~LI 9个亚组,照射波长650 nm,每组6个样本。A组细胞正常培养,B组仅接受TNF-α孵育;L组在TNF-α孵育12 h后进行LED光照处理,每日一次,分别在干预2、4、6次后(48、96、144 h),采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(商品名:噻唑蓝)比色法(简称MTT比色法)检测细胞活力,评估光照促进细胞增殖的量效关系。(2)诱导成骨分化实验。实验分组同细胞增殖实验,采用最佳光照剂量(功率密度1.4 m W/cm^(2),照射时间10 min)干预。在诱导成骨分化第7和21天时分别进行碱性磷酸酶染色、茜红素S染色检测C3H10T1/2细胞成骨分化情况,分析光照对成骨分化的影响。结果(1)红色LED的光照具有促进C3H10T1/2细胞增殖的生物学效应。其中1.4 m W/cm^(2)×10 min剂量光照组在各时间点都表现出显著性增殖促进作用。(2)使用1.4 m W/cm^(2)×10 min剂量对TNF-α干预成骨分化的细胞进行光照,能显著增加细胞成骨诱导后的碱性磷酸酶活性及矿化沉积。结论650 nm红光LED照射可部分抵抗TNF-α干预对间充质干细胞增殖、分化功能的抑制作用。在低剂量范围内,光照功率密度越大光生物调节作用效果越显著。

Objective To investigate the dose-effect relationship of 650 nm red LED irradiation for the proliferation and differentiation of C3H10T1/2 cells inhibited by TNF-α.Methods(1)Study on cell proliferation biology.C3H10T1/2 cells were randomly divided into three groups:the control group(group A),TNF-αintervention group(group B),and TNF-α+LED light group(group L).The cells in group L were further divided into 9 subgroups by light doses used,6 samples in each subgroup,and 650 nm irradiation wavelength was adopted.The cells in group A were cultured normally,and the cells in group B were only incubated with TNF-α.The cells in group L were treated with LED light after TNF-αincubation for 12 hours,once a day.After 2,4 and 6 times of intervention(48,96 and 144 h),the cell viability was measured with Thiazolyl Blue Tetrazolium Bromide(MTT)colorimetric method,and the dose-effect relationship between light intervention and cell proliferation was evaluated.(2)Osteogenic differentiation experiment.The experimental grouping was conducted in the same way as in experiment.The optimal light dose(power density:1.4 mW/cm^(2) and irradiation time:10 min)was used for light intervention.Osteogenic differentiation of C3H10T1/2 cells was detected with alkaline phosphatase staining and alizarin S staining 7 and 21 days after the osteogenic differentiation,and the effect of light on osteogenic differentiation was analyzed.Results(1)Red LED illumination promoted the proliferation of C3H10T1/2 cells.The 1.4 mW/cm^(2)×10 min dose group showed a significant promotion at each time point.(2)Following osteogenic induction,illumination of 1.4 mW/cm^(2)×10 min dose significantly promoted the alkaline phosphatase activity and mineralization that had been inhibited by TNF-α.Conclusions LED illumination at 650 nm will partially counteract the inhibitory effect of TNF-αintervention on the proliferation and differentiation of mesenchymal stem cells.For low doses,the greater the light power density,the greater the effect of photobiomodulation.