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目的探讨波长620 nm的发光二极管(lightemitting diode,LED)对大鼠间充质干细胞增殖的生物学效应。方法用密度梯度离心法分离SD大鼠骨髓间充质干细胞,再经贴壁筛选法筛选,取生长良好的第3代SD大鼠骨髓间充质干细胞为实验对象。将波长620 nm的LED置于细胞层上方2cm处,测得光功率密度为6.67 mW/cm~2按照不同的能量密度分为4组,分别为对照组(A组)能量密度为0 J/cm~2,照射时间0 s;B组能量密度0.5 J/cm~2,,照射时间75 s;C组能量密度1 J/cm~2,照射时间150 s和D组能量密度2 J/cm~2,照射时间300 s。每天照射1次,连续照射3 d,照射时注意避免各组相互干扰和其它来源光的影响。LED照射后采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测各组细胞增殖活性;EdU(5-Ethynyl-2’-deoxyundine)细胞增殖检测试剂盒检测细胞DNA复制活性;流式细胞仪检测各组细胞周期变化。结果 SD大鼠骨髓间充质干细胞经红光照射后3 d,B、C、D三组的细胞增殖活性和DNA复制活性明显增强,与A组比较差异具有显著意义(P<0.05)。S期和G2-M期细胞数量明显增多(P<0.05),其中能量密度0.5 J/cm~2组最显著。结论采用无热损伤剂量的620 nm红光照射SD大鼠骨髓间充质干细胞3 d后,可有效促进其体外增殖水平。
Objective To investigate the biological effects of liangtemitting diode (LED) with a wavelength of 620 nm on the proliferation of rat mesenchymal stem cells. Methods SD rat bone marrow mesenchymal stem cells were isolated by density gradient centrifugation and then screened by adherence screening method. The third generation SD rat bone marrow mesenchymal stem cells with good growth were used as experimental subjects. The 620 nm wavelength LED was placed 2 cm above the cell layer and the optical power density was 6.67 mW / cm ~ 2. The energy density of the control group (A group) was 0 J / cm 2 and irradiation time 0 s; the energy density of group B was 0.5 J / cm 2 and the irradiation time was 75 s; the energy density of group C was 1 J / cm 2, the irradiation time was 150 s and the energy density of group D was 2 J / cm ~ 2, irradiation time 300 s. Irradiation once a day, continuous irradiation 3 d, irradiation to avoid interference with each group and other sources of light effects. Cell proliferation activity was detected by cell counting kit-8 (CCK-8) assay after LED irradiation. EdU (5-Ethynyl-2’-deoxyundine) cell proliferation assay kit was used to detect DNA replication activity The changes of cell cycle in each group were detected by flow cytometry. Results The proliferation activity and DNA replication activity of SD rat bone marrow mesenchymal stem cells were significantly increased on the 3rd day after irradiation with B, C and D groups, which was significantly different from that of A group (P <0.05). S phase and G2-M phase cells increased significantly (P <0.05), of which the energy density 0.5 J / cm ~ 2 group the most significant. Conclusions The SD rat bone marrow mesenchymal stem cells irradiated with 620 nm red light without heat damage for 3 days can effectively promote the proliferation of SD rat bone marrow mesenchymal stem cells.