目的 探讨光生物调节治疗(PBM)不同波长激光诱导骨髓间充质干细胞(BMSCs)成软骨分化的作用差异。方法 实验采用第二代BMSCs,于传代后第1天开始进行近距离(7 cm)激光照射治疗。实验分为四组,分别为对照组、660 nm组、810 nm组、1 064 nm组。对照组不进行光生物治疗,激光诱导各组单次能量密度为4 J/cm2,照射时间15 min;成软骨诱导培养后,每3 d重复照射1次,持续21 d。通过CCK-8法评估细胞增殖活性,甲苯胺蓝染色检测糖胺聚糖(GAG)沉积,qRT-PCR和Western Blot分别分析成软骨标志基因(Aggrecan、Col2A1、GAG)的转录及蛋白表达水平。比较三组波长下的细胞增殖活性、糖胺聚糖(GAG)沉积 、 软骨标志基因(Aggrecan、Col2A1、GAG)的转录及蛋白表达水平。结果 CCK-8结果显示,与对照组相比,三种波长激光治疗下的BMSCs增殖率明显升高,其中1 064 nm组促增殖作用显著优于660 nm与810 nm组(P< 0.05);甲苯胺蓝染色显示,经光生物治疗后,BMSCs细胞外基质异染区域与对照组相比明显扩大,各组GAG分泌明显增加;与对照组相比,光生物治疗组Aggrecan、col2A1蛋白表达水平明显升高(P<0.05),Aggrecan、col2A1、GAG的mRNA表达水平显著提升(P<0.05),其中1 064 nm组激光作用效果最为显著。结论 特定波长(尤其是1 064 nm)的PBM可显著优化BMSCs的软骨分化效能,为组织工程中软骨再生策略的优化提供了实验依据。
Abstract
Objective To investigate the variances in the effects of photobiomodulation therapy (PBM) with different wavelength lasers on chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The second-generation BMSCs were used in the experiment, and laser irradiation treatment was initiated on the first day after passage at a close distance of 7 cm. The experiment was divided into four groups:a control group,a 660 nm group,an 810 nm group, and a 1064 nm group. The control group did not undergo photobiomodulation therapy. The single energy density of laser induction for each group was 4 J/cm2, and the irradiation duration was 15 minutes. After chondrogenic induction culture,the irradiation was repeated every 3 days for 21 days. Cell proliferation activity was evaluated by the CCK-8 method, and glycosaminoglycan (GAG) deposition was detected through toluidine blue staining. The transcription and protein expression levels of chondrogenic marker genes (Aggrecan, Col2A1, GAG) were analyzed by qRT-PCR and Western Blot,respectively. The cell proliferation activity, glycosaminoglycan (GAG) deposition, and the transcription and protein expression levels of chondrogenic marker genes (Aggrecan, Col2A1, GAG)in the three groups under the different wavelengths were compared. Results The CCK-8 results showed that compared with the control group,the proliferation rate of BMSCs under the three wavelengths of laser treatment significantly increased, among which the proliferation-promoting effect of the 1064 nm group was notably superior to that of the 660 nm and 810 nm groups (P＜0.05). The toluidine blue staining revealed that after photobiomodulation therapy,the extracellular matrix metachromatic area of BMSCs was significantly expanded compared to the control group,and the secretion of GAG in each group significantly increased. Compared with the control group, the protein expression levels of Aggrecan and Col2A1 in the photobiomodulation therapy groups were significantly elevated (P＜0.05), and the mRNA expression levels of Aggrecan, Col2A1, and GAG were significantly enhanced (P＜0.05), with the 1064 nm group demonstrating the most significant effect. Conclusions Specific wavelengths (especially 1064 nm) of PBM can significantly optimize the chondrogenic differentiation efficacy of BMSC,providing experimental basis for the optimization of cartilage regeneration strategies in tissue engineering.
关键词
光生物调节 /
低水平激光 /
成软骨分化 /
细胞实验 /
间充质干细胞
Key words
photobiomodulation /
low-level laser /
chondrocyte differentiation /
cell experiment /
mesenchymal stem cell
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