软骨细胞光生物调节作用的体外实验

Photobiomodulation on Chondrocyte Proliferation: in Vitro Evaluation

研究了低强度激光对兔软骨细胞增殖和变异的潜在性影响。选取3周龄新西兰白兔分离软骨细胞,在新生牛血清(NCS)体积分数分别为10%,5%,2.5%和无血清4种培养媒介中培养,采用功率6.5mW的He-Ne激光辐照(HNI)(5.74mW/cm2)软骨细胞。用四氮唑复合物法(XTT)检测细胞的活性。通过检测羟脯氨酸的(Hrp)含量了解细胞合成胶原的能力。在激光共聚焦显微镜下观察吖碇橙标记软骨细胞形态及DNA的表达。在营养缺乏培养状态中(5%,2.5%新生牛血清),辐照时间为16,30,45min的照射组细胞数量显著增加,具有明显的光生物调节作用(PBM);其中最佳辐照时间为30min,实际最佳辐照能量密度为9.42J/cm2;当光生物调节作用显著时,软骨细胞的DNA及胶原的合成也明显增强。He-Ne激光照射兔软骨细胞后在营养缺乏的媒介中增殖明显,所产生的光生物调节作用仅限在一定的剂量范围内产生,这一特性可为临床使用提供参考。

There have been controversial reports on photobiomodulation on cartilage healing so that the research on the potential effect of low intensity He-Ne laser irradiation （HNI） on the proliferation and variation of rabbit cartilage cells in vitro was systematically done in this paper. The chondrocytes isolated from the cartilage sample of 3-week-old New Zealand white rabbits were cultured with newborn calf serum （NCS） at 0%, 2.5%, 5%, and 10%, irradiated by HNI at 5.74 mW/cm^2 for 2, 8, 16, 30 and 45 min per day for 6 days, and then incubated till the 13th day. The proliferation and collagen synthesis were assessed by XTT assay and hydroxyproline （Hrp） content measurement, respectively. The DNA expression was observed by acridine orange stain and confocal laser scanning microscope. The chondrocyte morphology and ultrastructure have been observed on the 9th and 13th day after the first HNI by scan electron microscope. There is no significant photobiomodulation （PBM） on the proliferation of the chondrocytes （PPC） cultured with NCS at 10%. There is PPC cultured with NCS at 2.5% or 5%, and the effects are significant when the cells were irradiated by HNI for 16, 30 and 45 min, respectively, among which the radiation time 30 min corresponds the optimum dose 9.42 J/cm^2. Hrp secretion increased steadily with days in NHI group. When the photobiomodulation is significant, the chondrocyte DNA expression was significant, and its morphological characteristics have no significant differences from the one cultured with NCS at 10%. There might be photobiomodulation on the proliferation of the chondrocytes cultured in nutritional deficit condition and irradiated by HNI at limited dosage, which is of clinic importance.