光生物调节作用对软骨细胞基质分泌及超微结构变化的影响

Effect of photobiomodulation on extracellular matrix secretion and the chondrocytes ultrastructure

目的了解低强度He-Ne激光(HNL)照射对软骨细胞基质的分泌及其超微结构的影响。方法选取3周龄新西兰白兔分离培养软骨细胞,采用HNL照射第4代软骨细胞后,在营养缺乏的培养媒介中培养。收集激光照射后第2,4,6,8,10和12天的细胞培养液,用氯胺T消化法检测羟脯氨酸(Hrp)的含量;在HNL照射后第9天,用激光共聚焦显微镜观察吖啶橙染色后细胞内DNA及RNA含量的变化,在扫描电镜下观察软骨细胞的形态及细胞的分泌,在透射电镜下观察软骨细胞超微结构的改变。结果在营养缺乏的培养状态中,HNL对软骨细胞具有明显的光生物调节作用:(1)双因素重复测定资料的方差分析结果显示,HNL照射后软骨细胞合成胶原的能力在逐步增加,而对照组在培养至第2周开始Hrp含量明显下降;(2)吖啶橙染色荧光定量分析结果显示,HNL照射后第9天细胞的DNA含量较对照组增高;(3)HNL照射后的细胞在培养至第9天,扫描电镜下观察细胞表面仍然有较多的分泌物和突起,而对照组的细胞已伸展和变平,缺乏分泌;(4)透射电镜观察,HNL照射后第9天的细胞质内有丰富的粗面内质网,而对照组较多的细胞呈现出衰老现象,死亡细胞较多。结论HNL照射可能通过抑制营养缺乏引起内质网应激,促进Ⅱ型胶原和蛋白多糖等细胞外基质的分泌,并进一步促进细胞增殖。

Objective To study the effects of low intensity He-Ne laser irradiation （HNL） on the secretion of extracellular matrix and ultrastructure of the chondrocytes. Methods The chondrocytes isolated from the cartilage sample of 3-week old New Zealand white rabbits were cultured with newborn calf serum （NCS） , irradiated with HNL at 5.74 mW/cm^2 for 30 min daily for 6 days, and then incubated till the 13th day in nutrition-deficit medium. The type II collagen synthesis was assessed by a hydroxyproline （Hpr） content measurement on the 2nd, 4th, 6th, 8th, 10th and 12th days after the first HNL irradiation, respectively. On the 9th day after the first HNL irradiation, the DNA synthesis was observed by acridine orange staining and con-focal laser scanning microscope, and the cellular morphology and uhrastructure were observed by a scan electron microscope and a transmission electron microscope, respectively. Results The type II collagen synthesis increased steadily in HNL group. On the 9th day after the first HNL irradiation, the DNA synthesis and the matrix secretion such as type Ⅱ collagen and aggrecan were more in HNL group than those in the control group, and the structure of endoplasmic reticulum was clearer in HNL group than in control group. Conclusion HNL might inhibit nutrition-deficit induced endoplasmic reticulum stress of rabbit chondrocytes, promote the secretion of matrix such as type II collagen and aggrecan, and then promote their prolifera- tion at the observed irradiation dose.