桂枝加葛根汤对纤维环细胞的影响

Effects of intervertebral disc annulus fibrosus cells intervened in guizhigegentang

目的：探讨颈椎病的发病机理及桂枝加葛根汤防治该病的作用机制。方法：采用自制桂枝加葛根汤大鼠含药血清，对培养的椎间盘纤维环细胞进行不同浓度的药物干预，光镜和电镜观察未干预和桂枝加葛根汤含药血清不同浓度干预后纤维环细胞的细微和超微结构特点，利用荧光定量PCR检测椎间盘纤维环中主要蛋白多糖AggreeanmRNA的表达变化情况。结果：电镜观察结果显示，空白组细胞形态各异，大多呈椭圆形，部分线粒体空泡化，粗面内质网扩张；低剂量组见线粒体增多、浓缩密度增高，胞质内粗面内质网扩张，偶见脂滴或糖原；中剂量组见内质网扩张增多，密度增高，可见较多中等密度的蛋白质沉淀，线粒体增多；高剂量组见粗面内质网增多，扩张明显，镜下见较多高电子密度的蛋白质沉淀，可见较多线粒体及分泌颗粒；荧光定量PCR检测结果显示，中、高剂量组AggrecanmRNA表达的Ct值明显高于空白组fP〈0．05），低剂量组Ct值与空白组比较无明显差异，而高剂量组Ct值明显高于中剂量组（P〈0．05）。结论：（1）中、高剂量组细胞内出面内质网较空白组及低剂量组明显增多，出现电子密度较大的蛋白质沉淀，以高剂量组最为明显；（2）中、高剂量组桂枝加葛根汤大鼠含药血清能增强纤维环细胞AggrecanmRNA的表达，高剂量组效果最为明显；（3）桂枝加葛根汤在防止因纤维环细胞而引起的椎间盘退变具有重要作用。

Objeetive： To explore the pathogenesis of cervical syndrome and the mechanisms of guizhigegentang preventing cervical syndrome. Methods ： Intervertebral disc annulus fibrosus cells were Interfered whih different concentrations of drug that made by rats serum which contained guizhigegentang. Investigate the tiny and ultrastructure characteristics of induced and non-induced cells with an optical microscope and electron microscope. Then real-time quantitative PCR was used to detect changes of Aggrecan mRNA which is the main proteoglycan in intervertebral disc annulus fibrosus cells. Results the electron microscope showed： the cells in the blank group showed oval mostly, a few number of mitochondria and rough endoplasmic reticulum were vacuolation and expanded; in the low-dose chinese medicine group, the density was increased and the rough endoplasmic reticulum were expanded. Lipid drops and glycogen can be seen on occasion; in the middle-dose group, endoplasmic reticulum were increased and expanded, the density was increased, and much medium density of protein can be seen. The mitochondria were increased, the density was increased;in the high-dose group, the rough endoplasmic reticulum were increased and expanded obviously, lots of high electron density of protein can be seen and lots of lipid drops and glycogen can be seen. The real-time quantitative PCR shows： The Ct value of Aggrecan mRNA expression in middle-dose and high--dose group were significantly higher than the blank group （P〈 0.05）.And there was no no significant difference between low--dose group and the blank group, l^ut the Ct value of Aggrecan mRNA expression in high--dose group was significantly higher than the Middle-dose group（P〈0.05）. Conclusion： （1） The number of endoplasmic reticulum in the middle and high dose group was higher than in the blank group and low-dose group, and lots of greater density of protein can be seen, especially in the high dose group;（2） The middle-dose and high--dose group can enhance the expression of Aggrecan mRNA, and the high-dose group was more obvious;（3）Guizhigegentang can prevent the Intervertebral Disc changes which were caused by the degeneration of annulus fibrosus cells.