Effects Of Guizhi Plus Gegentang Decoction On Expression Of Proteoglycan In Intervertebral Disc Annulus Fibrosus Cells

Objective:To explore the mechanisms of Guizhi plus Gegen Decoction(GGD) preventing cervical syndrome. Establish rats'intervertebral disc annulus fibrosus cells culture system. Aannulus fibrosus cells were intervened by different concentrations of GGD. To observe the annulus fibrosus cells from molecular level and ultrastructural level.And explore the relationship of annulus fibrosus cells and Annulus degeneration.To deepen the mechanism of GGD in treating cervical spondylosis. Indirectly promoting the clinical application of Chinese medicine in the treatment of cervical spondylosis.Methods:(1)80 clean level adult SD rats randomly were divided into 4 groups by body quality (weight 200-230g). The blank group(20) were fed with 2mL saline water.The low-dose group(20) were fed with 1mL GGD.The middle-dose group(20) were fed with 2mL GGD. The high-dose group(20) were fed with 4mL GGD.Each group was fed for four days(twice a day).And to produce different concentrations of rats serum containing GGD. (2)Rats'intervertebral disc annulus fibrosus cells were isolated and cultured using adherence wall screening method. Toluidine blue staining, immunocytochemical staining and transmission electron microscope were used to identificating the phenotype.(3) The annulus fibrosus cells were intervened by GGD for 6 days. And the transmission electron microscope was used to see the morphology and structure changes of annulus fibrosus cells which intervened by GGD. Real-time quantitative PCR was used to test the expression changes of Aggrecan-mRNAResults:(1)Vitro Cultured and identification of annulus fibrosus cells:the cells have toluidine blue staining; the expression of collagen I and collagen II in annulus fibrosus cell was positive; Under the light microscope,cells were rounded, spindle or polygonal mostly,and a lot of rough endoplasmic reticulums can be seen in the cytoplasm; the features of cell structure were consistent with cartilage cell underthe transmission electron microscope. (2)Under the transmission electron microscope,the ultrastructural changes of annulus fibrosus cells intervened by different concentrations of GGD:the number of rough endoplasmic reticulums in the middle and high-dose groups was more than in the bank and low-dose groups,and greater electron density protein precipitation appears in the middle and high-dose groups,especially in the high-dose group. (3)the expression changes of Aggrecan-mRNA in the annulus fibrosus cells intervened by GGD:the Ct value of Aggrecan-mRNA in the middle and high-dose groups was obviously higher than in the bank and low-dose groups(P<0.05),and there was no significant difference between the bank group and the low-dose group.But the Ct value was obviously higher than the middle-dose group(P<0.05).Conclusion:(1)It was successful to cultured the cells in vitro using adherence wall screening method.And completed the identification of the cell.These provides a prerequisite for subsequent experiment. (2) GGD can make ultrastructural changes in the annulus fibrosus cells; the number of rough endoplasmic reticulums in the middle and high-dose groups was increased,and greater electron density protein precipitation appears.these suggesting that GGD can enhance the ability of protein secretion from the cell structure.(3)GGD can enhance the Aggrecan-mRNA expression of the annulus fibrosus cells.GGD can make a influence on production of Annulus fibrosus cells proteoglycan on the level of transcription.