| Objective:Using the vitro culture techniques of chondrocytes,and establishing the vitro cultured system of chondrocytes.Using the single traditional Chinese drug reseach methods to separate GuiLu ErXian Glue into GuiBan,LuJiao,RenShen and GouQi formula,observe the effect of GuiLu ErXian Glue and its separate on articular chondrocytes,search the main effective formula,and discuss the protect-ion mechanism of GuiLu ErXian Glue and its separate on impaired articular chondrocytes in cellul-ar and molecular lever,through the aspects of cartilage cell proliferation, expression of typeⅡcolla-gen and proteoglyca.Methods:1.To establish the vitro cultured system of chondrocytes:disassociated the original cartilage cell by the method of enzymatic digestion;observed each generation of cartilage cell by inverted micros-cope; observed the growth characteristics of cartilage cell with the method of MTT assay for grow curve;identified the cartilage cell by toluidine blue staining and Immunocytochemistry staining;2.Adopted MTT methed to assay the proliferation ability of GuiLu ErXian Glue and its separate medicated serum on rat chondrocytes and researched the effect of proliferation ability of GuiLu ErXian Glue and its separate medicated serum with different dosage and different action time,then screen the optimum condition its action;3.Immunocytochemistry was performed to observe the effect of GuiLu ErXian Glue and its se-parate medicated serum on typeⅡcollagen chondrocytes.4.PAS(Periodic Acid Schiff)was performed to observe the effect of GuiLu ErXian Glue and its separate medicated serum on neuter mucopolysaccharide.5.AB(Alcian Blue)was performed to observe the effect of GuiLu ErXian Glue and its separate medicated serum on acid mucopolysaccharide.Results:1.Disassociated and cultivated the original cartilage from 4-week old SD rats successfully; After 48h,the original chondrocytes were showed as polygon and irregular shape,with the rich cyto-plasm. The nucleus showed clear with oval shape. It's available to remain the phenotype of chondr-ocytes steadily within 3th generation from the performance of morphology, toluidine blue staining and Immunocytochemistry staining and the method of MTT assay for grow curve,and since the 4th generation,the chondrocytes begin to appear dedifferentiation phenomenon.2.The result of MTT assay is as below:The proliferation of each group in 24h,48h and 72h did not show the incremental effect along with the dosage,the moderate dosage of medicated serum aff- ected 48h had the strongest proliferation capacity on chondrocytes, so it is selected for the following experiment in order to further explore its mechanism.In 48h,The whole formula and OTL(Glucosa-mine hydrochloride Capsules,OTL) could obviously promote proliferation of chondrocyte(P<0.05 );GuiBan formula or LuJiao formula could both obviously promote proliferation of chondrocyte (P<0.05);RenShen formula or GouQi formula all did not show significant discrepancy(P>0.05); Compared with the whole formula,OTL show the better effectiveness(P<0.05);GuiBan formula or LuJiao formula all show significant discrepancy(P<0.05);RenShen formula or GouQi formula all show extremely significant discrepancy(P<0.01);Compared with each separate,GuiBan formula or LuJiao formula show significant discrepancy with RenShen formula or GouQi formula(P<0.05), LuJiao formula did not show obviously discrepancy compared with GuiBan formula,RenShen form-ula did not show obviously discrepancy compared with GouQi formula(p>0.05).3.The result of Immunocytochemistry show as follows:The typeⅡcollagen expression was visible well in seven groups'specimens,and the expression site was in the cytoplasm only,with yellow-brown particles.OTL and the whole formula could obviously promote the synthesis of typeⅡcollagen of chondrocyte(P<0.01),GuiBan formula and LuJiao formula all could effectively promote the synthesis of typeⅡcollagen of chondrocyte(P<0.05);RenShen formula and GouQi formula did not show significant promotion of typeⅡcollagen of chondrocyte(P>0.05);Compared with the whole formula,OTL show significant discrepancy(P<0.05);GuiBan formula or LuJiao formula all show significant discrepancy(P<0.05);RenShen formula or GouQi formula all show extremely significant discrepancy(P<0.01);Compared with each Sub-Formula,GuiBan formula or LuJiao formula show significant discrepancy with RenShen formula or GouQi formula(P<0.05),Lu-Jiao formula did not show obviously discrepancy compared with GuiBan formula,RenShen formula did not show obviously discrepancy compared with GouQi formula(p>0.05).4.The result of PAS staining is as follows:The neuter mucopolysaccharide expression was visible well in all seven groups'specimens,and the expression site was in the cartilage matrix and around the chondrocyte,with prunosus particles or foliated appearance.OTL and the whole formula could obviously promote the expression of neuter mucopolysaccharide of chondrocyte(P<0.01),Gu-iBan formula and LuJiao formula all could effectively promote the expression of neuter mucopolysaccharide of chondrocyte(P<0.05),RenShen formula and GouQi formula did not show obvious expression of neuter mucopolysaccharide of chondrocyte(P>0.05);Compared with the whole formula,OTL showed significant discrepancy(P<0.05);GuiBan formula or LuJiao formula all show significant discrepancy(P<0.05);RenShen formula or GouQi formula all show extremely significant discrepancy(P<0.01);compared with each Sub-Formula,GuiBan formula or LuJiao form-ula show significant discrepancy with RenShen formula or GouQi formula(P<0.05),LuJiao formula did not show obviously discrepancy compared with GuiBan formula,RenShen formula did not show obviously discrepancy compared with GouQi formula(p>0.05).5.The result of AB staining is as follow:The acid mucopolysaccharide expression was visible well in each group,and the expression site was in the cartilage Nucleus and around the Nucleus,with dark blue particles in Nucleus and lightblue around the Nucleus foliated appearance. OTL and the whole formula could obviously promote the synthesis of acid mucopolysaccharide of chondrocyte(P <0.01),GuiBan formula and LuJiao formula all could effectively promote the synthesis of acid mucopolysaccharide of chondrocyte(P<0.05),RenShen formula and GouQi formula did not show obvious synthesis of acid mucopolysaccharide of chondrocyte(P>0.05);Compared with the whole formula,OTL showed significant discrepancy(P<0.05);GuiBan formula or LuJiao formula all show significant discrepancy(P<0.05);RenShen formula or GouQi formula all show extremely significant discrepancy(P<0.01);Compared with each Sub-Formula,GuiBan formula or LuJiao formula show significant discrepancy with RenShen formula or GouQi formula(P<0.05),LuJiao formula did not show obviously discrepancy compared with GuiBan formula,RenShen formula did not show obviously discrepancy compared with GouQi formula(p>0.05).Conclusions:The cartilage cells of the rats separation cultured using MTT can maintain the phenotype of the cells well among three generations,which is consistent with the biocharacteristics of the cartilage cells.The proliferation of medicated serum of GuiLu ErXian Glue dose not show incremental effect along with the dosage,and the medicated serum with the mid-dose has the best effection which interfered the cartilage cells in 48h;The whole formula can facilitate the hyperplasy of the cartilage cells significantly and the synthetic of the type II collagen and the proteoglyca, which has more better effection than its separate,and it has the similar effection with the positive control drug OTL;According to compare GuiBan formula and LuJiao formula,they also can facilitate the hyperplasy of the cartilage cells significantly and the synthetic of the typeⅡcollagen and the proteoglyca,but RenShen formula and GouQi formula have no apparente promotion,which demonstrated that GuiBan formula and LuJiao formula educed the leading fuction in the whole formula,they are the major drugs in the whole formula. It hints that GuiBan formula and LuJiao formula may contain some active constituents,which can excite the hyperplasy of the cartilage cells significantly.It supposed that RenShen formula and GouQi formula make the augmentation of these active constituents with exciting according to a compatibility optimizationly,or all the formulas have some interactions to produce these active constituents, which demonstrated that RenShen formula and GouQi formula have the apparente synergetic effection in the whole formula.The concrete mechanism of action still needs the further research.GuiLu ErXian Glue can enhance the proliferation of cartilage straightly,and then facilitate the synthetic of the typeⅡcollagen and the proteoglyca,which may be a mechanism of GuiLu ErXian Glue delay the degeneration of cartilage,and prevention and cure the OA. |
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