Experimental Study Of Huatan Chushi Quyu Preparation On Cartilage And Synovial Cells Of Patients With Knee Osteoarthritis

Objective:To establish the knee cartilage cell separation, training system. Explore expectorative desiccant influence people degeneration knee cartilage cells I type, â…¡ type, X Collagen and related metabolic enzymes MMP-1, MMP-3, MMP-13, called a, IL-1, IL-6mRNA expressions. And delay the knee osteoarthritis articular cartilage degeneration mechanism and clinical curative effect.Methods:(1) With the improvement Hulth method to establish the rabbit knee osteoarthritis animal model, to observe the treatment group, meloxicam group (positive medicine group), model group and normal joint group of articular cartilage and synovial pathology and ultrastructural changes.(2) Using improved Hulth method to establish the knee osteoarthritis animal model treatment group, beauty observation los yesterday kang (group of positive medicine group), model group and normal joint group and joint in the fluid of serum IL-1, IL-6, TNF a and serum NO, SOD changes.(3) Using improved Hulth method to establish the knee osteoarthritis animal model observed treatment group, meloxicam group (positive medicine group), model group and normal serum and joint group articular cartilage and synovial MMP-in1, MMP-3mRNA expression level of the influence.(4) Mice expectoranting desiccant oral toxicity testing.(5) With the original training YangFa get knee joint organization of cartilage cells, ChuanDai training. Through the cart before the horse is a microscope cells form, draw the growth curve, red meat Iraq sappanwood dyeing, armour aniline blue stain and Collagen type I, II Collagen type, X the Collagen, MMP-1, MMP-13, TNF alpha immunofluorescence stain identification of cells. Will cartilage cells after ChuanDai separation, take the third generation cells, divided into seven group (group A:cartilage cells+DMEM control group, and group B: cartilage cells+5%expectorative desiccant serum; C:cartilage cells+5%serum; NS group D: cartilage cells+10%expectorative desiccant serum; group E:cartilage cells+10%serum; NS cartilage cells of group F:+20%expectorative desiccant serum; group G:cartilage cells+20%NS serum;), take4h,8h,24h,48h,72h, USES the RealTime-PCR method to detect each Collagen and metabolic enzymes mRNA expressions.Results:(1) The model group rabbit knee swelling rate was14.6%in treatment group of medicine rabbit knee swelling rate12.8%, walking ability more model group has obviously improved. The model groups of the rabbit knee limp serious, joint surface turn dark with grey, and have crack, erosion and ulcer formation. There exists different degree of synovial hyperplasia adhesion, joint fluid volume increased and show bubbles, and turbidity, Markins score JiaShouShu higher than the control group. The treatment group of articular cartilage and synovial morphological structure though not returned to normal, but Markins score a model group significantly reduced.(2) Model group rabbit serum and joint in the fluid of IL-1, IL-6, TNF a a pseudo scores significantly increased. And the treatment group are both above improved obviously, model group SOD activity during which is reduced, and NO significant rise content, the treatment group SOD activity of a model significantly increased NO NO significant differences, but also can see a trend to decrease.(3) Model group rabbit articular cartilage and synovial organization MMP-1, MMP-3mRNA expression level obviously higher than control group (p is less than0.01). The treatment group MMP-1,3in the joints and synovial mRNA in the expression level is model group reduced significantly (p is less than0.05).(4) Mice oral expectorative desiccant biggest tolerance for234g/kg for adult often of the amount of more than100times.(5) Expectorative desiccant medicated serum has raised the knee joint degeneration of the training of the generation of people in cartilage cells spindle, short spindle, part is the polygons, ChuanDai after three times in the growth of dendritic cells. Cell culture within three generations, most cartilage cells can maintain the spindle nuclei most maintain circle. In the morphology, immunofluorescence stain can maintain the stability in the phenotype, pure Iraq sappanwood red positive dyeing as cartilage cells matrix red, and the nuclei are blue purple, the cytoplasm and cell with purple around the new blue staining o, cell plasma cell membrane and are in dark blue, Collagen type â…¡ immunofluorescence stain visible cytoplasm and cell membrane for the green fluorescence, the nuclear area without fluorescence. Expectorative desiccant medicated serum all have cut the degeneration of cartilage cells MMP-1, MMP-3, MMP-13, IL-1, IL-6, TNF-a role, including eight hours of serum containing medicine10%cut MMP-1, MMP-3, MMP-13and IL-1role are relatively good,24hours of serum containing medicine10%cut IL-6role are relatively good,48hours of serum containing medicine10%cut TNF-a role of relatively good.(P<0.05)conclusion:indicates that the experiments successfully set up cartilage cells of isolation, culture system, and three generations grow well within cells. Proved expectorative desiccant treatment knee osteoarthritis, could slow joint degeneration and damage, no significant side effects.