The Investigation Of Nanobacteria In Human Arthritic Synovial Fluids Of Osteoarthritis And Its Morphological Effects And Mechanisms Of Action On Human Arthritic Chondrocyte Cell

PartⅠThe investigation of Nanobacteria in knee synovial fluids of osteoarthritis patients.Objective: To investigate the existence of Nanobacteria in knee synovial fluids of osteoarthritis patients.Methods: Synovial fluids which were obtained from the knee joints of 25 osteoarthritis patients and 22 healthy people were cultured to detect nanobacteria; Changes of electrolyte and glucose were dynamically detected by auto clinical chemistry analyzer every 14 days. After 8 weeks, Immunofluorescence was then conducted to identify the cultured nanobacteria by applying the cloning antibody of the nanobacteria. Finally, nanobacteria was observed with electron microscope.Results: In the process of cell culture, no remarkable changes of electrolyte and glucose occurred in the supernatant of the synovial fluids of healthy people and the concentration of K, Na, Cl, Mg, glucose were also no remarkable changes in he supernatant of the synovial fluids of 25 osteoarthritis patients (P＞0.05), while Ca and P decreased gradually in the supernatant of synovial fluids of osteoarthritis patients, which is demonstrated by the every-14-days-test results (P＜0.05). In Immunofluorescence investigations, nanobacteria which could emit green fluorescence were detected in Synovial fluids of 25 osteoarthritis patients and 1 healthy people, but it were not detected in Synovial fluids of 21 healthy people; The size range of Nanobacteria is 100-400nm by electron microscope analysis.Conclusions:(1) There are nanobacterias in synovial fluids of all osteoarthritis patients.(2) Only 1 was detected nanobacteria in synovial fluids of 22 healthy people.(3) The nanobacteria may be the key factor in the biological etiopathogenesis of Osteoarthritis. PartⅡEffects of biologic shape of nanobacteria on human arthritic chondrocyte cellObjective: To study the injury of nanobacteria on human arthritic chondrocyte cell.Methods: Changes of the morphology of chondrocyte cell after invasion by different concentration nanobacteria were observed under optical microscope. The survival and growth of the chondrocyte cell after invasion were investigated by MTT test. The spatial relations between chondrocyte cell and nanobacteria at different attacking times were identified by immunofluorescence. The Apoptosis process of human chondrocyte cell after invasion was monitored by flow cytometer.Results: The injury of chondrocyte cell is greatly dependent on the concentration of nanobacteria, and various injury degrees can be induced, such as distortion, ambiguous appearance, and injury of cell membrane, necrosis, even completely destroyed. The injury is getting worse with the higher concentration and in more time of invasion. MTT results show that compared with the situation under different concentration, the survival and growth of chondrocyte cells are obviously restrained by higher concentration nanobacteria (P＜0.05). Immunofluorescence results indicate nanobacteria can attack chondrocyte cell quickly, and enter cell with the time prolonged. Flow cytometer investigations show apoptosis of chondrocyte cell is little dependent on the concentration of nanobacteria in 24 hours (P＞0.05 )Conclusions: The chondrocyte cell can be injured by nanobacteria; The chondrocyte cell after invasion for a short period (24h) is not apoptosis; The injury is wores in higher concentration. PartⅢMechanisms of damage by nanobacteria on Human arthritic chondrocyte cellObjective: To investigate the mechanisms which nanobacteria injury on human arthritic chondrocyte cell by analyzing the free radical.Methods: Changes were detected by the content of SOD, MDA, NO and LDH in supernatant according to reagent instructions when 0.5Mcfarland nanobacteria attacked human arthritic chondrocyte cell for 0, 6, 12, 24, 48 and 72h respectively.Results: Activity of SOD in supernatant rise gradually(P＜0.005)and little change is observed for MDA content (P＞0.05). As for the No content, it gets higher and higher as the time passed and the peak content is obtained at 48h, then it decreases at 72h. Yet, their individual concentration rises drastically compared with the controll(P＞0.05). Activity of LDH rises gradually with time (P＜0.05).Conclusions: There may be no oxygen free radical participation while nanobacteria attack human arthritic chondrocyte cell; Under attack by nanobacteria the chondrocyte cells secrete NO and the No content gets higher and higher as the time passed; the NO may induce the necrosis of chondrocyte cells.