The Investigation Of Morphological Effects And Mechanisms Of Nanobacteria On Rabbit Artery Smooth Muscle Cell

Partâ… The culture and identification of Nanobacteriaof atherosclerotic plaqueObjective: To investigate whether the nanobacteria exist inatherosclerotic plaque or not and identify it.Methods: After 12 cases of atherosclerotic plaques and normalartery got from operation were grinded, the supernatant fluid was culturedfor eight weeks. The white powder got from the culture flask wasidentified by nanobacteria monoclonal antibody immunofluorescence andelectron microscope. Concentration of electrolyte and glucose weredynamically detected by auto clinical chemistry analyzer every weekafter nanobacteria reculture.Results: Nanobacteria was found in ten of twelve cases ofatherosclerotic plaques and one in twelve cases of normal artery, thedifference was significant through chisquare test (P＜0.05). Nanobacteriacan give out green fluorescence by indirect immunofluorescence. Thesize of nanobacreria was measured ranging from 100nm to 300nm byelectron microscope analysis. In the process of nanobacteria culture, noremarkable changes occurred to the concentration of K⁺,Na⁺,Cl^-,Mg²⁺ inthe supernatant and that of glucose, while Ca²⁺ and p^3- decreased gradually, which is demonstrated by the weekly tested results (P＜0.05).Conclusions: Nanobacteria was found in atherosclerotic plaquesand may play an important role in the atherosclerotic plaques instability.Indirect immunofluorescence and transmission electron microscope canbe used to identify nanobacteria. Nanobacteria consumed Ca²⁺ and P^3-instead of K⁺,Na⁺,Cl^-,Mg²⁺ and glucose Partâ…¡Effects of nanobacteria on morphology of rabbitartery smooth muscle cellsObjective: To study the injury of nanobacteria on artery smoothmuscle cells and whether the intervention of 1.95μg/ml Tetracycline candelay the injury or notMethods: Changes of the morphology of artery smooth muscle cellsafter invasion for 48 hours and 72 hours by different concentrationnanobacteria and intervention of Tetracycline were observed underoptical microscope. The survival and growth of artery smooth musclecells after nanobacteria invasion and intervention of Tetracycline wasinvestigated by MTT test. The apoptosis process of artery smooth musclecells after nanobacteria invasion and intervention of Tetracycline wasmonitored by flow cytometer.Results: The injury of artery smooth muscle cells was greatlydependent on the concentration of nanobacteria and time of invasion,various injury can be induced, such as distortion, ambiguous appearance,fracture of cell membrane, necrosis, even completely destroyed. Theinjury was getting worse with higher concentration and longer time ofinvasion. Such changes of cells as distortion, ambiguous appearance,fracture of cell membrane, necrosis, even completely destroyed can befound after intervention of Tetracycline as nanobacteria OD was higher than 0.1. After nanobacteria invaded for 48h, the difference in MTT wassignificant between trial group and control group except OD=0.001, and thedifference in trial group was significant (P＜0.05). After 72h, MTT in trialgroup was lower than that of control group, there was no difference betweenOD equaled to 0.1 and 0.3, but the difference was obvious in others(P＜0.05).MTT of 72h was higher than that of 48h as nanobacteria OD equaled to0.001, 0.005 (P＜0.05). MTT of 72h was lower than that of 48h asnanobacteria OD equaled to 0.01, 0.03, 0.05, 0.1 (P＜0.05), the differencein MTT between 48h and 72h was unobvious as nanobacteria OD equaled to0.3. After intervention of 1.95μg/ml Tetracycline, MTT in trial group waslower than that of intervention group, as nanobacteria OD equaled to 0.001,0.005, 0.01, 0.03, 0.05, 0.1. there was no difference between trial group andintervention group as nanobacteria OD equaled to 0.3. Little apoptosis ofartery smooth muscle cells was found in different concentration nanobacteriaand intervention of Tetracycline group by flow cytometer detection (P＞0.05).Conclusions: Artery smooth muscle cells can be injured bynanobacteria, the higher the concentration was, the graver the injury was. Thedeath of artery smooth muscle cell after invasion for a short period (24h) wasnecrosis rather than apoptosis. Tetracycline can delay the injury in smoothmuscle cells. Partâ…¢Mechanisms of injury of nanobacteria on rabbitartery smooth muscle cellsObjective: To investigate the mechanism of nanobacteria injury onrabbit artery smooth muscle cells by analyzing free radical.Methods: Changes of LDH, SOD, MDA and NO in supernatantwere detected following reagent instruction after 0.5Mcfarlandnanobacteria invaded artery smooth muscle cells for 0, 12, 24, 48 and 72hrespectively.Results: Activity of LDH rose gradually with longer time (P＜0.05).SOD Activity rose gradually (P＜0.05), and little change was observed inMDA content (P＞0.05). As for the NO content, it got higher and higher,and peak was obtained at 24h and then decreased, yet their individualconcentration rose drastically compared with the control (P＜0.05).Conclusions: There may be no free oxygen radical participationwhen nanobacteria invaded artery smooth muscle cells. Artery smoothmuscle cells can secrete SOD and NO. NO may play an important role inthe injury of nanobacteria on cells.