Isolation, Cultivation And Initial Identification Of Nanobacteria From Calcified Heart Valve Of Rheumatic Heart Disease

Objective The aim of this study was to isolate, culture and identify the nanobacteria from human calcified heart valve of rheumatic heart disease.Methods The study was composed of calcified heart valve group, normal heart valve group, negative control group of serum radiated by 30KGyγ-ray, positive control group of nanobacteral strain Se90, powdery hydroxyapatite ceramic group and blank control group of DMEM. Calcified heart valves and normal heart valves were homogenized and 0.22μm filtered. All groups were cultured in mammalian cell culture conditions. They were observed under microscope weekly. Collected aliquots of the cultures from different incubation periods were analyzed using spectrophotometer. Harvested pellet after three weeks were done immunofluorescence double staining of monoclonal antibody of nanobacterial and hoechst33258 and transmission electron microscope (TEM) observing.Results Movable tiny black particles were observed under microscope in calcified heart valve group, nanobacteral strain Se90 group and hydroxyapatite ceramic group. The optical density was significant among different groups and different time points (p=0.000, p=0.000) . Immunofluorescence double staining of monoclonal antibody of nanobacterial and Hoechst 33258 displayed tiny green fluorescence particles in calcified heart valve group, nanobacteral strain Se90 group and two samples of normal heart valve group. TEM analysis indicated the particles' size ranged from 88nm to 341nm in calcified heart valve group, which were similar to nanobacteral strain Se90 group. which were similar to nanobacteral strain Se90 group.Conclusions The nano particles were isolated and cultured from calcified heart valve of rheumatic heart disease in suitable conditions. The nano particles were nanobacterial by initial identification.