| Actinobacillus actinomycetemcomitans,which is the predominant pathogen of periodontitis,has been divided into serotype a,b,c,d and e based on the serologic study, serotype b is the most frequent and virulent serotype.Aa and its dissoluble product cloud participate in the occurrence and the development of periodontitis by means of destroying periodontal tissue, stimulating bone resorption,suppressing host immune.In the past, studies focus on the activation and apoptosis of cells,especially getting rid of the dissoluble product of Aa,in order to research the relation between host and the germ cell of Aa.However,it is not the main way of destroying that the germ cell breaks direct into the periodontal tissue,besides acute necrotizing ulcerative gingivitis, the germ cell has hardly any been found to break direct into the periodontal tissue,it is the most important mechanism that the dissoluble product of germ and the immunoreaction between host and germ result in the damage of periodontium.Therefore ,in this experiment,we pulverized Aa by ultrasonic pulverizer, take filtrate as the stimulation to incubate with peripheral blood polymorphonuclear leukocytes and observe the morphologic changes of destructive cells,so that,we cloud know more about the pathologic changes of periodontal tissue.In our research,we use Aa Y4(serotype b) as the stimulation.We did it in this way:1,Cultivation of Aa:Aa is cultivated on tryptone-soya agar medium supplemented with 5%fetal calf serum at 37℃(10%CO2).After 48 hours,the cultures are Gram-stained and examined by a series of biochemical reactions.2,Pulverization and filtration of Aa:the colonies of Aa are dealed with protease inhibitor lysis buffer,pulverized with ultrasonic pulverizer,60W,3min,then filtrated with 0.45μm filtration membrane ,and the sedimentation was removed.3,Isolation of PMNL:venous blood was drawn from 10 healthy volunteers(15~30years old).Peripheral blood PMNLwere isolationed by Percoll gradient centrifugation.4,Stimulating the PMNL with filtrate of ultrasonic pulverization from Aa:peripheral blood PMNL were cultured in 50ml culture flasks at a concentration of 107cells/ml with the filtrate in a humidified incubators at 37℃(5%CO2).The cells were detected for various intervals between 0 to 60 mins.The concentration of filtrate is 100μg/ml.And the control group is that the filtrate was added to the PMNL which has the antiserums.5,Making and observing the sample of transmission eletron microscope:the sample was dealed with 2.5%glutaral, 1%osmic acid,alcohol and epoxy.Then it cloud be observed by the eletron microscope(JEM-1200EX).There are obvious differences between the filtrate group and the control group at the points of 20min,40min and 60min.In the filtrate group,the degranulation of PMNL at 20min is more than before.Moreover,the differences between the two groups became greater at 40min and 60min.So we can draw a conclusion that the filtrate of ultrasonic pulverization from Aa can induce the degranulation of PMNL and the effect is time-dependent.In morphology,the differences between the two groups can be observed at 1min.At 5min,the degranulation of PMNL was showed in the filtrate group.Not all the cells in this group were induced degranulation,meanwhile,the PMNL in the control group looked like the normal one. Because most filtrate of ultrasonic pulverization from Aa is the dissoluble product of Aa,and the product is the most important thing that result in function obstacle of periodontal tissue.So stimulating PMNL by the filtrate can show the mechanism in periodontitis correctly.The filtrate of Aa can make PMNL lose its integrity,suppress the chemotaxis and phagocytosis of cells,therefore the PMNL can not enter the inflammatory tissue. So we conclude that the filtrate of ultrasonic pulverization from Aa can induce the degranulation of peripheral blood PMNL,also it can suppress the host immune function to promote the progress of periodontitis and provide theoretic basis for differential diagnosis,therapy and prognosi...
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