Experiment Study Of Biofilm Effect On The Etiopathogenesis Of Primary SS

Objective: To evidence the biofilm is one pathogensis of primary SS and to explore the etiopathogenesis of primary SS by studying of the immune reaction by alginate on parotid acinar cell in vitro; We try to develop an rabbit model of human-like primary SS by alginate.Methods: Rabbit parotid acinar cells were cultured in the reformed DMEM; Shape and structure of cultured cells were observed with phase contrast microscope and transmission electron microscope; PCK, α-amylase and Actin of the cultured parotid acinar cells were examined by immunohistochemical SP technique; The secretion function of the different generations was evaluated by the positive expression status of α-amylase .Rabbit was immunized by the conjugated alginate-BSA (1.0mg/kg) for 40-days routine immunity method; ELISA method was used to examine the titration (valence) of anti-alginate serum; The first generation parotid acinar cells were chosen to divided five groups (group A :contrast, group B: BAS, group C: alginate, group D: anti-alginate serum, group E: alginate +anti-alginate serum), Weexamined the proliferation of each part by MTT method at four times (lh, 6h,12h and 24h); In the meanwhile, The growth and shape of parotid acinar cells were observed under phase contrast microscope and scanning electron microscope.16 of the immunized rabbits by alginate were divided equally into Al and Bl groups, 12 normal rabbits were divided equally into A2 and B2 groups as control groups, Each rabbit was affused by 0.3ml(40fil/ml)alginate though parotid duct once time two days. After 7 times (group Al and group A2) and 14 times later(group Bl and group B2), The change of the flow of saliva, sialography, T subsets, Schirmer test and pathology were examined respectively in group Al, group B2 and their control groups.Results: The parotid acinar cells had the ability of proliferation in vitro. They could transmit 3 generations and get 4w long life. Microvilli, plasm crease and zymogen granules could be seen in the parotid acinar cell under the transmission electron microscope. The specific immunohistochemistry staining of PCK and a-amylase could be seen in the plaserm and (or) membrane of parotid acinar cells, but Actin not. The positive expression degrees of a-amylase in generation 1,2 ,3 and 4 were 79.6%, 76% ,47.7% and 2.4% respectiviely, which decreased gradually. There was a significant difference between the generation 3 and others ( P<0.05) . There was no significant difference between the generation 1 and generation 2 (/M).O5 ) .It showed that the cells of generation 1 and 2 maintained its characteristics and proliferation in vitro.Antibody-serum of alginate was acquired by routine immunity method, and the titration (valence) of anti-alginate serum reached 1:400; There was a significant difference by MTT between group E with four other groups at 24h( P<0.05 ), It showed that the proliferation of parotid acinar cells had been limited at 24h, other three times were no difference (iM).O5) ; Under phase contrast microscope we saw a few of acinar cells whose membrane destroyed at 12h, some in-cell contents were leaked out. Furthermore at 24h; under scanning electron microscope the hole of membrane could be seen early at 6h, most of acinar cells were broken and could not be recognized at 24h. It show that the immunity reaction by alginate could directly attack the membrane of parotid acinar cell.The saliva flow of group Al and Bl were lower then their control groups, there was a significant difference among these 4 groups^^cO.OS); The results of schirmer test for 4 groups were no significant difference; CD4 was increasing in the group Al and B1,CD8 was only increasing in group Bl, CD4/CD8 of group Bl was lower than group Al and others, There were significant difference among them CP<0.05) ; It showed different sialographic image in the two groups Aland Bl, the image of feather-like aside main duct could be seen in the group Al, the typical image of SS was showed in group Bl such as sialectasis, globular and cavity; The focal infiltrates of monocytes, mast cells and lymphocytes were seen around the glandular epithelium in group Al, It showed an allergic reaction image in the HE, A lot of epimyoepithelial islands was seen in the parotidgland tissues; There were many monocytes and cystic variant of this lesion exists replace the glandular epithelium in the group Bl.Conclution: The biofilm mostly be one pathogensis of primary SS, It may be one etiopathogenesis of primary SS that the immune reaction by alginate could directly attack the membrane of parotid acinar cell. We had developed a rabbit model of human-like primary SS by it.