The Study On The Biological Effect Of H.pylori On HPDLFs

[Objective]Establishing the model of human periodontal fiberblasts(hPDLFs)infected by Helieobacter pylori(H.pylori)in vitro,to observe the biological effect on the cell morphological,ultrastructure,total protein,gene and protein expression of ALP,OCN,COL-I of cultured hPDLFs infected by different concentrations of H.pylori,and explore the relationship between H.pylori and periodontitis and its clinical significance.[Methods]1.Culture and identify H.pylori(ATCC43504)by selective medium in vitro,and adjust the concentrations of H.pylori to 8×105cfu/ml,4×106cfu/ml,2×107cfu/ml,1 × 108cfu/ml.2.Culture hPDLFs in vitro and identify their morphological,adjust the concentration of 5th generation hPDLFs to 2 X 105/ml.3.Coculture four concentrations of H.pylori with 2×105/ml hPDLFs respectively for 0-5 day,establish the model of hPDLFs infected by in vitro,set up cell control group and bacteria control group simultaneously.After cocultured 6h,12h,24h,48h,the cells morphological were observed by inverted microscope;After cocultured 48h,the cells ultrastructure were observed by transmission electron microscope(TEM);After cocultured 0d,1d,3d,5d,the cells total protein were detected by using BCA assay kits;ALP,OCN,COL-I of gene and protein expression were assayed by Real-Time PCR(RT-PCR)and Western-blot method.The SPSS 13.0 software was used for statistics analysis.[Result]1.Inverted microscope shows:with the increasing of H.pylori concentration and the role of time,the cell morphology of adherent hPDLFs changed from spindle into round,adherent cells decreased,suspended cells increased and much cell debris appeared around.2.TEM shows:the ultrastructure of hPDLFs infected by H.pylori destroyed at varying degrees,cell nucleus was irregular,karyotheca was not intact,chromatin was partly clotted;cell organelle structure destroyed obviously,mitochondria was swelling or hyaline degeneration,mitochondrial cristae was sparse or vacuolar degeneration;rough endoplasmic reticulum membrane structure was destructive and there were varying degrees of degranulation3.The improved BCA assay shows:At 1th day,hPDLFs infected by H.pylori had higher total protein than the cell control group,the difference was not statistically significant(P>0.05);At 3th and 5th day,hPDLFs infected by H.pylori had lower total protein than the cell control group,the difference was statistically significant(P<0.05),and the decreased tendency heightened gradually.4.The RT-PCR and Western-blot assay both shows:the ALP,OCN and COL-I expression on gene and protein of hPDLFs infected by H.pylori were lower than the cell control group,the difference was statistically significant(P<0.05),and with the increasing of H.pylori concentration and the role of time,the decreased tendency heightened gradually.[Conclusion]1.H.pylori destroyed the cell morphological and ultrastructure of hPDLFs cultured in vitro,which indicated that H.pylori have cytotoxic effect on hPDLFs.2.The total protein of hPDLFs would be affected by the concentration and time of H.pylori infected.The division growth of hPDLFs would be improved when infected H.pylori by low concentration under short time,and with the increasing of H.pylori concentration and the role of time,the division growth of hPDLFs would be restrained.3.The capability of collagen synthesis and osteogenic differentiation of hPDLFs were correlated to the concentration and time of H.pylori infected.The normal function of hPDLFs would be affected by specific concentration and time of H.pylori infected.With the increasing of H.pylori concentration and the role of time,the biological effect on collagen synthesis and osteogenic differentiation of hPDLFs would be inhibited.