产黑色素卟啉单胞菌诱导骨吸收作用机制的研究
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摘要
实验目的
牙龈卟啉单胞菌(porphyromonas gingivalis,P.g)和牙髓卟啉单胞菌(porphyromonas endodontalis,P.e)是口腔卟啉菌属中两种重要的细菌。二者都是G-产黑色素的专性厌氧球杆菌,是目前公认的牙源性感染的优势致病菌,与牙周炎及根管感染导致的根尖周炎病变的发生和发展密切相关。很多研究表明,牙周炎和根尖周炎的临床症状及病变进程与牙周袋内及感染根管内的P.g、P.e密切相关。这些细菌能影响牙周及根尖组织中牙龈成纤维细胞、牙髓细胞、牙周膜细胞、血管内皮细胞等多种细胞炎症介子的表达,但其确切的致病机理尚未完全明确。目前已知P.g、P.e的致病物质有很多,包括脂多糖、外膜蛋白、荚膜、蛋白酶以及细菌的代谢产物等,这些致病物质可以通过多种途径逃脱机体的免疫防御反应,作用于牙周组织和根尖组织,导致牙周和根尖周围硬组织和软组织的分解破坏,致使牙齿松动乃至脱落。研究P.g、P.e在牙周和根尖周组织破坏中的作用,能明确牙槽骨吸收的病理机制,可以为临床上治疗牙槽骨过度吸收奠定理论基础,在牙源性感染的基础研究中有重大的意义。
牙周炎和根尖周炎的一个重要的病理改变是根尖及根周牙槽骨的吸收。牙槽骨是人体最活跃的骨组织,处于不断的自身修复和改建中。很多研究资料报告,刺激骨吸收的因素如PGE_2、IL-1,IL-6,TNF等都是作用于破骨细胞的前体细胞,在集落刺激因子的作用下,促进其向破骨细胞的分化和成熟并使其激活,同时抑制破骨细胞的凋亡。
关于P.g、P.e对成骨细胞代谢的影响,目前国内外研究报告都不多见。本实验通过研究P.g、P.e对成骨细胞的超微结构、细胞的活性及增殖能力、表达前炎症因子MCP-1(monocyte chemoattractant protein 1)以及骨代谢的最终调节因子OPG(osteoprotegerin)、RANKL(receptor activator of NF-κB ligand)的影响,进一步探讨P.g、P.e在牙周炎和根尖周炎发病中的致
Porphyromonas gingivalis (P. g) and Porphyromonas endodontalis(P. e)are Gram - negative, obligate anaerobic, asaccharolytic black - pigmented microorganisms, which are frequently detected in infected root canals and periodontitis , and they are thought to be the most important pathogens in periodontitis and periapical inflammation. They have gained special prominence in the search for etiologic organisms associated with periapical periodontitis and periodontitis . Many reports showed that the progress and the clinical symptom are closely related with P. g P. e,which exist in subgingival and infected roots. These bacterias can influences the expression of many inflammatory cytokines in various cells such as gingival fibro,pulp cell, blood vessel endothelial cell etal. However, the exact mechanisms are not fully undertanded . Many researches revealed that P. g and P, e possess many virulent factors, including fimbri, out - membrane protein, proteinase, lipopolysaecharide (LPS) , and so on. These virulent factors could escape the defense system in many ways to destroy periodontal supporting tissue and periapical tissue. It is significant to investigate the pathogene-sis of P. g and P. e in inducing the destructive effects in periapical periodontitis and periodontitis , and these will provide the theoretical basis for treating the bone resorption. One of the importment pathological chararters of periapical periodontitis and periodontitis is the bone resorption.There have been relatively few studies addressing the P. g P. e on osteo-blasts. The purpose of this study is to determine the effects of P. g P. e on osteo-blasts in the structure, the growth and proliferation , the ability of secreating
MCP - 1 and the expression of OPG or RANKLin gene level. In order to find out the possible pathogenesis of bone resorption induced by P. g P. e.Methods1 . Bacterial culture:The strains of P. g ATCC 33277 and P. e ATCC 35406 were grown in Brain Heart infusion Broth, supplemented with 10% , yeast - extract(5g/L) ,Hemin (5mg/L) , and Vitamin k3 ( 1mg/L). Under an atmosphere of 80 % N2,10 % H2,10% CO2 at 31X,. , until at an optical density. Bacterial cells were centrifu-gated on 3500 rpm/min for 10 min, the bacterial purity was determined by phase - contrast microscopy and Gram staining. And then were suspended in PBS at a density of 1010CFU/ml and stored at -70℃ until used.2. Cells culture:New born rats were killed, the calvariae were removed aseptically. The periosteal layers on both sides were carefully stripped off with tweezers under PBS, and washed with PBS for two times. Then the bone specimens were treated by 0.25% trypsinization for 15min at 37℃ ,and the supernatant was discarded. After that the specimens were minsed then were digested by 1 mg/ml collagenase II 31℃ for 90min, the supernatant was collected and centrifugedat at 1000r/ min for 5 min . The collagenase — release cells were plated in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37℃ in a humidified atmosphere of 5% CO_2 and 95% air. The medium was changed by 3 -4d and were serially passaged. The differential adherence method was used to purify the osteoblasts. The cells between the third and fifth passages were used in the following experiments.3. The morphologic observation and immunohistochernical identification oi osteoblasts :The property of osteoblasts were observed and photoed with phase contrast microscope on 100 200 400 times repectivelly. several pieces of glass were placed on the 6 - well plates, then the cells were seeded into them and incubated overnight at 37℃ with 5% CO2. The pieces of glass were dryed . Monoclonal
antibody against I collagen were employed to identificated osteoblasts by immu-nohistochemical method . The cell nucleus and cytoplasm were observed.4. Effects of bacterias on the proliferation of rat osteoblasts : Osteoblasts were collected and replated at a density of 105 cells/ml in 96 -well flat - bottomed plates for lOOjxl per well and incubated overnight at 37^1 with 5% CO2 to be confluent monolayers ,the solutions of P. g^ P. e at the density of 1010CFU/ml were diluted with DMEM containing 10% FCS into serial concentrations to stimulate osteoblastes , co - cultivated for 24h. the changes of proliferation to P. g AP. ewere determined by the percents of reduced AlamarBlue?.5. Observation of structure of cultured osteoblasts with transmission electron microscope:Osteoblasts were harvested by 0.25% Trypsinafter co - cultivation with 108 CFU/ml P. g ^P. e for 24h, they were washed with PBS . All of the cells samples were fixed and were sectioned in thickness. The changes of cell and were invested with transmission electron microscope.6. Detection of OPG and RANKL mRNA expression in osteoblasts by reverse transcriptase - polymerase chain reaction:Osteoblasts were cultured in flasks to reach confluent monolayers. P. g ^P. e at a density of 10\l09CFU/ml were added into the cells, osteoblasts in 10% FCS DMEM without acterias were employed as a negative control. The cells were collected after 24h co - cultured and stored at - d>0X . to investigate the kinetic effect of the bacterias, we used 10 CFU/ml P. g^P. e to stimulate osteoblastes for various times. The cells were collected at 0A6->12-N18^24h after co - cultivation, subsequently, total RNA were isolated from cells in each flask using TRIzol reagent according to the manufacturers instructions. The complementary DNA were synthesized from RNA using a cDNA Cycle Kit,which uses reverse transcriptase. The cDNA were amplified with TagDNA polymerase and specific primers in a thermal cycler. The PCR conditions were : initial denaturation 3 min,94cC : denaturation 1 min, 95X : annealing 1 min50X : extension 1 min, 72X ,fop 35 cycles: followed by a 7 min elongation step at 72^. Primer sequences were; OPG, forward, 5 ' -TTGGCTGAGTGTTCTGGT -3 ' ,re-verse5 ' -TTG GGA AAG TGG TAT GCT -3 ' ; RANKL, forward, 5 ' -
CAT CGG GTT CCC ATA AAG - 3 ' , reverse 5 ' - TGG ACA CCT GGA CGC TAA - 3 ' ; p - actin, forward, 5 ' - TGT ATG CCT CTG GTC GTA CCA C -3 ' , reverse5 ' - ACT GAG TAC TTG CGC TCA GGA G -3 ' PCR products were 423bp, 357bp and 690bp resperctively.7. Quantifying mRNA of OPG and RANKL;15 ill of the products including OPG 5ul, RANKL 5uland (3 - actin 5ul. PCR products were mixed ,then were electrophoresed for lh on 2% agarose gel and visualized by ethidium bromide staining. The bands for OPG and RANKL were noted, these bands were consistent with the size as designed by primers, when the band densities were measured and compared with the density of the band obtained for the housekeeping gene(3 — actin, relative proportions of mRNA syhthesis could be determined within each. The data were analyzed by using spssll. 0.8. Enzyme -linked immunosorbent assay( ELJSA) for MCP - 1 Production; Osteoblasts were cultured in 24 - well plates to reach confluent monolayers.P. g ^P. e at a density of 106and 108 CFU/ml were added into ostoblasts and co - cultured for 24h. The 108 CFU/ml P. g^P. e were stimulated the cells for 0hN 3hA6h^l2hN24h and 48h. the cells were discarded and the supernatants were collected . The concentration of MCP - 1 in samples were measured using ELJSA kit of MCP - 1 according to the manufactures instructions. The supernatyants were diluted by 10 times with diluents provided with the kit and the protein a-mounts of MCP - 1 secreted by osteoblasts were assayed with the kit. The concentration of MCP - 1 were calculated by the standard calibration curve.Results1. The effects of P. g and P. e on the growth and proliferation in cultured osteoblasts:The results of AlamarBlue? showed that P. g and P. e can decreased the proliferation of rat osteoblasts. When the cells were stimulated with 10 - 10 CFU/ml P. g and P. e, the proliferation of normal rat osteoblasts was higher than the infected cells. The proliferations were decreasing according with the concen-
trations of bacterias increasing. Statiscally significant differences were detected at concectration of 107CFU/ml (P<0.05)and 109CFU/ml(P <0.01).2 . The effects of P. g and P. e on the structures in cultured osteoblasts: The results of transmission electron microscope showed that P. g and P. e can damage the ultrastructures of osteoblasts. They can impair the nucleus and mitochondria, expande endoplasmic reticulum, form myelinated body.3. The effects of P. g and P. e on the expression of OPG and RANKL mR-NA in cultured osteoblasts:P. g and P. e induced to the express of OPG and RANKL in osteoblasts at gene level in a dose - and time — dependent manner. When osteoblasts were stimulated by 10\l09 CFU/ml P. gand P. efor 24h, the expression of RANKL mRNA were higher than the control. Statistical significant difference were observed . Kinetic studies showed that RANKL mRNA increased at 12h post stimulation , RANKLmRNA continued to increase . The basic expression of OPGmR-NA in cultured rat osteoblasts was very obvious, P. g^P, e inhibited its expression in dose - and time - dependent manner, the Ratio of OPG and RANKLmRNA was reversed.4. The effects of P. g and P. e on the MCP — 1 protein secreted by cultured rat osteoblasts:MCP - 1 protein secreted by rat osteoblasts infected with P. g and P. e were more than in normal control. The higher concentrion (10 CFU/ml P. g and P. e) were more violently than lower( 10°CFU/mlP. g and P. e) . In addition,the up - regulated effects were found in time - dependent manner. The MCP - 1 protein secreated by the cells were increased after being stimulated for 6h , and continued to increased to reach a maximal level at 24h, then decreased at 48h.Conclusions1 P. g and P. e can inhibit the proliferation of osteoblasts, and their effects were in dose - and time - dependent manner. The virulence could be bacterial cell component.2. P. g and P. e can damage the structure of osteoblasts and resulting in the
dissoving of the cells .3. P. g and P. e can increase the expression of RANKLmRNA in dose - and time - dependent manner, at the same time , inhibite the expression of OPGmR-NA . The bacterias could mediate the metabolism of alveolar at gene level, they were the important stimulating factors in bone resorption. And this may also point out new therapeutic approaches.4. P. g and P. e can up - regulate the MCP - 1 expression in osteoblasts. MCP - 1 are specific chemoattrctants for monocytes and neutropgils, and it seem to contribute to leukocyte recruitment which can increase the amount of preoste-oclast , accelerate the information of osteoclast.
牙龈卟啉单胞菌(porphyromonas gingivalis,P.g)和牙髓卟啉单胞菌(porphyromonas endodontalis,P.e)是口腔卟啉菌属中两种重要的细菌。二者都是G-产黑色素的专性厌氧球杆菌,是目前公认的牙源性感染的优势致病菌,与牙周炎及根管感染导致的根尖周炎病变的发生和发展密切相关。很多研究表明,牙周炎和根尖周炎的临床症状及病变进程与牙周袋内及感染根管内的P.g、P.e密切相关。这些细菌能影响牙周及根尖组织中牙龈成纤维细胞、牙髓细胞、牙周膜细胞、血管内皮细胞等多种细胞炎症介子的表达,但其确切的致病机理尚未完全明确。目前已知P.g、P.e的致病物质有很多,包括脂多糖、外膜蛋白、荚膜、蛋白酶以及细菌的代谢产物等,这些致病物质可以通过多种途径逃脱机体的免疫防御反应,作用于牙周组织和根尖组织,导致牙周和根尖周围硬组织和软组织的分解破坏,致使牙齿松动乃至脱落。研究P.g、P.e在牙周和根尖周组织破坏中的作用,能明确牙槽骨吸收的病理机制,可以为临床上治疗牙槽骨过度吸收奠定理论基础,在牙源性感染的基础研究中有重大的意义。
牙周炎和根尖周炎的一个重要的病理改变是根尖及根周牙槽骨的吸收。牙槽骨是人体最活跃的骨组织,处于不断的自身修复和改建中。很多研究资料报告,刺激骨吸收的因素如PGE_2、IL-1,IL-6,TNF等都是作用于破骨细胞的前体细胞,在集落刺激因子的作用下,促进其向破骨细胞的分化和成熟并使其激活,同时抑制破骨细胞的凋亡。
关于P.g、P.e对成骨细胞代谢的影响,目前国内外研究报告都不多见。本实验通过研究P.g、P.e对成骨细胞的超微结构、细胞的活性及增殖能力、表达前炎症因子MCP-1(monocyte chemoattractant protein 1)以及骨代谢的最终调节因子OPG(osteoprotegerin)、RANKL(receptor activator of NF-κB ligand)的影响,进一步探讨P.g、P.e在牙周炎和根尖周炎发病中的致
Porphyromonas gingivalis (P. g) and Porphyromonas endodontalis(P. e)are Gram - negative, obligate anaerobic, asaccharolytic black - pigmented microorganisms, which are frequently detected in infected root canals and periodontitis , and they are thought to be the most important pathogens in periodontitis and periapical inflammation. They have gained special prominence in the search for etiologic organisms associated with periapical periodontitis and periodontitis . Many reports showed that the progress and the clinical symptom are closely related with P. g P. e,which exist in subgingival and infected roots. These bacterias can influences the expression of many inflammatory cytokines in various cells such as gingival fibro,pulp cell, blood vessel endothelial cell etal. However, the exact mechanisms are not fully undertanded . Many researches revealed that P. g and P, e possess many virulent factors, including fimbri, out - membrane protein, proteinase, lipopolysaecharide (LPS) , and so on. These virulent factors could escape the defense system in many ways to destroy periodontal supporting tissue and periapical tissue. It is significant to investigate the pathogene-sis of P. g and P. e in inducing the destructive effects in periapical periodontitis and periodontitis , and these will provide the theoretical basis for treating the bone resorption. One of the importment pathological chararters of periapical periodontitis and periodontitis is the bone resorption.There have been relatively few studies addressing the P. g P. e on osteo-blasts. The purpose of this study is to determine the effects of P. g P. e on osteo-blasts in the structure, the growth and proliferation , the ability of secreating
MCP - 1 and the expression of OPG or RANKLin gene level. In order to find out the possible pathogenesis of bone resorption induced by P. g P. e.Methods1 . Bacterial culture:The strains of P. g ATCC 33277 and P. e ATCC 35406 were grown in Brain Heart infusion Broth, supplemented with 10% , yeast - extract(5g/L) ,Hemin (5mg/L) , and Vitamin k3 ( 1mg/L). Under an atmosphere of 80 % N2,10 % H2,10% CO2 at 31X,. , until at an optical density. Bacterial cells were centrifu-gated on 3500 rpm/min for 10 min, the bacterial purity was determined by phase - contrast microscopy and Gram staining. And then were suspended in PBS at a density of 1010CFU/ml and stored at -70℃ until used.2. Cells culture:New born rats were killed, the calvariae were removed aseptically. The periosteal layers on both sides were carefully stripped off with tweezers under PBS, and washed with PBS for two times. Then the bone specimens were treated by 0.25% trypsinization for 15min at 37℃ ,and the supernatant was discarded. After that the specimens were minsed then were digested by 1 mg/ml collagenase II 31℃ for 90min, the supernatant was collected and centrifugedat at 1000r/ min for 5 min . The collagenase — release cells were plated in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37℃ in a humidified atmosphere of 5% CO_2 and 95% air. The medium was changed by 3 -4d and were serially passaged. The differential adherence method was used to purify the osteoblasts. The cells between the third and fifth passages were used in the following experiments.3. The morphologic observation and immunohistochernical identification oi osteoblasts :The property of osteoblasts were observed and photoed with phase contrast microscope on 100 200 400 times repectivelly. several pieces of glass were placed on the 6 - well plates, then the cells were seeded into them and incubated overnight at 37℃ with 5% CO2. The pieces of glass were dryed . Monoclonal
antibody against I collagen were employed to identificated osteoblasts by immu-nohistochemical method . The cell nucleus and cytoplasm were observed.4. Effects of bacterias on the proliferation of rat osteoblasts : Osteoblasts were collected and replated at a density of 105 cells/ml in 96 -well flat - bottomed plates for lOOjxl per well and incubated overnight at 37^1 with 5% CO2 to be confluent monolayers ,the solutions of P. g^ P. e at the density of 1010CFU/ml were diluted with DMEM containing 10% FCS into serial concentrations to stimulate osteoblastes , co - cultivated for 24h. the changes of proliferation to P. g AP. ewere determined by the percents of reduced AlamarBlue?.5. Observation of structure of cultured osteoblasts with transmission electron microscope:Osteoblasts were harvested by 0.25% Trypsinafter co - cultivation with 108 CFU/ml P. g ^P. e for 24h, they were washed with PBS . All of the cells samples were fixed and were sectioned in thickness. The changes of cell and were invested with transmission electron microscope.6. Detection of OPG and RANKL mRNA expression in osteoblasts by reverse transcriptase - polymerase chain reaction:Osteoblasts were cultured in flasks to reach confluent monolayers. P. g ^P. e at a density of 10\l09CFU/ml were added into the cells, osteoblasts in 10% FCS DMEM without acterias were employed as a negative control. The cells were collected after 24h co - cultured and stored at - d>0X . to investigate the kinetic effect of the bacterias, we used 10 CFU/ml P. g^P. e to stimulate osteoblastes for various times. The cells were collected at 0A6->12-N18^24h after co - cultivation, subsequently, total RNA were isolated from cells in each flask using TRIzol reagent according to the manufacturers instructions. The complementary DNA were synthesized from RNA using a cDNA Cycle Kit,which uses reverse transcriptase. The cDNA were amplified with TagDNA polymerase and specific primers in a thermal cycler. The PCR conditions were : initial denaturation 3 min,94cC : denaturation 1 min, 95X : annealing 1 min50X : extension 1 min, 72X ,fop 35 cycles: followed by a 7 min elongation step at 72^. Primer sequences were; OPG, forward, 5 ' -TTGGCTGAGTGTTCTGGT -3 ' ,re-verse5 ' -TTG GGA AAG TGG TAT GCT -3 ' ; RANKL, forward, 5 ' -
CAT CGG GTT CCC ATA AAG - 3 ' , reverse 5 ' - TGG ACA CCT GGA CGC TAA - 3 ' ; p - actin, forward, 5 ' - TGT ATG CCT CTG GTC GTA CCA C -3 ' , reverse5 ' - ACT GAG TAC TTG CGC TCA GGA G -3 ' PCR products were 423bp, 357bp and 690bp resperctively.7. Quantifying mRNA of OPG and RANKL;15 ill of the products including OPG 5ul, RANKL 5uland (3 - actin 5ul. PCR products were mixed ,then were electrophoresed for lh on 2% agarose gel and visualized by ethidium bromide staining. The bands for OPG and RANKL were noted, these bands were consistent with the size as designed by primers, when the band densities were measured and compared with the density of the band obtained for the housekeeping gene(3 — actin, relative proportions of mRNA syhthesis could be determined within each. The data were analyzed by using spssll. 0.8. Enzyme -linked immunosorbent assay( ELJSA) for MCP - 1 Production; Osteoblasts were cultured in 24 - well plates to reach confluent monolayers.P. g ^P. e at a density of 106and 108 CFU/ml were added into ostoblasts and co - cultured for 24h. The 108 CFU/ml P. g^P. e were stimulated the cells for 0hN 3hA6h^l2hN24h and 48h. the cells were discarded and the supernatants were collected . The concentration of MCP - 1 in samples were measured using ELJSA kit of MCP - 1 according to the manufactures instructions. The supernatyants were diluted by 10 times with diluents provided with the kit and the protein a-mounts of MCP - 1 secreted by osteoblasts were assayed with the kit. The concentration of MCP - 1 were calculated by the standard calibration curve.Results1. The effects of P. g and P. e on the growth and proliferation in cultured osteoblasts:The results of AlamarBlue? showed that P. g and P. e can decreased the proliferation of rat osteoblasts. When the cells were stimulated with 10 - 10 CFU/ml P. g and P. e, the proliferation of normal rat osteoblasts was higher than the infected cells. The proliferations were decreasing according with the concen-
trations of bacterias increasing. Statiscally significant differences were detected at concectration of 107CFU/ml (P<0.05)and 109CFU/ml(P <0.01).2 . The effects of P. g and P. e on the structures in cultured osteoblasts: The results of transmission electron microscope showed that P. g and P. e can damage the ultrastructures of osteoblasts. They can impair the nucleus and mitochondria, expande endoplasmic reticulum, form myelinated body.3. The effects of P. g and P. e on the expression of OPG and RANKL mR-NA in cultured osteoblasts:P. g and P. e induced to the express of OPG and RANKL in osteoblasts at gene level in a dose - and time — dependent manner. When osteoblasts were stimulated by 10\l09 CFU/ml P. gand P. efor 24h, the expression of RANKL mRNA were higher than the control. Statistical significant difference were observed . Kinetic studies showed that RANKL mRNA increased at 12h post stimulation , RANKLmRNA continued to increase . The basic expression of OPGmR-NA in cultured rat osteoblasts was very obvious, P. g^P, e inhibited its expression in dose - and time - dependent manner, the Ratio of OPG and RANKLmRNA was reversed.4. The effects of P. g and P. e on the MCP — 1 protein secreted by cultured rat osteoblasts:MCP - 1 protein secreted by rat osteoblasts infected with P. g and P. e were more than in normal control. The higher concentrion (10 CFU/ml P. g and P. e) were more violently than lower( 10°CFU/mlP. g and P. e) . In addition,the up - regulated effects were found in time - dependent manner. The MCP - 1 protein secreated by the cells were increased after being stimulated for 6h , and continued to increased to reach a maximal level at 24h, then decreased at 48h.Conclusions1 P. g and P. e can inhibit the proliferation of osteoblasts, and their effects were in dose - and time - dependent manner. The virulence could be bacterial cell component.2. P. g and P. e can damage the structure of osteoblasts and resulting in the
dissoving of the cells .3. P. g and P. e can increase the expression of RANKLmRNA in dose - and time - dependent manner, at the same time , inhibite the expression of OPGmR-NA . The bacterias could mediate the metabolism of alveolar at gene level, they were the important stimulating factors in bone resorption. And this may also point out new therapeutic approaches.4. P. g and P. e can up - regulate the MCP - 1 expression in osteoblasts. MCP - 1 are specific chemoattrctants for monocytes and neutropgils, and it seem to contribute to leukocyte recruitment which can increase the amount of preoste-oclast , accelerate the information of osteoclast.
引文
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11.张凤秋,杨连甲,吴织芬.牙龈卟啉菌蛋白酶的结构和作用.《牙体牙髓 牙周病学杂志》2004:14(6)352-354
12.汤亚玲、潭红、周学东。牙髓卟啉单胞菌的生物特性与致病性《国外医学》口腔医学分册 2003:30(2);108-109
13. Kim CS, Choi SH, Choi BK, etal The effect of recombinant human bone morphogenetic protein-4 on the osteoblastic differentiation of mouse calvarial cells affected by porphyromonas gingivalis. J Periodontol 2002: 73; 1126-1132
14. Matsushita K, Tajima T, Tomita K, etal. Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodntopathic black- pigmented bacteria in patients with multilesional periapical periodontitis. J Endodon 1999; 25(12); 795-79
15. Yang CL, Tsai CH, Huang FM, et al. Induction of interleukin-6 gene expressin by pro -inflammatory cytokines and black -pigmented bacteroides in human pulp cell cultures. International Endodontic Journal 2003 36: 352-357
16. Yanling jiang,and Herbert schilder. An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells. J Endodontics 2002, 28 (4) 279-282
17. Kazuhiko Nakata, Masahiro Yamasaki, Takahiro Iwata, etal. Anaerobic Bacterial extracts influence production of matrix metalloproteinases and their inhibitors by human dental pulp cells. J Endod 2000; 26; 410-413
18 . Hyun Jung ko and Sung sam hm. Production of macrophage inflammatory protein MIP - 1αand MIP - 1β by human polymorphonuclear neutrophils stimulated with porphyromonas endodontalis iipopolysaccharide. J Endodontics 2002; 28; 754-757
19. Jing Y, Russll TR, Schilder H, Graves DT. Endodontic pathogens stimulate monocyte chemoattractant protein- 1 and interleukin- 8 in mononuclear cells. J Endod 1998; 24; 86-90
20. Amasaki M, Nakata K, Imaizumi I, etal. Cytotoxixc effect of endodontic bacteria on periapical fibroblasts. J Endod 1998; 24; 534-539
21. Volejni kova S, Laskari M, Marks SCJr, etal. Monocyte recruitment and expression of monocyte chemoattractant protein-1 are developmentally regu lated in remodeling bone in the mouse. Am J Pathol. 1997: 150(5): 1711-1721.
22. Darveau RP, Belton CM, reife RA, etal. Local chemokine paralysis, a novel pathogenic mechanism for porphyromonas gingivalis. infect immune 1998; 66; 1660-1665
23.毛松,陈森,何安光。牙龈卟啉单胞菌对脐静脉血管内皮细胞表达IL-8和MCP-1mRNA的影响.《牙体牙髓牙周病学杂志》2002;12(2):72-75
24. Steffen Mj, holt SC, ebersole J1. Porphyromonas gingivalis induction of mediator and eytokine secretion by human gingival fibroblasts. Oral microbial immunol; 2000; 15; 172-1803
25. Yu - chao chang, Chung - chih lai, Shun - fa yang, etal. Stimulation of matrix metalloproteinases by black -pigmented baeteroides in human pulp and periodontal ligament cell cultures. J endodon 2002; 28; 90-931
26. Nair, S. P, S. meghji, M. Wilson, etal. Bacterially induced bone destruction: mechanisms and misconceptions. Infect immune. 1996; 64: 2371-2380
27. Kusumoto Y, Hirano H, Saitoh K, etal . Human gingival epithelial cells produce chemotactie factorsb interleukin- 8 and monocyte chemoattractant protein -1 after stimulation with porphyromonas gingivalis via toll -like receptor 2. J periodontal. 2004; 75930; 370-379
28.朱小玲,孟焕新,陈智滨.快速进展性牙周炎牙龈组织中单核/巨噬细胞浸润与MCP-1表达.《中华口腔医学杂志》1999;34;214-216
29. Nassar H, Chou Hh, Khlgatian M, etal. Role for fimbria and lysine -specific cysteine proteinase gingipain K in expression of interleukin 8 and monocyte chemoattractant protein in porphyromonas gingivalis -infected endothe lial cells infect immune 2002; 70; 264-276.
30. Matsushita K, Tajima T, Tomita K, etal. Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodontnpathic black- pigmented bacteria in patients with multilesional periapical periodontitis. J Endod 1999; 25(12) 795-799
31. Yang SF, Hsieh YS, Huang FM, etal. Effect of black -pigmented bacteria on the plasminogen - plasmin system in human pulp and osteoblastic cels. Oral Surg Oral Med Oral Pathol . 2003:95(5) ; 621 -625
32. Graves DT, Jiang Y, Valente AJ. The expression of monocyte chemoattrac- tant protein -1 and other chemokines by osteoblasts. front biosci. 1999 ( 4); 571-580
33 . Graves DT, Jiang Y, Valente AF. Regulated expression of MCP - 1 by osteoblastic cells in vitro and in vivo. Histol Histopathol. 1999 ;14(4) ; 347 -1354
34. Illians SR, Jiang Y, Cochran D, etal. Regulated expression of monocyte chemoattractant protein -1 in normal human osteoblastic cells. Am J physi- ol. 1992 ;263(10 194-19916
35. Dosner LJ, Miligkos T, Gilles JA , etal. Monocyte chemoattractant protein - 1 induces monocyte recruitment and that is associated with an increase in numbers of osteoblasts. Bone. 1997: 21(4) ;321 -327
36. Graves DT, Alsulaimani F, Ding Y, etal. Developmentally regulated monocyte recruitment and bone resorption are modulated by functional deletion of the monocytic chemoattractant protein -1 gene. Bone. 2002:31(2) ;282 -28
37. Odell LJ, Baumgartner JC, Xia T ,etal. Survey for collagenase gene partC in Porohyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections. J Endod 1999 ;25(8) ; 555 -558
38. Lacey DL, Timms E, Tan H - L, etal. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation . Cell 1998 ; 93 :165 -176
39. Simonet WS, Lacey DL, Dunstan CR, etal. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997; 89; 309 -319
40. Yasuda H, Shima N, Nakagawa N, etal. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci (USA) 1998; 95; 3597 - 3602
41. Jun Nukaga, Makoto Kobayashi, Toshimasa S hinki, etal. Regulatory
effects of interleukin- 1β and prostaglandin E2 on expression of receptor activator of nuclear factor-kb ligand in human periodontal ligament cells. J Periodontol 2004 75; 249-259
42. Hosoya S, Matsushima K. Stimulation of interleukin -1β production of human dental pulp cells by porphyromonas endodontalis lipopolysaccharide. J Endodon. 1997; 23; 39-42
43. Lin SK, Yen-ping KM, Wang JS, etal. Differential regulation of interleukin-6 and inducible cyclooxygenase gene expression by cytokines through prostaglandin- dependent and independent mechanisms in human dental pulp fibroblasts. J Endodon 2002; 28: 197-201
44.沈辉,潘时中。骨保护素与骨质疏松《国外医学》内分泌分册2004;24(4)230-233
45. Wong BR, Rho J, Arron J, etal. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c - jun N - terminal kinase In T Cells. J Biol Chem 1997; 272; 25190-25194
46. Anderson DM, Maraskovsky E, Billingsley WL, etal. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature. 1997; 390; 175-179
47. Wong B, Josien R, Choi Y. TRANCE is a TNF family member that regulates dendritic cells and osteoclast function. J Leukoc Biol 1999; 65; 715-724
48. Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun. 1999; 256 (3); 449-455
49.张小磊,彭彬。TNF配体家族的新成员-RANKL的研究进展《国外医学》口腔医学分册2004:31(3)179-180
50.于世凤,李斌斌。骨保护因子及其配体调控骨吸收的研究进《国外医学》口腔医学分册 2004:31(4)249-252
51. Palmqvist Py, Persson E, Conaway H, eta1. IL - 1, leukemia.inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF - Kbhgand, osteoprotegerin, and receptor activator of NF - kB in mouse calvariae . J immunol 2002: 169: 3353-336223.
52. Sun-Kyeong Lee, Judy Kalinowski, Sanadra Jastrzebski, etal. 1,25(Oh)2 Vitamin D3 -Stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced IL- 1α and receptor activator of NF -KB ligand production in osteoblasts. J Immunol 2002: 169: 2374-2380
53. Palmqvist PY, Persson E, Herschel Conaway H. , etal. IL -6, Leukemia inhibitory factor, and oncoastatin M Stimulate bone resorption and regulate the expression of receptor activator of NF-Kbligand , osteoprotegerin, and receptor activator of NF - KB in mouse calvariae. J Immnunol 2002; 169: 3353-3362
54. Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Disease. 2002; 8(3); 147-159
55. Yasuda H, Shima N, Nakagawa N, etal. A novel molecular mechanism modulating osteoclast differentiation and function. Bone. 1999 25 (1); 109-113
56. Jing Y and Schilder H. An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells. J endodontics. 2002 28; (4) 279-282
57.张晓磊,彭彬.大鼠实验性根尖周病变中RANKL的免疫组化研究《中华口腔医学杂志》2004 39(3):180
58. Ogasawara T, Yoshimine Y, Kiyoshima T, etal. in situ expression of RANKL, RANK, Osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue. J Periodontal Res. 2004: 39 (1); 42-49
59. Takeshi Kikuchi, Tetsuya Matsuguchi, Naotake Tsuboi, etal. Gene expression of osteociast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via toli-like receptors. I Immunol. 2001; 166; 3574-3579
60. Shimizu-Ishiura M, Kawana F, Sassaki T. Osteoprotegerin administrationreduces femoral bone loss in ovariectomized mice via impairment of osteoelast structure and function. J Electron Mierose 2002; 51; 315-325
61. Zou W, Hakim I, Tsehoep K, etal. Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoelast differentiation by an autoerine mechanism. J Cell Bioehem. 2001; 83(1) 70-83
2. Hashioka K, Yamasaki M, Nakane A, etal. The relationship between clinical symptoms and anaerobic bacteria from infecting root canals. J Endodon. 1992; 18; 558-561
3. Machado JC, Siqueira jF, Hirata R, etal. Detection of porphyromonas endodontalis in infected root canals by 16S rRNA gene -directed polymerase chain reaction. J Endodon 2000; 26 (12); 729-732
4. Siqueira JF, Rocas IN, Oliveira JC, etal. Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed polymerase chain reaction. J Endodon 2001; 27(3); 164-167
5. Mayanas G, Sano T, Shimauchi H, etal. Detection frequency of periodontitis-associated bacteria by polymerase chain reaction subgingival and supragingival plaque of periodontitis and herlthy subjects. Oral microbial immunol. 2004 19(60; 379-385
6. Bogen G, Slots J. Black-pigmented anaerobic rods in closed periapical lesions. Int Endod J 1999; 32; 204-210
7. Murakami Y, Hanazawa S, Tanahashi H, etal. A possible mechanism of maxillofacial abscess formation: involvement of Porphyromonas endodontalis lipopolysacch-aride via the expression of inflammatory cytokines. Oral microbiology and immunology. 2001; 16; 321-325
8. Kubortiwe M, Ameno A, Kimtma KR, etal. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbio immunol. 2004: 19(30; 168-176
9.徐屹,李雪,黄萍。龈下牙周微生物检测技术的进展情况.《国外医学》口腔医学分册 2004;31(6)435-437
10. Rosen G, Shoshani M, Naor R, etal. The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease. Oral Microbiol immunol. 2001; 16; 326-331
11.张凤秋,杨连甲,吴织芬.牙龈卟啉菌蛋白酶的结构和作用.《牙体牙髓 牙周病学杂志》2004:14(6)352-354
12.汤亚玲、潭红、周学东。牙髓卟啉单胞菌的生物特性与致病性《国外医学》口腔医学分册 2003:30(2);108-109
13. Kim CS, Choi SH, Choi BK, etal The effect of recombinant human bone morphogenetic protein-4 on the osteoblastic differentiation of mouse calvarial cells affected by porphyromonas gingivalis. J Periodontol 2002: 73; 1126-1132
14. Matsushita K, Tajima T, Tomita K, etal. Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodntopathic black- pigmented bacteria in patients with multilesional periapical periodontitis. J Endodon 1999; 25(12); 795-79
15. Yang CL, Tsai CH, Huang FM, et al. Induction of interleukin-6 gene expressin by pro -inflammatory cytokines and black -pigmented bacteroides in human pulp cell cultures. International Endodontic Journal 2003 36: 352-357
16. Yanling jiang,and Herbert schilder. An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells. J Endodontics 2002, 28 (4) 279-282
17. Kazuhiko Nakata, Masahiro Yamasaki, Takahiro Iwata, etal. Anaerobic Bacterial extracts influence production of matrix metalloproteinases and their inhibitors by human dental pulp cells. J Endod 2000; 26; 410-413
18 . Hyun Jung ko and Sung sam hm. Production of macrophage inflammatory protein MIP - 1αand MIP - 1β by human polymorphonuclear neutrophils stimulated with porphyromonas endodontalis iipopolysaccharide. J Endodontics 2002; 28; 754-757
19. Jing Y, Russll TR, Schilder H, Graves DT. Endodontic pathogens stimulate monocyte chemoattractant protein- 1 and interleukin- 8 in mononuclear cells. J Endod 1998; 24; 86-90
20. Amasaki M, Nakata K, Imaizumi I, etal. Cytotoxixc effect of endodontic bacteria on periapical fibroblasts. J Endod 1998; 24; 534-539
21. Volejni kova S, Laskari M, Marks SCJr, etal. Monocyte recruitment and expression of monocyte chemoattractant protein-1 are developmentally regu lated in remodeling bone in the mouse. Am J Pathol. 1997: 150(5): 1711-1721.
22. Darveau RP, Belton CM, reife RA, etal. Local chemokine paralysis, a novel pathogenic mechanism for porphyromonas gingivalis. infect immune 1998; 66; 1660-1665
23.毛松,陈森,何安光。牙龈卟啉单胞菌对脐静脉血管内皮细胞表达IL-8和MCP-1mRNA的影响.《牙体牙髓牙周病学杂志》2002;12(2):72-75
24. Steffen Mj, holt SC, ebersole J1. Porphyromonas gingivalis induction of mediator and eytokine secretion by human gingival fibroblasts. Oral microbial immunol; 2000; 15; 172-1803
25. Yu - chao chang, Chung - chih lai, Shun - fa yang, etal. Stimulation of matrix metalloproteinases by black -pigmented baeteroides in human pulp and periodontal ligament cell cultures. J endodon 2002; 28; 90-931
26. Nair, S. P, S. meghji, M. Wilson, etal. Bacterially induced bone destruction: mechanisms and misconceptions. Infect immune. 1996; 64: 2371-2380
27. Kusumoto Y, Hirano H, Saitoh K, etal . Human gingival epithelial cells produce chemotactie factorsb interleukin- 8 and monocyte chemoattractant protein -1 after stimulation with porphyromonas gingivalis via toll -like receptor 2. J periodontal. 2004; 75930; 370-379
28.朱小玲,孟焕新,陈智滨.快速进展性牙周炎牙龈组织中单核/巨噬细胞浸润与MCP-1表达.《中华口腔医学杂志》1999;34;214-216
29. Nassar H, Chou Hh, Khlgatian M, etal. Role for fimbria and lysine -specific cysteine proteinase gingipain K in expression of interleukin 8 and monocyte chemoattractant protein in porphyromonas gingivalis -infected endothe lial cells infect immune 2002; 70; 264-276.
30. Matsushita K, Tajima T, Tomita K, etal. Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodontnpathic black- pigmented bacteria in patients with multilesional periapical periodontitis. J Endod 1999; 25(12) 795-799
31. Yang SF, Hsieh YS, Huang FM, etal. Effect of black -pigmented bacteria on the plasminogen - plasmin system in human pulp and osteoblastic cels. Oral Surg Oral Med Oral Pathol . 2003:95(5) ; 621 -625
32. Graves DT, Jiang Y, Valente AJ. The expression of monocyte chemoattrac- tant protein -1 and other chemokines by osteoblasts. front biosci. 1999 ( 4); 571-580
33 . Graves DT, Jiang Y, Valente AF. Regulated expression of MCP - 1 by osteoblastic cells in vitro and in vivo. Histol Histopathol. 1999 ;14(4) ; 347 -1354
34. Illians SR, Jiang Y, Cochran D, etal. Regulated expression of monocyte chemoattractant protein -1 in normal human osteoblastic cells. Am J physi- ol. 1992 ;263(10 194-19916
35. Dosner LJ, Miligkos T, Gilles JA , etal. Monocyte chemoattractant protein - 1 induces monocyte recruitment and that is associated with an increase in numbers of osteoblasts. Bone. 1997: 21(4) ;321 -327
36. Graves DT, Alsulaimani F, Ding Y, etal. Developmentally regulated monocyte recruitment and bone resorption are modulated by functional deletion of the monocytic chemoattractant protein -1 gene. Bone. 2002:31(2) ;282 -28
37. Odell LJ, Baumgartner JC, Xia T ,etal. Survey for collagenase gene partC in Porohyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections. J Endod 1999 ;25(8) ; 555 -558
38. Lacey DL, Timms E, Tan H - L, etal. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation . Cell 1998 ; 93 :165 -176
39. Simonet WS, Lacey DL, Dunstan CR, etal. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997; 89; 309 -319
40. Yasuda H, Shima N, Nakagawa N, etal. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci (USA) 1998; 95; 3597 - 3602
41. Jun Nukaga, Makoto Kobayashi, Toshimasa S hinki, etal. Regulatory
effects of interleukin- 1β and prostaglandin E2 on expression of receptor activator of nuclear factor-kb ligand in human periodontal ligament cells. J Periodontol 2004 75; 249-259
42. Hosoya S, Matsushima K. Stimulation of interleukin -1β production of human dental pulp cells by porphyromonas endodontalis lipopolysaccharide. J Endodon. 1997; 23; 39-42
43. Lin SK, Yen-ping KM, Wang JS, etal. Differential regulation of interleukin-6 and inducible cyclooxygenase gene expression by cytokines through prostaglandin- dependent and independent mechanisms in human dental pulp fibroblasts. J Endodon 2002; 28: 197-201
44.沈辉,潘时中。骨保护素与骨质疏松《国外医学》内分泌分册2004;24(4)230-233
45. Wong BR, Rho J, Arron J, etal. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c - jun N - terminal kinase In T Cells. J Biol Chem 1997; 272; 25190-25194
46. Anderson DM, Maraskovsky E, Billingsley WL, etal. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature. 1997; 390; 175-179
47. Wong B, Josien R, Choi Y. TRANCE is a TNF family member that regulates dendritic cells and osteoclast function. J Leukoc Biol 1999; 65; 715-724
48. Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun. 1999; 256 (3); 449-455
49.张小磊,彭彬。TNF配体家族的新成员-RANKL的研究进展《国外医学》口腔医学分册2004:31(3)179-180
50.于世凤,李斌斌。骨保护因子及其配体调控骨吸收的研究进《国外医学》口腔医学分册 2004:31(4)249-252
51. Palmqvist Py, Persson E, Conaway H, eta1. IL - 1, leukemia.inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF - Kbhgand, osteoprotegerin, and receptor activator of NF - kB in mouse calvariae . J immunol 2002: 169: 3353-336223.
52. Sun-Kyeong Lee, Judy Kalinowski, Sanadra Jastrzebski, etal. 1,25(Oh)2 Vitamin D3 -Stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced IL- 1α and receptor activator of NF -KB ligand production in osteoblasts. J Immunol 2002: 169: 2374-2380
53. Palmqvist PY, Persson E, Herschel Conaway H. , etal. IL -6, Leukemia inhibitory factor, and oncoastatin M Stimulate bone resorption and regulate the expression of receptor activator of NF-Kbligand , osteoprotegerin, and receptor activator of NF - KB in mouse calvariae. J Immnunol 2002; 169: 3353-3362
54. Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Disease. 2002; 8(3); 147-159
55. Yasuda H, Shima N, Nakagawa N, etal. A novel molecular mechanism modulating osteoclast differentiation and function. Bone. 1999 25 (1); 109-113
56. Jing Y and Schilder H. An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells. J endodontics. 2002 28; (4) 279-282
57.张晓磊,彭彬.大鼠实验性根尖周病变中RANKL的免疫组化研究《中华口腔医学杂志》2004 39(3):180
58. Ogasawara T, Yoshimine Y, Kiyoshima T, etal. in situ expression of RANKL, RANK, Osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue. J Periodontal Res. 2004: 39 (1); 42-49
59. Takeshi Kikuchi, Tetsuya Matsuguchi, Naotake Tsuboi, etal. Gene expression of osteociast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via toli-like receptors. I Immunol. 2001; 166; 3574-3579
60. Shimizu-Ishiura M, Kawana F, Sassaki T. Osteoprotegerin administrationreduces femoral bone loss in ovariectomized mice via impairment of osteoelast structure and function. J Electron Mierose 2002; 51; 315-325
61. Zou W, Hakim I, Tsehoep K, etal. Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoelast differentiation by an autoerine mechanism. J Cell Bioehem. 2001; 83(1) 70-83