【作者】 李秀兰；

【导师】 艾红军；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2005， 博士

【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PGE₂、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更多还原

【Abstract】 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 secreatingMCP - 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% CO₂ 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 . Monoclonalantibody 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 thedissoving 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.更多还原