摘要
抗生素在口腔颌面部感染的治疗中发挥着重要作用。研究发现牙周致病菌可侵入牙龈上皮细胞和成纤维细胞内,以逃避宿主的防御机制;有些抗生素可在上述细胞内及龈沟液中积聚,其浓度高于相应血药浓度,而有些药物不能在细胞内积聚,胞内药物浓度相近或低于相应血药浓度,这使得抗生素在应用中益发复杂。研究细胞对不同抗生素摄取机制对指导临床用药及提高药效的发挥具有重要作用。牙周细胞对药物的摄取及跨细胞转运是人工种植牙给药系统以及牙根管给药系统理论的重要基础之一,本课题组前期研究已发现牙周组织细胞可选择性摄取部分常用抗生素,为进一步探讨这两种给药系统的可行性及规律,本文对牙周细胞摄取抗生素的机制进行了初步研究,并建立和验证人牙周膜和牙龈成纤维细胞药物转运模型。全文分两大部分。
第一部分人牙周膜和牙龈成纤维细胞摄取抗生素机制的初步研究
实验一人牙周膜和牙龈成纤维细胞对甲硝唑及替硝唑摄取机制的研究
目的:研究人牙周膜成纤维细胞(HPDLF)及人牙龈成纤维细胞(HGF)对甲硝唑和替硝唑的摄取,探讨摄取的方式及通过牙根管给药的可行性。材料与方法:采用高效液相色谱法研究HPDLF及HGF分别与甲硝唑及替硝唑孵育后不同时间细胞内药物的浓度,细胞内药物浓度与细胞外药物浓度的关系,以及温度、pH和转运抑制剂丙磺舒和腺嘌呤对细胞摄取甲硝唑和替硝唑的影响。结果:孵育3min后HPDLF及HGF胞内甲硝唑和替硝唑浓度即可达到细胞外水平;细胞内甲硝唑和替硝唑浓度随着细胞外甲硝唑和替硝唑浓度的增加而呈线性增加;温度、pH和转运抑制剂对HPDLF及HGF摄取甲硝唑和替硝唑无明显影响。结论:HPDLF及HGF可快速摄取甲硝唑和替硝唑,证实了根管给药的可行性;甲硝唑及替硝唑通过简单扩散方式进入HPDLF及HGF细胞内;甲硝唑与替硝唑在进入HPDLF及HGF的动力学之间没有显著差别,同时HPDLF与HGF间在摄取甲硝唑及替硝唑时也没有差别。
实验二有机离子转运体mRNA在人牙龈成纤维细胞上的表达
目的:检测有机阳离子转运家族及有机阴离子转运家族在HGF的表达,并探讨其在人牙龈成纤维细胞主动转运中的作用。方法:采用RT-PCR法检测有机阳离子转运体有机阴离子转运体家族成员的mRNA在HGF上的表达。结果:HGF有OCT1、OCT2、OCTN1及OAT1-4 mRNA的表达。结论:证实了HGF存在有机阳离子转运体及有机阴离子转运体的推测。
第二部分人牙周膜及牙龈成纤维细胞药物转运模型的建立及对部分口腔常用抗生素的跨细胞转运
实验一人牙周膜及牙龈成纤维细胞药物转运模型的建立与验证
目的:借鉴Caco-2细胞模型,建立HPDLF及HGF药物转运模型,用以研究牙根管给药系统及人工种植体牙给药系统中牙周细胞对药物的转运。方法:将原代培养的HPDLF及HGF分别接种到Transwe 11聚酯滤膜上,连续以光学显微镜观察细胞的生长排列情况,测定跨膜电阻,并以渗漏标志物荧光素钠等指标进行检测。结果:HPDLF及HGF接种1周后细胞基本汇合,到2周时细胞连接紧密且完整,细胞呈复层生长,跨膜电阻分别达56.14±7.4 3及57.34±7.62Ω·cm~(-2),经荧光素钠孵育30分钟检测细胞旁渗漏小于1%。
结论:所建模型在2周后细胞排列情况可模拟体内牙周膜和牙龈生理状态,且符合模型建立要求,可作为研究牙周细胞转运药物的体外模型。
实验二人牙周膜成纤维细胞及牙龈成纤维细胞药物转运模型对甲硝唑、替硝唑、米诺环素及头孢噻肟钠的转运
目的:利用HPDLF及HGF药物转运模型研究HPDLF及HGF对于部分口腔常用抗生素的跨细胞转运,探讨对不同药物的转运规律。方法:在建立的HPDLF及HGF药物转运模型,以高效液相色谱法测定HPDLF及HGF对甲硝唑等药物的跨细胞转运。结果:甲硝唑、替硝唑及米诺环素经7-8min即可完全被HPDLF及HGF跨细胞转运,头孢噻肟钠分别经50及46min左右即可完全被HPDLF及HGF跨细胞转运。结论:HPDLF及HGF可跨细胞转运甲硝唑、替硝唑、米诺环素及头孢噻肟钠;进一步证实了人工种植牙给药系统以及牙根管给药的可行性。
Some periodontitis pathogen can invade oral epithelial cells and gingival fibroblasts,evading the host response.Some antibiotics can attain higher levels in the cells described above and in gingival crevice fluid than that in blood,while some antibiotics couldn't be accumulated in cells.The study on the uptake mechanism of antimicrobials by cells will provide a guide to clinical appilication of antimicrobials and improve the therapeutic effect of antimicrobials.Uptake and transcellular transport of agents by human periodontal ligaments fibroblasts(HPDLF) and human gingival fibroblasts(HGF) are theoretical basis of dental implant administration system and dental root canal administration system. To explore the feasibility of local drug administration routes described above,we studied the uptake mechanism of antibiotics by HPDLF and HGF preliminarily, and developed HPDLF and HGF drug transport models.The study includes two parts.
Part 1 Preliminary Study on the Uptake Mechanism of Antibiotics by HPDLF and HGF
Experiment 1 Study on the Uptake Mechanisms of Metronidazole and Tinidazole by HPDLF and HGF
Objective:To investigate the uptake of metronidazole and tinidazole byHPDLF and HGF.Methods:After HPDLF and HGF incubated with metronidazole and tinidazole respectively,the intracellular agents concentrations were measured at different times with high performance liquid chromatography(HPLC).The relationship between the intracellular agents concentrations and extracellurlar agents concentrations,and the effect of temperature,pH and transport inhibitors on the uptake of agents by HPDLF and HGF were studied.Results:The intracellular metronidazole and tinidazole concentrations reached the extracellular levels in 3 minutes after incubation;The intracellular metronidazole and tinidazole concentrations increased linearly with the extracellular level; Temperature,pH and transport inhibitors had no significant effect on the uptake of agents by HPDLF and HGF.Conclusions:HPDLF and HGF can take up metronidazole and tinidazole rapidly via simple diffusion,which proves the feasibility of drug delivery through the dental implant administration system and root canal.
Experiment 2 Expression of Organic Ion Transporters mRNA in Human Gingival Fibroblasts
Objective:To detect the expression of organic cation transporters(OCTs) and organic anion transporters(OATs) mRNA in human gingival fibroblasts(HGF) and.Methods:RT-PCR was used to detect the expression of OCTs and OATs mRNA in HGF.RESULTS:The expression of OCT1、OCT2、OCTN1 and OAT1-4 mRNA in HGF was found.CONCLUSION:The findings of the test verify the speculation that there exist OCTs and OATs in the HGF.
Part 2 Development of HPDLF and HGF Drug Transport Cell Models and Transcellular Transport of Antibiotics
Experiment 1 Development and Validation of HPDLF and HGF Drug Transport Cell Models
Objective:To develop HPDLF and HGF drug transport cell models.
Methods:HPDLF and HGF were cultivated on polycarbonate filter membrane of transwell respectively.After1,2,3 and 4 weeks of culture,transepithelia electrical resistance(TEER) was detected and the growth of HPDLF and HGF were observed by light microscope.After 2 weeks of culture,the models were observed by the scanning electron microscope(SEM),transmission electron microscope (TEM),and the permeability was measured with fluorescein sodium.RESULTS: HPDLF and HGF cells connected each other tightly and completely after 2 weeks. Observation of section of filter membrane revealed a stratified cell growth of HPDLF and HGF 2 weeks after inoculation,and TEERs of HPDLF and HGF were 56.14±7.43 and 57.34±7.62Ω·cm~(-2) respectively.Being incubated for 30 minutes, less than 1%of fluorescein sodium reached the inferior filter membrane 2 week after inoculation.CONCLUSION:The tests of permeability and TEER were consistent with the demands of development of drug transport cell models. HPDLF and HGF drug transport cell models could be used to study drug transport by HPDLF and HGF in vitro.
Experiment 2 Transcellular Transport of Metronidazole,Tinidazole, Minocycline and Cefotaxime Sodium in HPDLF and HGF Drug Transport Cell Models
Objective:To investigate the transcellular transport of metronidazole,tinidazole, minocycline and cefotaxime sodium in drug transport cell models of HPDLF and HGF.Methods:The transcellular transport of metronidazole,tinidazole, minocycline and cefotaxime were measured with HPLC.Results: Metronidazole,tinidazole and minocycline could be transported through the HPDLF and HGF cell layers completely in 7-8min.Cefotaxime sodium could be transported through the HPDLF and HGF cell layers completely in 50 and 46 min..Conclusion:The transcellular transport of agents by HPDLF and HGF were observed in our study,which proves the feasibility of drug delivery through the root canal.
引文
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