米诺环素对伴放线共生放线杆菌的抑菌作用及对人牙周膜成纤维细胞的生物学作用
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摘要
实验一米诺环素对伴放线共生放线杆菌抑菌作用的研究
目的：检测MINO对Aa的最小抑菌浓度,避免因MINO使用剂量不足造成无法清除Aa的感染并造成菌株的耐药。
方法：复苏菌种,涂布平板,测定Aa生长对数期、生长曲线。琼脂稀释法测定MINO对Aa的MIC。
结果：细菌接种后24h细菌的生长浓度与OD值呈对数线性关系,1-24h时增殖缓慢,48-84h增殖活跃,96h有下降的趋势。MINO对Aa的MIC是8mg／L。
结论：MINO对Aa具有抑菌作用,MIC是8mg／L。
实验二米诺环素对人牙周膜成纤维细胞的生物学作用
目的：探索hPDLF的体外培养方法并进行鉴定。通过检测MINO对hPDLF细胞增殖、细胞周期和对总蛋白、Ⅰ型胶原(COLI)、碱性磷酸酶(ALP)、骨钙素(OCN)的影响,探讨MINO牙周局部给药的效应浓度范围。
方法：选用因正畸需要拔出的前磨牙牙周膜组织为原代培养的组织来源,组织块法、酶消化法、盖玻片固定相结合进行hPDLF原代培养,波形丝蛋白、角蛋白、S-100因子免疫荧光染色进行细胞鉴定。加入矿化液诱导后钙结节染色探讨成骨特性。将不同浓度的MINO(0、10、20、40、100、200mg／L)分别作用于第5代hPDLF,孵育1、4、7、10、14d后MTT法检测细胞增殖；孵育4d后,流式细胞仪检测细胞周期；BCA法检测细胞总蛋白；荧光定量PCR和Western-blot法检测COLI、ALP、OCN的基因表达和蛋白表达。结果采用SPSS 13.0软件包进行分析。
结果：体外培养的hPDLF波形丝蛋白染色阳性,角蛋白、S-100因子染色阴性,证实为hPDLF。hPDLF在矿化液诱导下可形成矿化结节,表现出成骨特征。以第5代hPDLF为研究对象：10～200mg／L MINO显著促进其增殖和有丝分裂(P<0.05),100mg／L为最大效应浓度；20～200mg／L MINO呈浓度依赖性促进总蛋白和COLI基因表达和蛋白表达(P<0.05),10mg／L MINO总蛋白和COLI基因表达和蛋白表达高于对照组,但无统计学差异(P>0.05)；40～200 mg／L时MINO抑制ALP和OCN基因表达和蛋白表达(P<0.05),10～20mg/L MINO对ALP和OCN基因和蛋白表达无抑制作用(P>0.05)。
结论：组织块法、酶消化法、盖玻片固定相结合进行hPDLF原代培养可获得大量高纯度的hPDLF,细胞活性好,增殖能力强。hPDLF可通过矿化液诱导表达成骨特性。在10～20mg／L的浓度范围内,MINO促进hPDLF增殖和有丝分裂,促进总蛋白和COLI,对ALP和OCN无抑制作用。根据本研究结果综合考虑,10～20mg／L可作为MINO牙周局部给药的效应浓度范围。
ExperimentI The study on the bactriostasis of MINO against Aa
Objective:Detect MIC of MINO on Aa, to avoid the incapability of clearing the infection caused by Aa and the resistance due to the inadequate dose.
Methods:First recovery the strain, then spread plate. After those, logphase and growthcurve of Aa were measured. MIC was determined by agar dilution method.
Results:After bacterination for 24h, the log-linear relationship was presented between growth concentration of bacteria and OD value.The growth trends among different periods were different.During the first to 24h, it proliferated slowly, then actively during 48h to 84h. But it became downward in 96h. MIC was 8mg/L. Conclusion:The bactriostasis of MINO on Aa was efficient, and MIC was 8mg/L.
ExperimentⅡBiological effect of MINO on hPDLF
Objective:Explore culture methods for hPDLF in vitro and the effective concentration range of MINO for periodontal local administration.
Methods:Periodontal membrane tissue was used as original tissue for the primary culture.The tissue was from premolor teeth which needed to be extracted because of orthodontics. Three methods, including explant method, enzymatic digestion method and cover-glass, were combined and used for hPDLF primary culture. Vimentin filament, keratin and S-100 factor dealed by immunofluorescence were used for cell identification. The osteogenic property was researched after calcium nodule dyeing induced by mineral solution.The cell growth curve was plotted by MTT method.Steps were as follows:Different concentrations of MINO (0,10,20,40,100,200mg/L) were on the 5th generation hPDLF. After incubation for 1、4、7、10、14d, cell proliferation were measured by MTT method. After incubation for 4d, the change of cell cycle was dected by flow cytometry.Cells total protein was dected by using BCA assay and COLI, ALP, OCN of gene expression and protein expression were assayed by PCR and Western-blot method. The SPSS 13.0 software package was used for statistics analysis.
Results:The immunofluorescence results of vitro culture were positive for vimentin filament, negative for keratin and S-100 factor. These were confirmed to be hPDLF. hPDLF could form mineralization nodules in the induction of mineral solution, and performed osteogenic property. For the 5th generation hPDLF, MINO could accelerate proliferation, and mitosis when its concentration interval was 10～200 mg/L and the difference from other interval was statistically significant. The maximum effect concentration was 100mg/L.for concentration interval 20～200mg/L, MINO could significantly improve total protein and COLI expression on gene and protein aspect(P<0.05). Though 10mg/L group had higher total protein and COLI expression on gene and protein aspect than the control group, the difference was not statistically significant(P>0.05). For concentration interval 40～200 mg/L, MINO inhibited the ALP and OCN gene expression and protein expression(P<0.05).For concentration interval 10～20 mg/L, MINO didn't inhibite the ALP and OCN gene expression and protein expression(P>0.05).
Conclusion:A large quantities of high-purity hPDLF could be obtained by the primary culture method for hPDLF combined with explant method, enzymatic digestion method and cover-glass.Besides the large quantities, the cells had high activity and multiplication capacity. hPDLF expressed osteogenic property by the induction of mineral solution.10～20mg/L, MINO could accelerate proliferation and mitosis, improve total protein and COLI gene expression and protein expression, but have no inhibition effect on ALP and OCN. General considerating according to the finding 10～20mg/L could be the effective concentration range of MINO for periodontal local administration.
目的：检测MINO对Aa的最小抑菌浓度,避免因MINO使用剂量不足造成无法清除Aa的感染并造成菌株的耐药。
方法：复苏菌种,涂布平板,测定Aa生长对数期、生长曲线。琼脂稀释法测定MINO对Aa的MIC。
结果：细菌接种后24h细菌的生长浓度与OD值呈对数线性关系,1-24h时增殖缓慢,48-84h增殖活跃,96h有下降的趋势。MINO对Aa的MIC是8mg／L。
结论：MINO对Aa具有抑菌作用,MIC是8mg／L。
实验二米诺环素对人牙周膜成纤维细胞的生物学作用
目的：探索hPDLF的体外培养方法并进行鉴定。通过检测MINO对hPDLF细胞增殖、细胞周期和对总蛋白、Ⅰ型胶原(COLI)、碱性磷酸酶(ALP)、骨钙素(OCN)的影响,探讨MINO牙周局部给药的效应浓度范围。
方法：选用因正畸需要拔出的前磨牙牙周膜组织为原代培养的组织来源,组织块法、酶消化法、盖玻片固定相结合进行hPDLF原代培养,波形丝蛋白、角蛋白、S-100因子免疫荧光染色进行细胞鉴定。加入矿化液诱导后钙结节染色探讨成骨特性。将不同浓度的MINO(0、10、20、40、100、200mg／L)分别作用于第5代hPDLF,孵育1、4、7、10、14d后MTT法检测细胞增殖；孵育4d后,流式细胞仪检测细胞周期；BCA法检测细胞总蛋白；荧光定量PCR和Western-blot法检测COLI、ALP、OCN的基因表达和蛋白表达。结果采用SPSS 13.0软件包进行分析。
结果：体外培养的hPDLF波形丝蛋白染色阳性,角蛋白、S-100因子染色阴性,证实为hPDLF。hPDLF在矿化液诱导下可形成矿化结节,表现出成骨特征。以第5代hPDLF为研究对象：10～200mg／L MINO显著促进其增殖和有丝分裂(P<0.05),100mg／L为最大效应浓度；20～200mg／L MINO呈浓度依赖性促进总蛋白和COLI基因表达和蛋白表达(P<0.05),10mg／L MINO总蛋白和COLI基因表达和蛋白表达高于对照组,但无统计学差异(P>0.05)；40～200 mg／L时MINO抑制ALP和OCN基因表达和蛋白表达(P<0.05),10～20mg/L MINO对ALP和OCN基因和蛋白表达无抑制作用(P>0.05)。
结论：组织块法、酶消化法、盖玻片固定相结合进行hPDLF原代培养可获得大量高纯度的hPDLF,细胞活性好,增殖能力强。hPDLF可通过矿化液诱导表达成骨特性。在10～20mg／L的浓度范围内,MINO促进hPDLF增殖和有丝分裂,促进总蛋白和COLI,对ALP和OCN无抑制作用。根据本研究结果综合考虑,10～20mg／L可作为MINO牙周局部给药的效应浓度范围。
ExperimentI The study on the bactriostasis of MINO against Aa
Objective:Detect MIC of MINO on Aa, to avoid the incapability of clearing the infection caused by Aa and the resistance due to the inadequate dose.
Methods:First recovery the strain, then spread plate. After those, logphase and growthcurve of Aa were measured. MIC was determined by agar dilution method.
Results:After bacterination for 24h, the log-linear relationship was presented between growth concentration of bacteria and OD value.The growth trends among different periods were different.During the first to 24h, it proliferated slowly, then actively during 48h to 84h. But it became downward in 96h. MIC was 8mg/L. Conclusion:The bactriostasis of MINO on Aa was efficient, and MIC was 8mg/L.
ExperimentⅡBiological effect of MINO on hPDLF
Objective:Explore culture methods for hPDLF in vitro and the effective concentration range of MINO for periodontal local administration.
Methods:Periodontal membrane tissue was used as original tissue for the primary culture.The tissue was from premolor teeth which needed to be extracted because of orthodontics. Three methods, including explant method, enzymatic digestion method and cover-glass, were combined and used for hPDLF primary culture. Vimentin filament, keratin and S-100 factor dealed by immunofluorescence were used for cell identification. The osteogenic property was researched after calcium nodule dyeing induced by mineral solution.The cell growth curve was plotted by MTT method.Steps were as follows:Different concentrations of MINO (0,10,20,40,100,200mg/L) were on the 5th generation hPDLF. After incubation for 1、4、7、10、14d, cell proliferation were measured by MTT method. After incubation for 4d, the change of cell cycle was dected by flow cytometry.Cells total protein was dected by using BCA assay and COLI, ALP, OCN of gene expression and protein expression were assayed by PCR and Western-blot method. The SPSS 13.0 software package was used for statistics analysis.
Results:The immunofluorescence results of vitro culture were positive for vimentin filament, negative for keratin and S-100 factor. These were confirmed to be hPDLF. hPDLF could form mineralization nodules in the induction of mineral solution, and performed osteogenic property. For the 5th generation hPDLF, MINO could accelerate proliferation, and mitosis when its concentration interval was 10～200 mg/L and the difference from other interval was statistically significant. The maximum effect concentration was 100mg/L.for concentration interval 20～200mg/L, MINO could significantly improve total protein and COLI expression on gene and protein aspect(P<0.05). Though 10mg/L group had higher total protein and COLI expression on gene and protein aspect than the control group, the difference was not statistically significant(P>0.05). For concentration interval 40～200 mg/L, MINO inhibited the ALP and OCN gene expression and protein expression(P<0.05).For concentration interval 10～20 mg/L, MINO didn't inhibite the ALP and OCN gene expression and protein expression(P>0.05).
Conclusion:A large quantities of high-purity hPDLF could be obtained by the primary culture method for hPDLF combined with explant method, enzymatic digestion method and cover-glass.Besides the large quantities, the cells had high activity and multiplication capacity. hPDLF expressed osteogenic property by the induction of mineral solution.10～20mg/L, MINO could accelerate proliferation and mitosis, improve total protein and COLI gene expression and protein expression, but have no inhibition effect on ALP and OCN. General considerating according to the finding 10～20mg/L could be the effective concentration range of MINO for periodontal local administration.
引文
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[15]Schwarz F, Nuesry E, Bieling K, et al.Influence of an erbium, chromium-doped yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser on the reestablishment of the biocompatibility of contaminated titanium implant surfaces. J Periodontol.2006,77(11):1820-1827
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[17]Giannini R, Vassalli M, Chellini F, et al. Neodymium:yttrium aluminum garnet laser irradiation with low pulse energy:a potential tool for the treatment of peri-implant disease.Clin Oral Implants Res.2006,17(6):638-643
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[21]唐志辉,曹采方.超声洁治和局部用甲硝唑治疗轻、中度种植体周围炎.中华口腔医学杂志.2002,37(3)：173-175
[22]王宏宇,张立立.两种局部用药治疗种植体周围炎的临床疗效观察感染炎症.口腔颌面修复学杂志.2007,8(3)：169
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[24]Renvert S,Lessem J,Dahlen G, et al. Mechanical and repeated antimicrobial therapy using a local drug delivery system in the treatment of peri-implantitis:a randomized clinical trial.Journal of Periodontology.2008,79(5):836-844
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[26]张春宝,张蓉,马轩祥.牙周治疗对种植体周围龈沟液中IL-1β、 IL-6和TNF-α表达的影响.牙体牙髓牙周病学杂志.2005,106(7)：17-19
[27]Salvi GE, Persson GR, Heitz-Mayfield LJ, et al. Adjunctive local antibiotic therapy in the treatment of peri-implantitis Ⅱ:clinical and radiographic outcomes. Clin Oral Implants Res.2007,18(3):281-285
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