【作者】 刘宇；

【导师】 刘洪臣；

【作者基本信息】 中国人民解放军军医进修学院 ， 口腔临床医学， 2007， 博士

【摘要】 近年来多项研究表明，牙周致病菌在宿主组织细胞的胞内感染使牙周炎的治疗复杂化。具有合适的细胞内药代动力学和细胞内药效学性质的治疗药物的筛选非常重要。另外，有研究者提出通过根管与牙周组织的通道，使药物进入牙周组织局部以及全身的假说，这需要了解口腔组织、特别是牙周膜组织细胞对各种药物的转运。目前还未见人牙周膜成纤维细胞对抗生素转运的研究的报道，因此本课题系统研究了人牙周膜成纤维细胞对8种口腔常用抗生素的吸收、转运，为局部用药的筛选和通过根管局部及全身给药假说提供实验依据。本研究共分为八部分：实验一HPDLF对甲硝唑的跨膜转运目的：研究HPDLF对甲硝唑的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：正畸拔除的前磨牙利用组织块法原代培养HPDLF，常规培养MC3T3-E1细胞；采用免疫组化鉴定细胞类型，描绘生长曲线；细胞经甲硝唑孵育，超声破碎，高效液相色谱法测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：成功原代培养HPDLF；HPDLF波形丝蛋白染色阳性，MC3T3-E1细胞骨钙素染色阳性。HPDLF和MC3T3-E1细胞的生长曲线近似“S”形，前者的生长速度慢于后者。20μg/ml的甲硝唑孵育5、10分钟，HPDLF胞内均不能检测出药物存在，而MC3T3-E1细胞药物含量分别是0.165±0.034ng/μg、0.206±0.065ng/μg。结论：HPLC可以用于甲硝唑含量的测定。甲硝唑不存在HPDLF的跨膜转运，MC3T3-E1细胞对甲硝唑则存在跨膜转运。实验二HPDLF对替硝唑的跨膜转运目的：研究HPDLF对替硝唑的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：替硝唑溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：100μg/ml替硝唑孵育1、5、10分钟时HPDLF胞内药物含量分别是0.067±0.049ng/μg、0.099±0.024ng/μg、0.078±0.015ng/μg，200μg/ml替硝唑孵育5分钟时HPDLF胞内替硝唑含量是0.142±0.011ng/μg；100μg/ml替硝唑孵育5分钟时，MC3T3-E1细胞胞内替硝唑含量是0.082±0.027ng/μg。结论：HPLC可以用于细胞内替硝唑的测定。替硝唑存在HPDLF的跨膜转运，这种转运与细胞外药物浓度有关，并存在着细胞差异。实验三HPDLF对盐酸四环素的跨膜转运目的：研究HPDLF对盐酸四环素的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：盐酸四环素溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：10μg/ml四环素孵育1、5、10分钟时HPDLF胞内药物含量分别是0.192±0.008ng/μg、0.212±0.082ng/μg、0.620±0.075ng/μg(p＜0.01)；20μg/ml四环素孵育5分钟时HPDLF胞内含量是0.503±0.056ng/μg，显著高于10μg/ml组数据(p＜0.05)；10μg/ml四环素孵育5分钟时，MC3T3-E1细胞胞内含量是0.666±0.560ng/μg。结论：HPLC可以用于细胞内盐酸四环素的测定。盐酸四环素存在HPDLF的跨膜转运，这种转运与细胞外药物浓度有关，并存在着细胞差异。实验四HPDLF对米诺环素的跨膜转运目的：研究HPDLF对米诺环素的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：米诺环素溶液孵育细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：20μg/ml米诺环素孵育1、5、10分钟时HPDLF胞内含量分别是1.661±0.520ng/μg、1.949±0.415ng/μg、2.154±0.728ng/μg(p＜0.05)，40μg/ml米诺环素孵育5分钟时HPDLF胞内含量是2.155±0.728ng/μg；20μg/ml米诺环素孵育1、5、10分钟时MC3T3-E1胞内含量分别是1.532±0.396ng/μg、3.331±0.538ng/μg、2.811±0.798ng/μg(p＜0.05)，10μg/ml、40μg/ml米诺环素孵育5分钟时MC3T3-E1细胞内含量是0.751±0.265ng／μg、3.188±0.636ng/μg(p＜0.01)。结论：HPLC可以用于细胞内米诺环素的测定。米诺环素存在HPDLF的跨膜转运，这种转运与细胞外药物浓度有关，并存在着细胞差异。实验五HPDLF对罗红霉素的跨膜转运目的：研究HPDLF对罗红霉素的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：罗红霉素溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：20μg/ml的罗红霉素孵育HPDLF及MC3T3-E1细胞1、5、10分钟时均无法检测出胞内药物的存在，40μg/ml的药物作用5分钟，同样无法检测。结论：HPLC可以用于罗红霉素含量的测定。在本实验条件下，不能检测出HPDLF以及MC3T3-E1细胞对罗红霉素的跨膜转运。实验六HPDLF对诺氟沙星的跨膜转运目的：研究HPDLF对诺氟沙星的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：诺氟沙星溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：10μg/ml诺氟沙星孵育1、5、10分钟时HPDLF胞内药物含量分别是0.095±0.032ng/μg、0.096±0.052ng/μg、0.104±0.078ng/μg；10μg/ml诺氟沙星孵育1、5、10分钟时MC3T3-E1胞内药物含量分别是0.033±0.018ng/μg、0.034±0.013ng/μg、0.082±0.029ng/μg(p＜0.05)，20μg/ml诺氟沙星孵育5分钟时MC3T3-E1细胞内药物含量是0.070±0.057ng/μg。结论：HPLC可以用于细胞内诺氟沙星的测定。HPDLF对诺氟沙星存在跨膜转运，这种转运与细胞外药物浓度有关，还存在着细胞差异。实验七HPDLF对盐酸林可霉素的跨膜转运目的：研究HPDLF对盐酸林可霉素的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：盐酸林可霉素溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：10μg/ml的盐酸林可霉素孵育HPDLF及MC3T3-E1细胞1、5、10分钟时均无法检测出胞内药物的存在，20μg/ml的药物作用5、10分钟，同样无法检测。结论：HPLC可以用于盐酸林可霉素的测定。本实验条件下不能检测出HPDLF以及MC3T3-E1细胞对盐酸林可霉素的跨膜转运。实验八HPDLF对头孢噻肟钠的跨膜转运目的：研究HPDLF对头孢噻肟钠的跨膜转运，为根管全身给药假说提供实验依据。材料与方法：头孢噻肟钠溶液孵育HPDLF和MC3T3-E1细胞，超声破碎细胞后，HPLC测定不同时间点的胞内药物含量，考马斯亮蓝法测定细胞蛋白总量。结果：100μg/ml的头孢噻肟钠孵育1、5、10分钟时HPDLF胞内含量分别是0.104±0.030ng/μg、0.151±0.007ng/μg、0.161±0.046ng/μg；孵育5分钟时，MC3T3-E1胞内含量0.096±0.027ng/μg。结论：HPLC可以用于细胞内头孢噻肟钠的测定。头孢噻肟钠存在HPDLF的跨膜转运，这种转运存在着细胞差异。上述研究结果表明，高效液相色谱法测量稳定、分离度好、精密度高、重复性好，可以用于上述多种药物含量的检测。利用该方法，可检测到人牙周膜成纤维细胞对替硝唑、盐酸四环素、米诺环素、诺氟沙星、头孢噻肟钠存在跨细胞膜的转运，而对甲硝唑、罗红霉素和盐酸林可霉素不存在跨细胞膜的转运。人牙周膜成纤维细胞对药物的转运与细胞外液的药物浓度有关。人牙周膜成纤维细胞与MC3T3-E1细胞系对同一种药物的转运存在差异，这表明药物的跨细胞膜转运存在细胞类型和/或种属差异性。本研究在一定程度上证实了根管全身给药假说的可行性。另外，选择能够转运至胞内的药物更有利于牙周炎的治疗。更多还原

【Abstract】 The invasion of bacteria to host cells made it more difficult to deal with the periodontal dieases. The concentrations of some medicine in gingival crevicular fluid were higher than those in serum. To verify the hypothesis of delivering medicine to the periodontium and whole body through the root canal, we carried out experiments to study the biological transport of 8 kinds of antibiotics by human periodontal ligaments fibroblasts (HPDLF). There are:Part 1: the biological transport of metronidazole by HPDLF Objective: To investigate biological transport of metronidazole by HPDLF. Methods: Premolars extracted for orthodontic reasons were used in this study. HPDLF were primarily cultured by tissue explant. HPDLF were incubated in metronidazole solutions. After disrupting cells by supersonic wave, the intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: HPDLF were primarily cultured successfully and passaged stably. The intracellular metronidazole content of HPDLF couldn’t be detected, incubated in 20 μg/ml antibiotics solution. The MC3T3-E1 cells intracellular contents were 0.165±0.034 ng/μg, 0.206±0.065 ng/μg at 5 min, 10 min respectively. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Metronidazole can’t be transported by HPDLF. The transport is cell specific.Part 2: the biological transport of tinidazole by HPDLFObjective: To investigate biological transport of tinidazole by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.067±0.049 ng/μg, 0.099±0.024 ng/μg, 0.078±0.015 ng/μg at 1min, 5 min, 10 min respectively, incubated in 100 μg/ml antibiotics solution. The HPDLF intracellular content was 0.142±0.011 ng/μg at 5 min, incubated in 200 μg/ml antibiotics solution. The MC3T3-E1 intracellular content was 0.082±0.027 ng/μg at 5 min, incubated in 100 μg/ml tinidazole solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Tinidazole can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 3: the biological transport of tetracycline hydrochloride by HPDLF Objective: To investigate biological transport of tetracycline hydrochloride by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.192±0.008 ng/μg, 0.212±0.082 ng/μg, 0.620±0.075 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution (p<0.01). The HPDLF intracellular content was 0.503±0.056 ng/μg at 5 min , incubated in 20 μg/ml antibiotics solution and was siginificantly higher than that of 10 μg/ml group (p<0.05). The MC3T3-E1 intracellular content was 0.666±0.560 ng/μg at 5 min, incubated in 10 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Tetracycline hydrochloride can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 4: the biological transport of minocycline by HPDLF Objective: To investigate biological transport of minocycline by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in minocycline solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 1.661±0.520 ng/μg, 1.949± 0.415 ng/μg, 2.154± 0.728 ng/μg at 1min, 5 min, 10 min respectively, incubated in 20 μg/ml antibiotics solution (p<0.05). The HPDLF intracellular content was 2.155±0.728 ng/μg at 5 min, incubated in 20 μg/ml antibiotics solution. The MC3T3-E1 intracellular contents were 1.532±0.396 ng/μg, 3.331±0.538 ng/μg, 2.811±0.798 ng/μg at 1min, 5 min, 10 min respectively, incubated in 20 μg/ml antibiotics solution (p<0.05). The MC3T3-E1 intracellular contents were 0.751±0.265 ng/μg, 3.188±0.636 ng/μg at 5 min respectively, incubated in 10 μg/ml and 40μg/ml antibiotics solution (p<0.01). Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Minocycline can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 5: the biological transport of roxithromycin by HPDLF Objective: To investigate biological transport of roxithromycin by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The intracellular antibiotics content of HPDLF and MC3T3-E1 cells couldn’t be detected at 1min, 5 min, 10 min, incubated in 20 μg/ml and 40 μg/ml antibiotics solutions. Conclusion: HPLC is an accurate, sensitive method for measurement of roxithromycin. With the limitations, roxithromycin can’t be detected of transport by HPDLF.Part 6: the biological transport of norfloxacin by HPDLF Objective: To investigate biological transport of norfloxacin by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in norfloxacin solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.095±0.032 ng/μg, 0.096±0.052 ng/μg, 0.104±0.078 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution. The MC3T3-E1 intracellular contents were 0.033±0.018 ng/μg, 0.034±0.013 ng/μg, 0.082±0.029 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution (p<0.05). The MC3T3-E1 intracellular contents were 0.070±0.057 ng/μg at 5 min , incubated in 20 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Norfloxacin can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 7: the biological transport of lincomycin hydrochloride by HPDLF Objective: To investigate biological transport of lincomycin hydrochloride by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The intracellular antibiotics content of HPDLF and MC3T3-E1 cells couldn’t be detected at 1min, 5 min, 10 min , incubated in 10 μg/ml and 20 μg/ml antibiotics solutions. Conclusion: HPLC is an accurate, sensitive method for measurement of lincomycin hydrochloride. With the limitations, lincomycin hydrochloride can’t be detected of transport by HPDLF.Part 8: the biological transport of cefotaxime sodium by HPDLF Objective: To investigate biological transport of cefotaxime sodium by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions.The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.104±0.030 ng/μg, 0.151±0.007 ng/μg, 0.161±0.046 ng/μg at 1min, 5min, 10min respectively, incubated in 100 μg/ml antibiotics solution. The MC3T3-E1 intracellular content was 0.096±0.027 ng/μg at 5 min, incubated in 100 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Cefotaxime sodium can be transported by HPDLF. The transport was time-dependent, cell specific.更多还原