苯妥英钠对人牙周膜成纤维细胞生物学活性影响的实验研究

目的研究苯妥英钠（PHT）对体外培养的人牙周膜成纤维细胞（hPDLF）生物学功能的影响,并探讨其用于促进牙周组织再生的可能性。方法将不同质量浓度的PHT（1、5、20、100、500、2 500 mg/L）加入体外培养的第4代hPDLF,检测其对细胞增殖活性、蛋白合成、碱性磷酸酶（ALP）活性的影响;Von Kossa染色法检测PHT（20、100、500 mg/L）对第4代hPDLF矿化结节形成能力的影响;应用ELISA法测定PHT（20、100 mg/L）对第3代hPDLF中骨形态发生蛋白- 2（BMP- 2）表达的影响。结果在20、100 mg/L的质量浓度时,PHT可显著促进hPDLF的增殖及分化（P<0.01）;在质量浓度100 mg/L时,PHT可增强hPDLF的蛋白合成（P<0.05）;同时,PHT在质量浓度100 mg/L可显著促进hPDLF的矿化能力及BMP- 2的表达（P<0.01）。但高质量浓度（2 500 mg/L）的PHT则严重抑制细胞的生物学活性。结论适当质量浓度的PHT对hPDLF的增殖和分化有促进作用,但过高质量浓度的PHT具有细胞毒性,不利于牙周组织的修复和再生。     

牛血小板衍化生长因子对人牙周膜成纤维细胞的生物学效应

目的：观察在牛血小板衍化生长因子作用下人牙周膜成纤维细胞ＤＮＡ和胶原蛋白合成的情况。方法：采用体外细胞培养技术和核素掺入法。结果：２０ｎｇ／ｍｌ～６０ｎｇ／ｍｌ牛血小板衍化生长因子可明显促进人牙周膜成纤维细胞ＤＮＡ合成，４０ｎｇ／ｍｌ浓度使细胞ＤＮＡ合成在２４ｈ达最高峰；对细胞的胶原蛋白合成无明显促进作用。结论：牛血小板衍化生长因子对人牙周膜成纤维细胞ＤＮＡ合成有促进作用，在牙周组织的创伤修复中可能起重要作用。     

In vitro growth of periodontal fibroblasts on treated cementum.

The aim of the present study was to assess the ability in vitro of phosphoric and citric acids, applied on human root cementum, to neutralize noxious plaque and calculus and to allow the growth of human gingival fibroblasts. Fibroblasts grown on cementum treated with phosphoric acid appeared typically elongated and aligned parallel to the root surface. Fibroblasts grown on cementum treated with citric acid, in both normal and periodontally diseased teeth, lost their elongated shape, acquiring polygonal borders with irregular cytoplasmic extrusions, and the cell density was significantly lower. These findings suggest that phosphoric acid cleaning of both normal and diseased root surfaces may result in an oriented, high rate of fibroblastic growth with more effective periodontal cellular proliferation than that observed after citric acid treatment.     

The effects of extracts from periodontopathic bacteria on human periodontal fibroblasts stimulated with mineralization supplements

Bacterial effects on in vitro mineralization of human periodontal fibroblasts (HPF) have not yet been examined in great detail. In our study, we investigated the effects of soluble extracts of the periodontopathic bacteria Porphyromonas gingivalis, Bacteroides forsythus and, Treponema denticola on cell proliferation, mineralization, as well as on osteoblastic markers present in HPF cultured in vitro, such as alkaline phosphatase (ALP) activity and collagen content. Periodontal fibroblasts stimulated by B-glycerophosphate, ascorbic acid and dexamethasone (BAD) or by dexamethasone and ascorbic acid (DA) were compared to unstimulated cells. During the cultivation period, the stimulation of HPF by combined dexamethasone and ascorbic acid (DA) had a strong inductive effect on proliferation, ALP activity and collagen formation. The extracts obtained from the periodontal pathogens had a suppressing effect on the proliferation rate of HPF. The extracts from P. gingivalis, B. forsythus and T. denticola caused a decrease in ALP activity within 24 h of application. While extracts obtained from P. gingivalis and B. forsythus induced a reduction in collagen content in BAD- and DA-stimulated HPF cells, T. denticola extracts led to an increase in collagen. Our data suggest that specific periodontopathic bacteria may suppress tissue regeneration in vivo not only by activating host defense mechanisms but also directly via a suppression of growth and differentiation of HPF and a reduction in the extracellular collagen matrix. For the process of pocket formation, not even the direct influence of viable bacteria seems to be necessary. Additionally, long-distance effects of bacteria harboured in periodontal pockets or in root canals may be of importance.     

人牙周膜成纤维细胞对诺氟沙星的跨膜转运

目的 研究人牙周膜成纤雏细胞(human periodontal ligament fibroblasts,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.方法 诺氟沙星溶液孵育HPDLF和MC3T3-E1细胞.超声破碎细胞后,高效液相色谱法(high performance liquid chromatography,HPLC)测定胞内药物含量,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、lO分钟时HPDLF胞内药物含量分别是0.095±0.032ng/μg0.096±0.052ng/μg、0.104±O.078ng/μg;1Oμg/mL诺氟沙星孵育l、5、10分钟时MC3T3-El胞内药物含量分别是0.033±0.018ng/μg、0.034±0.013ng/μg、O.082±0.029ng/μg,10分钟组数据显著性高于前两组数据,20μg/mL诺氟沙星孵育5分钟时MC313-El细胞内药物含量是0.070±0.057ng/μg.结论 HPLC可用于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及孵育时间有关,还存在着细胞差异.     

人牙周膜成纤维细胞对罗红霉素的跨膜转运

目的 研究人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)对罗红霉素的跨膜转运,为通过根管局部及全身给药假说提供实验依据.方法 罗红霉素溶液孵育HPDLF和MC3T3-E1细胞,超声破碎细胞后,高效液相色谱法(high performance liquid chromatography,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 20 μg/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,40 μg/ml的药物作用5分钟,同样无法检测.结论 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.     

人牙周膜成纤维细胞对米诺环素的跨膜转运

目的研究人牙周膜成纤维细胞（HPDLF）对米诺环素的跨膜转运,为通过根管局部及全身给药的假说提供实验依据。方法用米诺环素溶液孵育HPDLF和MC3T3- E1细胞,超声破碎细胞后,高效液相色谱法（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HPLC可以精确测量细胞内米诺环素的含量。细胞种类和孵育时间对细胞内米诺环素含量有显著性影响（P<0.01）,胞内米诺环素含量随着时间延长而增加,两种细胞的胞内药物含量不同;胞外米诺环素浓度增加时,细胞内药物含量增加。结论米诺环素存在HPDLF的跨膜转运,这种转运与细胞外药物浓度及孵育时间有关,并存在着细胞差异。     

Protein production by human gingival fibroblasts is enhanced by guanidine EDTA extracts of cementum

Nonconfluent cultures of human gingival fibroblasts were exposed to both guanidine and guanidine EDTA extracts of cementum for 48 hours. To compare the effects of cementum extracts on fibroblasts with other mineralized tissue extracts, cells were also exposed to guanidine and guanidine EDTA extracts of dentin and alveolar bone. The cells were radioactively labeled during the last 24 h. Total protein production was measured via the incorporation of radioactive proline. Collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine EDTA extracts elicited statistically significant increases in total protein production compared to controls. At 50 μg/ml of extract, the increases in protein production were 340%, 143% and 338% for bone, cementum and dentin, respectively. Similar results were obtained for collagen production. In contrast, the guanidine extracts had no effect on either protein or collagen production by human gingival fibroblasts. These data indicate that the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identifying such proteins and understanding their biological functions will enhance our knowledge of the mechanisms that regulate connective tissue regeneration.     

人牙周膜成纤维细胞对盐酸林可霉素跨膜转运的研究

目的:研究人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)对盐酸林可霉素的跨膜转运,为根管牙周局部及全身给药提供实验依据.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:10 μg/ml的盐酸林可霉素孵育HPDLF及MC3T3-E1 细胞 1 min、5 min、10 min时均无法检测出胞内药物的存在,20 μg/ml的药物作用5 min、1O min,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.     

人牙周膜成纤维细胞对四环素的跨膜转运

目的 研究人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)对四环素的跨膜转运,为通过根管局部或全身给药假说提供实验依据.方法 盐酸四环素溶液孵育HPDLF和MC3T3-E1细胞,超声破碎细胞后,高效液相色谱法测定胞内药物含量,考马斯亮蓝法测定细胞蛋白质量.结果 10 mg/L四环素孵育1、5、10 min后细胞内四环素含量与细胞蛋白质量的比值分别为(0.192±0.008)、(0.212±0.082)、(0.620±0.075)ng/μg.20 mg/L四环素孵育5 min后细胞内四环素含量与细胞蛋白质量的比值为(0.503±0.056)ng/μg.10 mg/L四环素孵育5 min后MC3T3-E1细胞内四环素含量与细胞蛋白质量的比值为(0.666±0.560)ng/μg.结论 HPDLF可跨膜转运四环素.转运与孵育时间及细胞外药物浓度相关.     

人牙周膜成纤维细胞对头孢噻肟钠的跨膜转运

目的:研究人牙周膜成纤维细胞(human periodontal ligament fibroblasts, HPDLF)对头孢噻肟钠的跨膜转运,为根管牙周局部及全身给药假说提供实验依据.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:100 μg/ml的头孢噻肟钠孵育1、5、10 min时HPDLF胞内含量分别是(0.104±0.030) ng/μg、(0.151±0.007) ng/μg、(0.161±0.046) ng/μg;孵育5 min时, MC3T3-E1胞内含量(0.096±0.027) ng/μg.结论:HPLC可用于细胞内头孢噻肟钠的测定.头孢噻肟钠在HPDLF内存在跨膜转运,这种转运存在着细胞差异.     

人牙周膜成纤维细胞增殖与压力关系的初步观察

目的：在体外培养环境下观察压力对人牙周膜成纤维细胞（HPLF）增殖的影响。方法：取第4－6代培养的HPLF,应用可控压力细胞加载装置分别间断性施加30、60、90kPa的压力,分别在培养3、5、7d后用MTT法测定各组的A值。结果：HPLF在30、60、90kPa的压力培养环境下MTT反应的A值显著小于对照组,压力值越大,A值越小。结论：30、60、90kPa间断性压力显著抑制PLF细胞增殖,该抑制作用随压力值增大而增强。     

Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro

BACKGROUND, aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. METHOD: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. RESULTS: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. CONCLUSION: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.     

Cytological and cytogenetic effects of the fluoroquinolone ofloxacin on human periodontal ligament fibroblasts

The fluoroquinolone ofloxacin (OFLX) is one of the candidates of antibacterial agents to be topically used against periodontitis. To estimate the maximum concentration of OFLX which exerts little or no adverse effect on the periodontal ligament, cytological and cytogenetic effects of OFLX on human periodontal ligament fibroblasts (Pel cells) were examined. Treatment of Pel cells with < or =0.3 mM OFLX for 24 or 48 h had little inhibitory effect on cellular growth and survival. DNA, RNA and protein syntheses in Pel cells did not decrease in response to treatment with < or =0.3 mM OFLX. The constitutive level of alkaline phosphatase mRNA was retained in cells treated with < or = 0.03 mM OFLX for 24 or 48 h. The level of type I procollagen mRNA was not affected by treatment with < or = 0.003 mM OFLX for 24 or 48 h. Cytogenetic effects of OFLX were evaluated by the ability of OFLX to induce chromosome aberrations in Pel cells. Treatment with OFLX at 0.3-3.0 mM for 6, 24, or 48 h failed to induce chromosome aberrations in Pel cells. The failure of OFLX to induce chromosome aberrations was seen even in the presence of exogenous metabolic activation using a 5% rat liver post-mitochondrial supernatant mixture. These results indicate that treatment of Pel cells with < or =0.003 mM OFLX has few or no adverse effects on the cytological and cytogenetic endpoints examined, suggesting that there would be little adverse effect on growth and differentiation of the periodontal ligament, as well as little cytogenetic activity, if OFLX were to be topically administered to the gingival crevice at the minimal inhibitory concentration (MIC90) against periodontopathic bacteria (< or = 0.0027 mM). It is important to note, however, that extrapolation of these findings to in vivo conditions has yet to be undertaken.     

Quantitative comparison of cytocidal effects of tetracyclines and fluoroquinolones on human periodontal ligament fibroblasts

The cytocidal effects of tetracyclines and fluoroquinolones on human periodontal ligament fibroblasts (Pel cells) were studied. Pel cells were treated for 24 h with tetracycline (TC), demeclocycline (DMC), minocycline (MINO), chlortetracycline (CTC), tosufloxacin (TFLX), enoxacin (ENX), sparfloxacin (SPFX), lomefloxacin (LFLX) or ofloxacin (OFLX), and allowed to form colonies. The cytocidal effects of the antibacterial agents, as determined by a decrease in colony-forming efficiency, increased as the dose increased. However, CTC was an exception. As a quantitative measure of the cytocidal effect, LD50, the concentration which results in a 50% decrease in colony-forming efficiency relative to control cells, was extrapolated from the dose-response curves. The rank-order of cytocidal effects (LD50) was DMC [symbol: see text] MINO [symbol: see text] TC > CTC. DMC, MINO and TC were at least 5.6-6.6 times more cytocidal than CTC. The cytocidal effects of the fluoroquinolones were in the following order: TFLX > ENX > SPFX > LFLX > OFLX. TFLX, ENX, SPFX and LFLX were 36.3, 11.4, 7.7 and 3.1 times more cytocidal than OFLX, respectively. Little cytocidal effect was observed when the cells were treated with either < or = 0.03 mM tetracyclines or < or = 0.01 mM fluoroquinolones. The results provide useful estimates concerning the relative toxicities against human periodontal ligament of antibacterial agents used to treat periodontitis.     

透明质酸对尼古丁作用下人牙周膜成纤维细胞增殖影响研究

目的观察不同浓度的尼古丁对人牙周膜成纤维细胞(HPDLFs)增殖的影响,并探讨透明质酸对尼古丁作用下对HPDLFs的保护作用。方法体外成功培养的HPDLFs,按不同的处理分为4组:(1)单纯细胞组(对照组);(2)尼古丁组:浓度分别为10、100、250、500、1 000μg/m L;(3)HA组:浓度为0.1%和0.2%;(4)尼古丁组+HA组(总10子组)。观察4组细胞增殖率,ALP的活性,Runx2、ColⅠ、OPN、OCN的mRNA的水平,细胞钙矿化能力。结果 (1)与对照组相比,各浓度尼古丁组均可以抑制HPDLFs的增殖率、ALP活性,Runx2、ColⅠ、OPN、OCN的mRNA水平,未见钙化矿化结节。(2)与对照组相比,HA组均可以促进HPDLFs的增殖率、ALP活性和Runx2、ColⅠ、OPN、OCN的mRNA水平并观察到了钙化矿化结节。(3)与尼古丁组相比,HA+尼古丁组对HPDLFs的增殖率和ALP活性起到促进作用,对Runx2、ColⅠ、OPN、OCN的mRNA的表达水平有提高并均观察到钙化矿化结节;各组差异具有统计学意义(P<0.05)。结论 HA能够介导HPDLFs增殖,增加ALP活性和矿化组织相关蛋白,包括Runx2、ColⅠ、OPN和OCN等,并且抵抗尼古丁对HPDLFs的毒性作用,从而起到保护作用。