间歇性机械牵张力对人牙周膜成纤维细胞增殖动力学的影响

目的　观察间歇性机械牵张力作用下,人牙周膜成纤维细胞(hPDLFs)增殖动力学的变化,探究机械力介 导的牙周组织改建的可能机制。方法　体外培养hPDLFs,取第4~7代细胞,通过四点弯曲细胞力学加载仪对细胞 加载不同力值大小的间歇性机械牵张力(1 000,2 000,3 000μstrain),分别在不同时间点收集细胞,流式细胞仪测定 细胞DNA含量,计算增殖指数(PI)。结果　机械牵张力影响hPDLFs增殖活性。1 000μstrain和2 000μstrain机械牵 张力组与对照组相比,均能促进细胞增殖,hPDLFs DNA含量增多(P<0·05),增殖指数升高(P<0·05);3 000μstrain 机械牵张力组与对照组比较,增殖指数无差异(P>0·05),但抑制细胞DNA合成(P<0·05)。结论　一定力值范围 的机械牵张力能促进hPDLFs的增殖活性,过大力值的机械牵张力反而抑制细胞增殖。     

机械压力对人牙周膜成纤维细胞增殖活力的影响

目的:探讨不同力值和不同时间的压力对人牙周膜成纤维细胞增殖活力的影响。方法:采用细胞加载装置,对细胞分别施加0、0.25、0.5、1.0 MPa的压力30 min;施加1.0 MPa的压力,分别持续10、30、50 min,通过四唑盐比色实验(MTT法)检测细胞受力后6、12、18、24、30、36 h的OD值。结果:0.25、0.5 MPa组的OD值与对照组无显著性差异(P>0.05);1.0 MPa压力下,加载30 min和加载50 min组有显著性差异(P<0.05),表现为受力后6 h细胞OD值即明显降低,12 h进一步降低,与对照组相比差异均有统计学意义(P<0.05),24 h后细胞OD值与对照组无差异。结论:人牙周膜成纤维细胞的增殖活力和压力值以及压力作用时间存在一定的关系;细胞的增殖活力随着压力值的增加、压力作用时间的延长而有所降低,但这种影响是可逆的。     

尼古丁对人牙周膜成纤维细胞增殖能力的影响

目的：体外研究尼古丁对人牙周膜成纤维细胞增殖能力的影响。方法：用不同浓度的尼古丁与体外培养的人牙周膜成纤维细胞作用不同时间,用MTT比色法测定细胞的生长情况,流式细胞分析法测定尼古丁对细胞周期的影响。结果：各浓度的尼古丁组均能抑制人牙周膜成纤维细胞的生长,但不同浓度尼古丁对牙周膜细胞的抑制作用无明显差异;各浓度组尼古丁均能降低G1的比例,高浓度尼古丁（5×10^-1g/L）组G1期比例降至84.62%,G2/M期升高至12.83%。结论：尼古丁能抑制人牙周膜成纤维细胞的生长,并影响其细胞周期的进程。     

rhBMP-4与rhIGF-I联合应用对人牙周膜成纤维细胞增殖及分化能力的影响

目的:探讨rhBMP-4与rhIGF/-I联合应用对人牙周膜成纤维细胞增殖及分化能力的影响.方法:将人牙周膜成纤维细胞分别与10 ng/ml rhBMP-4、10 ng/ml rhIGF-I、10 ng/ml rhBMP-4 10 ng/ml rhIGF-I、无血清DMEM共同培养3 d,用MTT法测定细胞增殖情况,用碱性磷酸酶试剂盒检测细胞碱性磷酸酶的活性,用羟脯氨酸试剂盒检测细胞分泌I型胶原的量.结果:第3天,10 ng/ml rhBMP-4 10 ng/ml rhIGF-I组人牙周膜成纤维细胞的牛长增殖能力最强,并与其它3组比较有显著性差异(P＜0.05),但其增强细胞碱性磷酸酶的活性及促进细胞分泌I型胶原的能力并不比两者单独作用强.结论:rhBMP-4与rhIGF-I联合应用,能协同促进人牙周膜成纤维细胞增殖,但不能协同促进细胞分化.     

缺氧对人牙周膜成纤维细胞增殖和超微结构影响的实验研究

目的:体外培养人牙周膜成纤维细胞(HPLFS),观察缺氧对其生长增殖能力的影响.方法: 分离培养 HPLFS,随机分为 21% O2对照组和10%、5%、2% O2缺氧组,采用四唑盐(MTT)比色法检测 HPLFS增殖情况,透射电镜下观察细胞超微结构改变.结果:MTT 法检测,与对照组比,12 h、24 h,缺氧对细胞增殖随缺氧程度呈依赖性增强,但重度缺氧(2% O2)24 h组具有统计学差异;48 h、72 h,重度缺氧组细胞增殖与对照组相比明显降低,差别具有统计学意义.透射电镜下,重度缺氧24 h细胞胞质内粗面内质网和线粒体明显增多,细胞突起增多;72 h细胞发生退变,溶酶体增多.结论:长期重度缺氧条件下,牙周膜的改建和修复功能降低,可能是高原牙周疾病多发、牙周组织破坏较重的重要原因.     

缺氧对人牙周膜成纤维细胞增殖和碱性磷酸酶活性的影响

目的:观察不同程度缺氧对人牙周膜成纤维细胞(HPLFS)增殖、分化的影响.方法:随机将HPLFS分为4组;210mL/LO2对照组、100、50、20mL/LO2缺氧组(轻中重缺氧组),用MTT法、碱性磷酸酶试剂盒分别检测HPLFS的增殖和碱性磷酸酶(ALP)的活性.结果:第24小时,重度缺氧可促进HPLFS细胞增殖,第48、72小时,重度缺氧则明显抑制r细胞增殖;中、重度缺氧对细胞碱性磷酸酶活性表达随缺氧时间则明显受到抑制.结论:缺氧时间、程度对HPDLFs增殖和ALP活性表达存在效应关系,长期中、重度缺氧不利HPLFS的生长及向成骨分化能力,提示牙周组织改建修复功能降低.     

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。     

糖基化终产物对牙周膜成纤维细胞增殖的影响

目的：探讨糖基化终产物在糖尿病牙周病形成中的作用。方法：原代培养牙周膜成纤维细胞,将糖基化终产物修饰的人血清白蛋白及未修饰人血清白蛋白与牙周膜成纤维细胞在体外共同培养,MTT法测定细胞增殖,用碱性磷酸酶测定法测定ALP。结果：糖基化终产物修饰的人血清白蛋白能以浓度依赖的方式抑制牙周膜细胞的增殖（p〈0.05）,而未修饰人血清白蛋白可使细胞增殖,但两种白蛋白均不诱导ALP的表达。结论：糖基化终产物可通过抑制牙周膜成纤维细胞的增殖,降低糖尿病病人牙周组织对损伤的修复能力,导致牙周病。     

缺氧环境对牙周膜成纤维细胞增殖及凋亡的影响

目的：研究缺氧环境对牙周膜成纤维细胞的增殖及凋亡相关蛋白表达的影响。方法采用氯化钴模拟法构建人牙周膜成纤维细胞缺氧模型。观察缺氧条件下牙周膜成纤维细胞形态变化,采用CCK-8检测细胞增殖情况,并用Western Blot方法检测凋亡相关因子p53、Bcl-2及caspase-7的表达变化。结果随着时间延长,缺氧组细胞数量减少,体积逐渐变小,细胞胞浆浓缩,细胞存活率降低。 Western Blot 结果显示缺氧组 p53、Bcl-2及caspase-7蛋白表达量随着缺氧时间延长逐渐升高。缺氧组p53蛋白、caspase-7表达高于常氧组,Bcl-2表达低于常氧组。结论缺氧环境可影响牙周膜成纤维细胞形态、增殖活性及凋亡相关蛋白的表达。     

环孢素A联合脂多糖对牙周膜成纤维细胞增殖的影响

目的探讨环孢素A(CsA)联合牙龈卟啉单胞菌脂多糖(Pg-LPS)对人牙周膜成纤维细胞(hPDLFs)增殖的影响。方法改良组织块法培养hPDLFs,分别用CsA(10、100、200、400ng/ml),Pg-LPS(10、100、1000ng/ml),CsA(100ng/ml)加Pg-LPS(100ng/ml),CsA(100ng/ml)加Pg-LPS(1000ng/ml)作用于生长良好的第3～5代细胞,MTT法测其药物作用用下的增殖情况。结果细胞经上述浓度CsA或Pg-LPS作用后形态无明显变化,CsA在100ng/ml浓度情况下,促细胞增殖作用最明显;高浓度Pg-LPS(1000ng/ml)显著抑制细胞增殖,100ng/mlCsA能够显著降低Pg-LPS(1000ng/ml)对细胞增殖的抑制效应;CsA(100ng/ml)与Pg-LPS(100ng/ml)联合作用于细胞时,对细胞的增殖无明显影响。结论在一定的浓度下,CsA促PDLF增殖作用明显;CsA能对抗脂多糖(LPS)抑制细胞增殖的效应。     

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。     

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.     

釉基质衍生物对脂多糖作用下牙周膜成纤维细胞增殖和凋亡的影响

目的研究釉基质衍生物（enamel matrix derivatives,EMD）对牙龈卟啉单胞菌脂多糖（Porphyromonas gingivalis lipopolysaccharide,Pg-LPS）作用下的人牙周膜成纤维细胞（human periodontal ligament fibroblasts,hPDLFs）增殖和凋亡的影响。方法从正畸治疗需拔除的前磨牙中,分离培养人hPDLFs,传至第4代;实验组培养液中分别加入100μ/mLEMD、10μg／,mL Pg-LPS或者100μ/mL EMD＋10 μg／mL Pg-LPS,对照组不加入刺激物;以四甲基偶氮唑盐[3-（4,5-dimethy1-2-thiazolyl）-2,5-diphenyl．2H-tetrazoliumbromide,MTT]法检测hPDLFs的增殖,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法分析细胞凋亡。结果原代hPDLFs生长良好,加入刺激后第3天时,第4代hPDLFs的Mrrr值由高至低分别是：EMD组、对照组、EMD＋LPS组、LPS组。刺激48h后,LPS组凋亡率（12．10％±2．80％）大于EMD＋LPS组（8．07％±2．04％）、EMD组（3．68％±1．73％）和对照组（3．87％±1．27％）（P〈0．05）。结论EMD能减弱Pg-LPS对hPDLFs增殖的影响,并且降低Pg-LPS所导致的hPDLFs凋亡,可能是其促进牙周组织再生的重要机制。     

尼古丁对牙周膜成纤维细胞的细胞毒性研究

目的体外研究尼古丁对牙周膜成纤维细胞（PDLFs）的细胞毒性作用,探讨尼古丁对牙周病的影响。方法用不同浓度的尼古丁作用于PDLFs,采用MTT比色法于24、48、72h测量细胞的活性：采用流式细胞仪技术于48h检测细胞周期和凋亡率。结果尼古丁抑制PDLFs细胞的生长．随着浓度的增加和时间的延长,抑制率明显增强;细胞周期分布发生了改变,G0／G1期的细胞DNA含量与正常对照组相比．明显增加．而G2期和S期则逐渐减少,同时出现了较高的凋亡率．周期阻滞及凋亡率随着浓度的递增而增加。结论尼古丁可能通过导致PDLFs细胞凋亡、抑制该细胞的增殖并使其DNA合成减少而导致牙周病的发生。     

In vitro clonogenic capacity of periodontal ligament fibroblasts cultured with Emdogain

Abstract – The aim of the present study was to evaluate the efficiency of Emdogain (EMD) in preserving the size of the periodontal ligament progenitor pool (clonogenic capacity) and in promoting their proliferation. Periodontal ligament fibroblasts (PDLF) were obtained from explants of young permanent healthy tooth. After initial outgrowth (10 days to 2 weeks following explantation), the culture medium of experimental flasks was replaced with medium supplemented with 100 μg ml^?1 EMD, whereas the other served as controls and were fed with regular medium. Following 5 weeks, the cells were washed (3×), harvested (trypsin + EDTA), and evaluated for their viability. Viable cells from each group were inoculated into six 96‐well plates at a concentration of one viable cell per two wells and were allowed to grow for 5 weeks. The percentage of cells with clonogenic capacity was determined as the number of colonies formed/number of cells seeded × 100 in the experimental and control groups. Three degrees of dish area coverage were utilized: up to 25%, between 25% and 75% and higher than 75%. This experiment was repeated four times from four different donors. A total of 2328 cells were evaluated, half of which, were cultured with EMD. The mean percentage of cells (from all donors) who exhibited any clonogenic capacity in the presence of EMD was comparable with that of cells cultured in the absence of EMD: 26.6 ± 14.3% when compared with 34.6 ± 20.6% respectively (P = 0.186). Similarly, the percentage of clones that proliferated to cover up to 25% of the well area was comparable in the two groups 7.5 ± 8.6 for EMD‐treated clones and 7.1 ± 7.8 for untreated clones (P = 0.674). The percentage of clones that proliferated to cover 25% up to 75% of the well area was greater EMD‐treated clones as compared with the untreated cells: 8.1 ± 6.7% vs 3.8 ± 3%. However this difference was not statistically significant (P = 0.277). In contrast, the percentage of clones that covered more than 75% of the well area was significantly lower in the EMD‐treated clones when compared with the untreated clones (10.9 ± 11.1 vs 23.8 ± 14.7; P = 0.022) . In conclusion, EMD decreased the percentage of PDLF with capabilities of arising colonies with 75–100% confluency probably by increasing their differentiation.     

Cyclic stretching force induces apoptosis in human periodontal ligament cells via caspase-9

The response of periodontal ligament (PDL) cells to mechanical stimulation is important in the periodontal tissue remodelling. Our previous study showed that cyclic stretching force on PDL cells induced early apoptosis. However, the mechanism of stretching force-induced cell death is unclear. In the present study, we examined whether PDL cells undergo apoptosis by stretching force using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end-labellling method (TUNEL) and investigated the mechanism by which cyclic stretching force initiated apoptosis. We found that PDL cells became aligned regularly and the number of apoptotic cells increased significantly in a time-and force-dependent manner after the application of cyclic stretching force. Caspase-3 activity increased in proportion to the magnitude of the stretching force, and this effect was reduced significantly by a caspase-9 inhibitor, whereas a caspase-8 inhibitor had no such effect. We therefore concluded that the in vitro application of cyclic stretching force can induce apoptosis in PDL cells by activating the caspase-3 via the caspase-9 signalling cascade. Our findings may provide a novel insight into the mechanism of apoptosis induced by stretching force in PDL cells.     

氯霉素对人牙周膜细胞增殖及超微结构的影响

目的：比较0．25％氯霉素和生理盐水对人牙周膜细胞增殖和超微结构的影响,以提高脱位牙再植的疗效。方法：采用组织块法体外培养人牙周膜细胞,取第5代培养细胞,分别加入氯霉素、生理盐水和DMEM培养液作用1h,在作用后0、12、24、48h进行细胞计数．观察对牙周膜细胞增殖的影响。采用透射电镜观察人牙周膜细胞超微结构的变化。数据采用SAS9．1软件包进行方差分析。结果：在0、12、48h时间点．氧霉素组和生理盐水组的细胞数显著低于DMEM培养液对照组（P〈0．0001）;在24h时间点,生理盐水组的细胞数显著低于对照组（P〈0．05）。各时间点氯霉素和生理盐水两组细胞计数无显著差异（P〉0．05）。电镜观察,氯霉素可导致人牙周膜细胞线粒体空泡化．内质网减少。结论：氯霉素对人牙周膜细胞增殖的影响与生理盐水没有区别,但对人牙周膜细胞的超微结构有损伤作用。     

Oestrogen inhibits osteoclast formation induced by periodontal ligament fibroblasts

Objective

Since tooth-associated fibroblasts are taken to participate in the formation of osteoclasts and it is unknown whether oestrogen affects this process, the effects of 17β-estradiol (17β-E₂) were studied on osteoclastogenesis induced by human periodontal ligament fibroblasts (PLFs) and gingival fibroblasts (GFs).

Methods

Human peripheral blood mononuclear cells (PBMCs) were seeded on monolayers of PLFs and GFs and cocultured for 14 days in the presence or absence of various concentrations of 17β-E₂. The number of tartrate resistant acid phosphatase (TRACP)-positive osteoclast-like cells (OCs) was assessed. In addition, we analysed the PBMC-induced withdrawal of the fibroblasts. mRNA expression was determined of oestrogen receptor (ER)-α, ER-β, receptor activator nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) by PLFs and GFs.

Results

PBMCs induced a higher number and larger fibroblast-free areas if cocultured with PLFs than with GFs. Concomitantly, the number of TRACP-positive OCs was significantly higher in PLF cocultures. 17β-E₂ inhibited the formation of OCs in PLF cocultures. 17β-E₂ did not alter the expression of RANKL, OPG, and ER-α mRNAs in either fibroblast cell population.

Conclusion

Our data indicate that PLFs may promote osteoclastogenesis more strongly than GFs. 17β-E₂ inhibits the PLF-induced formation of osteoclast-like cells. Thus, the inhibitory effect of oestrogen on osteoclast formation appears to be cell type dependent.     

辛伐他汀对人牙周膜细胞增殖和分化的影响

目的:研究辛伐他汀对人牙周膜细胞增殖和成骨分化的影响.方法:将第4 代人牙周膜细胞在条件矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(10-9、10-8、10-7、10-6 mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,4-硝基苯基磷酸二钠盐(4-nitrophenyl phosphate,hexahydrate,PNPP)偶氮法检测碱性磷酸酶(alkaline phosphatase,ALP)活性.结果:辛伐他汀各浓度组均能促进人牙周膜细胞的增殖和分化,其中10-8、10-7、10-6 mol/L组与对照组比较,差异有统计学意义(P<0.05),10-7 mol/L的辛伐他汀组促进细胞增殖和ALP活性的作用最明显.结论:适宜浓度的辛伐他汀可有效促进人牙周膜细胞的增殖和成骨分化.