周期性张应力对成纤维细胞增殖能力的影响

目的探讨周期性张应力对体外培养的小鼠胚胎成纤维细胞增殖特性的影响。方法将体外培养的L929细胞分为加力组与对照组,加载周期性张应力的细胞为加力组,不加力的为对照组。应用细胞应力加载系统对L929细胞加载8%形变率、0.1 Hz的周期性张应力,分别在加力1 h、2 h、4 h和8 h后应用流式细胞仪分析细胞周期;四甲基偶氮唑盐比色法分析细胞的增殖活性。结果加力组加力1 h(q=5.532,P<0.01)与2 h(q=6.569,P<0.01),S期细胞比率(S-phase fraction,SPF)较对照组出现了下降,并伴有细胞数量的增加(P>0.05);加力组在加力4 h后,SPF较对照组下降(P>0.05),细胞数量增加(P>0.05);加力组在加力8 h后,SPF较对照组开始出现增高(q=4.287,P<0.05),细胞数量显著增加(q=5.202,P<0.01)。结论 8%形变率、0.1 Hz的周期性张应力引起细胞增殖活性的变化,表现为随应力加载时间的延长,细胞的增殖活性出现先被抑制、后被促进的改变。     

周期性张应力对人牙周膜细胞生物学活性的影响

目的:探讨周期性张应力对体外培养的人牙周膜细胞生物活性的影响。方法:对体外培养的人牙周膜细胞施加不同大小的周期性牵张力(6%、12%、20%细胞表面拉伸率),24h后检测其对细胞的总蛋白合成、ALP活性、Col-Ⅰ和OCN分泌的影响。实验结果用SPSS 13.0进行统计分析。结果:较小的牵张力对人牙周膜细胞生物活性无显著影响,随力值的增大,牵张力能够呈强度依赖性抑制人牙周膜细胞的活性,减少总蛋白合成及ALP活性,抑制Col-Ⅰ及OCN的分泌。结论:周期性张应力可以抑制人牙周膜细胞的生物学活性,并呈强度依赖性。     

周期性张应力对人牙周膜细胞Periostin表达的影响

目的:研究在机械力作用下人牙周膜细胞(h PDLCs)内Periositn(PN)m RNA和蛋白的表达变化。方法:采用组织块酶消化法培养h PDLCs,经鉴定后将第4代细胞随机分为4个实验组和1个对照组(非加力组),4个实验组分别采用Flexcell-5000 Tension System应力加载系统对h PDLCs加载6、12、24、48 h的周期性张应力;然后采用q RT-PCR、Western blot分别检测各组h PDLCs中PN m RNA和蛋白的表达变化。结果:在正常h PDLCs中表达PN m RNA和蛋白;与对照组相比,加力6 h后,PN m RNA和蛋白的表达水平均明显降低(P<0.05);加力12 h后,PN m RNA和蛋白的表达均逐渐升高,并持续至24 h达最高水平(P<0.05);而在加力48 h时,PN m RNA和蛋白表达均明显下降,并恢复至对照组的表达水平(P>0.05)。结论:机械力可诱导h PDLCs中PN的表达增强及活化,且具有时间依赖性;提示PN可能参与了细胞内生物力学信号的转导。     

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。     

周期性张应力对牙周膜细胞内基质金属蛋白酶-8和13表达的影响

目的：观察周期性张应力作用下牙周膜细胞（human periodontal ligament cells,HPDLC）中基质金属蛋白酶（MMPs）的表达变化。方法：通过建立体外应力加载系统,对培养在弹性膜六孔板的HPDLC施加0.1Hz,硅胶膜形变率分别为6%、12%、18%的周期性张应力,分别在加载2、6、12h后利用逆转录聚合酶链反应（RT-PCR）和Western Blot技术检测HPDLC的MMP-8,MMP-13的表达变化。结果：HPDLC在加载周期性牵张力后,细胞生长方向顺应力方向而发生改变,MMP-8和MMP-13的表达明显增加。结论：周期性循环张力可诱导HPDLC中MMP-8、MMP-13表达增强,为MMP-8、MMP-13可能参与正畸力下牙周组织的改建提供了依据。     

动态张、压应力刺激下人牙周膜成纤维细胞细胞骨架变化

目的:观察不同动态张、压应力刺激下人牙周膜成纤维细胞细胞骨架的变化.方法:用Forcel四点弯曲加载装置对体外培养的人牙周膜成纤维细胞分别施加不同动态的张、压应力(强度为1 000、2 000、4 000 μstrain,加力时间为0、1、2、4、8、12 h),经荧光倒置显微镜观察施加不同动态张、压应力后细胞形态变化,细胞骨架荧光染色强度测定分析细胞骨架F-actin表达量的变化.结果:加力后细胞骨架形态和微丝蛋白发生规律性变化;细胞F-actin荧光染色强度正常→下降→正常;细胞骨架对张、压应力作用的反应敏感程度无明显差异.结论:在一定的张、压应力范围内人牙周膜成纤维细胞细胞骨架的形态结构具有一定的稳定性.     

人外周血单个核细胞对人牙周膜成纤维细胞增殖和分化的影响

目的:观察人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)对人牙周膜成纤维细胞(human periodontal ligament fibroblast cells,HPLFs)增殖分化的影响,为进一步探讨正畸牙齿移动的生物学机制奠定基础。方法:建立人PBMCs与HPLFs共培养系统,通过细胞计数及生化检测法观察人外周血单个核细胞对人牙周膜成纤维细胞增殖和分化的影响。结果:3 d和5 d时,共培养组HPLFs细胞计数分别为4.5×104及8.5×104,明显高于对照组,且两组分别与对照组HPLFs细胞计数有显著性差异(P<0.05)。transwell共培养组与对照组相比,在3 d时,两组HPLFs分泌型ALP活性有显著性差异(P<0.05),5 d及7 d时差异尤其显著(P<0.01),tran-swell共培养组HPLFs分泌型ALP活性低于对照组。结论:人PBMCs能促进HPLFs的增殖,但抑制HPLFs分泌型ALP活性。     

机械牵张作用对牙周膜成纤维细胞增殖的影响

目的 :观察机械牵张作用对牙周膜成纤维细胞 (PDLFs)增殖能力的影响。方法 :用自行研制的细胞加载系统对PDLFs施以频率为 6次 /min(每次为 5s牵拉、5s松弛 ) ,幅度 12 %的牵张力 ,于加载 2 4、48、96h后 ,通过细胞计数、流式细胞仪检测观察PDLFs增殖能力的改变情况。结果 :在不同的时间点 ,加力组及对照组的细胞数均不断增加 ,在 48h及 96h时加力组结果明显高于对照组。流式细胞仪结果显示在不同时间点加力组处于DNA合成期的细胞数明显高于对照组。结论 :在对培养的人PDLFs施加频率为 6次 /min、幅度 12 %的牵张力时 ,对细胞的增殖起一定的促进作用     

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.     

Apoptotic gene expression by human periodontal ligament cells following cyclic stretch

Xu C, Hao Y, Wei B, Ma J, Li J, Huang Q, Zhang F. Apoptotic gene expression by human periodontal ligament cells following cyclic stretch. J Periodont Res 2011; 46: 742–748. © 2011 John Wiley & Sons A/S Background and Objective: Periodontal ligament cells play an important role in maintaining homeostasis of periodontal tissue upon mechanical force loading caused by mastication or orthodontic force. Previous studies revealed force‐driven periodontal ligament cell death via apoptosis, but the force‐sensing genes assigned to the apoptotic pathway have not been fully characterized. The present study aimed to identify force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells. Material and Methods: Human periodontal ligament cells were exposed to 20% stretch strain for 6 or 24 h, and the differential expression of 84 genes implicated in the apoptotic pathway were quantified by real‐time PCR array technology. Results: Ten and 11 genes showed upregulated expression after 6 and 24 h stretches, respectively, and there were two downregulated genes in response to both 6 and 24 h stretches. These genes included those encoding the tumor necrosis factor ligand family (TNFSF8), tumor necrosis factor receptor family (FAS, TNFRSF10B, TNFRSF11B, TNFRSF25 and CD27), the Bcl‐2 family (BAG3, BAK1, BCL2L11 and BCLAF1), the caspase family (CASP5 and CASP7), the inhibitor of apoptosis proteins family (BIRC3, BIRC6 and NAIP), the caspase recruitment domain family (RIPK2 and PYCARD) and the death domain family (DAPK1), as well as an oncogene (BRAF). Conclusion: This study identified several force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells and should facilitate future studies on force‐driven apoptosis by providing putative target genes.     

烟草对牙周成纤维细胞影响的实验观察

目的 观察尼古丁和烟草浸提液(smokeless tobacco extract，ST)对牙周膜成纤维细胞(periodontal ligament fibroblasts，PDLFs)的形态、超微结构及增殖贴附的影响。方法 用不同浓度的尼古丁和ST作用于人PDLFs，细胞爬片作HE染色，光学显微镜观察细胞的形态；透射电镜作超微结构观察；MTT法分别于第5天和2h测细胞的增殖和贴附情况。结果 随着尼古丁和ST浓度的增加，细胞排列的极性消失，细胞变小并由长梭形变成椭圆形或圆形；超微结构显示，细胞器成分减少，尤其是粗面内质网和高尔基体减少，含胶原蛋白的分泌小泡减少，波形丝、微管分解消失。细胞核有程度不同的减小或核畸变；细胞的增殖和贴附能力呈浓度依赖性抑制。结论 尼古丁和ST可改变PDLFs的形态和结构，抑制细胞的胶原合成，改变细胞的支架结构，从而改变细胞的贴附和增殖，提示烟草在牙周病的发病中有重要作用。     

Effect of HBSS storage time on human periodontal ligament fibroblast viability

Abstract – Hank’s balanced salt solution (HBSS) is recommended for the storage of avulsed teeth. The objective of this study was to evaluate if the HBSS storage time influences its ability to maintain the viability of human periodontal ligament fibroblasts (PDLF) by the analysis of cell metabolic function using MTT assay. PDLF were kept at 20°C for 3, 6, 24, 48, 72, 96 and 120 h in recently prepared HBSS (HBSS), HBSS stored for 6 months (HBSS 6 M), HBSS stored for 12 months (HBSS 12 M), and in Save‐A‐Tooth system’s HBSS (Save). Minimum essential medium (MEM) at 37°C and tap water at 20°C served as positive and negative controls, respectively. Cell viability was determined by the tetrazolium salt‐based colorimetric (MTT) assay. Data were statistically analyzed by the Kruskal–Wallis and Scheffé tests (α = 5%). Starting with the 6 h time‐point, HBSS was significantly more effective than HBSS 6 M, HBSS 12 M and Save in maintaining cell viability. HBSS 6 M effectiveness was similar to that of HBSS 12 M for up to 48 h, becoming higher at 72 h. In conclusion, the storage time of HBSS had a negative influence on its ability to maintain PDLF viability.     

定量观察力值对人牙周膜细胞骨改建相关细胞因子表达的影响

目的 定量观察不同力值的机械牵张力对体外培养的人牙周膜细胞骨改建相关细胞因子的影响.方法 利用北京理工大学研制的细胞加力装置对体外培养的人牙周膜成纤维细胞(hPDLFs)施加不同大小的周期性牵张力(5％、10％细胞表面拉伸率),检测其对细胞的骨保护素(osteoprotegerin,OPG)、骨保护素配体(receptor activator of nuclear factor kappa-Bligand,RANKL)及成骨相关转录因子RUNX2、Osterix表达的影响.实验结果用SPSS 13.0进行统计分析.结果 加力后RUNX2、Osterix在5％组表达量呈现时间依赖性增加,但变形量10％组增加出现的较早且之后会出现平台期;细胞受力后OPG在5％和10％组表达量均呈现时间依赖性增加,两组间不存在显著性差异;细胞受力后RANKL的表达量在两组中均受到抑制.结论 周期性牵张力刺激对人牙周膜细胞成骨相关因子RUNX2、Osterix和破骨相关因子OPG、RANKL的表达有一定的影响,并且在不同力值刺激下这些细胞因子的表达随时间变化的形式不同.     

人牙周膜成纤维细胞原代培养的研究

目的：研究如何提高人中膜成纤维细胞原代培养的成功率。方法：以牙槽窝刮取组织块法替代传统的牙根刮取法，以组织块自然贴壁法替代传统的干燥贴壁法。结果：48h后细胞游出率为40％，而成功传代率为20％，结论：通过对取材和培养方法的改良，可以显著提高人牙周膜成纤维细胞原代培养的成功率。     

Effect of milk renewal on human periodontal ligament fibroblast viability in vitro

Abstract – Milk has been studied extensively and has gained wide acceptance as a suitable storage medium capable of maintenance of avulsed teeth that cannot be replanted immediately. The objective of this study was to evaluate whether the renewal of milk as a storage medium every 24 h for up to 120 h is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability in vitro. Plates with confluent PDLF were soaked in minimum essential medium (MEM) at 37°C (positive control) and in skimmed milk (22 wells) and water (negative control) for 24, 48, 72, 96, and 120 h at 5 and 20°C. The skimmed milk was renewed every 24 h in 11 of the wells of each plate. After these periods, cell viability was determined by the tetrazolium salt‐based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal–Wallis and Scheffé tests (α = 5%). At 24 h, milk and MEM performed similarly. However, from 48 h onwards, MEM was significantly better than renewed and not renewed milk at both temperatures. Regardless of temperature (5 or 20°C), renewal of milk with fresh milk did not affect its ability to maintain PDLF viability.     

Effect of temperature and storage media on human periodontal ligament fibroblast viability

Abstract – Many solutions have been examined as possible storage media for avulsed teeth. The purpose of this study was to compare the effectiveness of several storage media to preserve cultured periodontal ligament fibroblasts (PDLF) under different temperatures. The media tested were: sterile Hank’s balanced salt solution (sHBSS), non‐sterile HBSS (nHBSS), skimmed milk, Save‐A‐Tooth^®, Minimum Essential Medium (MEM) and water (negative control). MEM at 37°C was used as positive control. PDLF were obtained from explants of extracted healthy human teeth. Plates containing confluent PDLF were soaked in the various media for 3, 6, 24, 48 and 72 h at 37°C and 20°C. After incubation, viability of the cells was determined using the tetrazolium salt‐based colorimetric (MTT) assay and the Trypan Blue exclusion test after 6, 24, 48 and 72 h of incubation at 20°C. The results were analyzed statistically using Kruskal–Wallis, Scheffé and Mann–Whitney (α = 5%) tests. Results from the MTT assay at 37°C and 20°C showed that skimmed milk was the best storage medium for up to 24 and 48 h, respectively, followed by nHBSS and sHBSS. Results from the Trypan Blue exclusion test showed that the best storage media were milk, sHBSS and nHBSS, with no statistical differences, for any time period. The Save‐A‐Tooth^® had a detrimental effect on cells after 24 h. The influence of temperature on the effectiveness of the storage media tested showed at 20°C a decreasing order of efficacy as follows: milk > sHBSS and nHBSS > MEM > Save‐A‐Tooth^® > water while at 37°C it was: MEM > nHBSS > milk > sHBSS > Save‐A‐Tooth^® > water. In conclusion, incubation temperature altered the effectiveness of the storage media and skimmed milk at 20°C was better than HBSS in maintaining PDLF viability.     

氟化钠对人牙周膜细胞增殖及矿化能力的影响

目的:观察氟化钠对体外培养的人牙周膜细胞增殖及矿化能力的影响,为氟添加入牙周组织工程药物中的应用提供依据.方法:原代培养并鉴定人牙周膜细胞,应用CCK8检测不同浓度NaF对hPDLCs增殖的影响,并筛选出4个浓度用于矿化实验.矿化条件下,将0、1×10-5、5×10-4和1×10-3 mol/L的NaF作用hPDLCs后,通过碱性磷酸酶(ALP)染色、茜素红染色和实时荧光定量PCR检测矿化能力及成骨相关基因的表达.采用SPSS20.0软件包对数据进行单因素方差分析.结果:5×10-5、1×10-4、5×10-4 mol/L的NaF均能促进hPDLCs增殖,且以5×10-4 mol/L效果最佳(P＜0.05).而1×104 mol/L的NaF碱性磷酸酶染色阳性面积最大、茜素红染色矿化结节数量最多(P＜0.05).RT-PCR结果根据时间、指标变化程度较大.结论:5×10-5、1×10-4、5×10-4 mol/L的NaF能促进hPDLCs的增殖能力,1×10-5 mol/L的NaF能提高hPDLCs的碱性磷酸酶活性及钙结节形成.