碱性成纤维细胞生长因子对体外培养人牙髓细胞增殖和迁移的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）对体外培养人牙髓细胞增殖、迁移活性的影响。方法以常规组织块培养法体外获得人牙髓细胞,实验组分别加入终浓度为1.0、5.0、10.0和50.0ng/ml的bFGF,对照组仅加入等量的空白培养基,采用CCK-8法分别测定450nm下各组光吸收度A值,研究bFGF对牙髓细胞增殖活性的影响;应用Transwell小室培养法,研究bFGF对牙髓细胞迁移活性的影响。结果与对照组相比,bFGF在1.0～50.0ng/ml浓度下可促进牙髓细胞的增殖（P〈0.05）,bFGF最低显效浓度为1.0ng/ml,最佳显效浓度为10.0ng/ml。bFGF在1.0～50.0ng/ml浓度下诱导细胞迁移（P〈0.05）。结论 bFGF可诱导体外培养人牙髓细胞的增殖和迁移,在牙髓牙本质复合体修复中可能发挥重要作用。     

miR-138靶向PLD2基因对口腔癌细胞增殖、迁移的影响

目的：探讨miR-138靶向PLD2基因抑制口腔癌细胞增殖、迁移的机制。方法：口腔癌细胞转染miR-138后,采用RT-PCR检测细胞中miR-138的表达水平,MTT法检测细胞增殖能力,流式细胞术检测细胞转染miR-138后细胞周期的分布,Transwell迁移实验检测细胞的迁移能力;采用Western 免疫印迹实验检测胃癌细胞MMP-9、PLD2及Cyclin D1的表达水平,采用荧光素酶报告实验分析miR-138与PLD2基因的靶向关系。采用SPSS 21.0软件包对数据进行统计学分析。结果：口腔癌细胞转染miR-138后,miR-138相对表达水平为4.28±0.16,显著高于空白对照及miR-NC组（P<0.05）。荧光素酶报告基因实验发现,口腔癌细胞转染miR-138后,PLD2野生质粒荧光素酶相对活性低于其他组（P<0.05）,miR-138组的PLD2的mRNA表达水平低于空白对照及miR-NC 组（P<0.05）。口腔癌细胞转染miR-138后,miR-373组的口腔癌细胞的增殖能力低于空白对照及miR-NC组（P<0.05）。流式细胞术实验发现,miR-138组的G₀/G₁期比例为（64.39±6.49）%,显著高于空白对照组及miR-NC组（P<0.05）;miR-138组S期比例为（13.28±3.16）%,显著低于空白对照组及miR-NC组（P<0.05）;各组G₂/M期比例差异无统计学意义（P>0.05）。Transwell实验发现,口腔癌细胞转染miR-138后,迁移细胞数量为138.46±24.37,显著低于空白对照组及miR-NC组（P<0.05）。Western免疫印迹实验发现,miR-138组MMP-9的相对水平为0.14±0.04、Vimentin的相对水平为0.17±0.02、Cyclin D1的相对水平为0.15±0.03,均显著低于空白对照组及miR-NC组（P<0.05）。结论：miR-138可靶向调控PLD2基因表达,对口腔癌的增殖、迁移能力产生抑制作用。     

SREBP2对口腔鳞癌细胞增殖和迁移的影响及其调控机制

目的:探讨胆固醇调节元件结合蛋白2(SREBP2)在口腔鳞癌(OSCC)中的调控机制,通过敲低和过表达SREBP2,分析其对口腔鳞癌细胞CAL27增殖、迁移、侵袭的影响及作用机制.方法:采用实时定量反转录PCR(qRT-PCR)检测SREBP2在OSCC中的表达量.构建siRNA和过表达质粒并转染CAL27细胞,通过细...     

唑来膦酸对成纤维细胞作用的影响

目的 分析唑来膦酸对成纤维细胞的影响,探讨唑来膦酸对软组织损伤及引起创口软组织愈合困难的可能机制。方法 体外常规培养NIH 3T3成纤维细胞。流式细胞凋亡实验检测加入唑来膦酸后成纤维细胞凋亡的变化。MTT法检测加入唑来膦酸条件下成纤维细胞增殖活性的变化,细胞划痕实验观察唑来膦酸对成纤维细胞迁移能力的影响。采用SPSS 17.0软件包对数据进行统计学分析。结果 在药物浓度为5~50 μmol/L范围时,随着药物浓度升高,唑来膦酸促进成纤维细胞凋亡,抑制增殖、迁移的作用逐渐增强。结论 唑来膦酸能对软组织造成损伤,可引起拔牙创软组织愈合困难。     

c-fos调控p16/CyclinD1信号途径并促进口腔鳞状细胞癌增殖和迁移

目的:探讨c-fos对口腔鳞状细胞癌增殖和迁移的影响及作用机制.方法:免疫组化法分别检测口腔鳞状细胞癌组织标本和正常口腔黏膜组织标本(n=60)中的CyclinD1、p16和c-fos.根据不同处理将HN6和SCC9细胞分为空白对照组、siRNA-scramble组、siRNA-c-fos组.检测各组细胞中CyclinD1和p16 mRNA和蛋白表达量的变化,以及细胞增殖和迁移能力的变化.结果:与正常组比较,病例组中c-fos和CyclinD1表达增加,p16表达降低;与空白对照组相比,siRNA-c-fos组HN6和SCC9细胞中CyclinD1表达显著降低,p16表达明显上升,细胞的增殖能力和迁移能力都显著下降.结论:c-fos可以通过p16/CyclinD1信号途径增强口腔鳞状细胞癌增殖和迁移.     

肿瘤相关成纤维细胞对口腔鳞癌细胞生长和迁移的影响

目的：研究肿瘤相关成纤维细胞（ cancer associated fibroblasts, CAFs）对口腔鳞癌细胞生物学行为的影响,初步探讨CAFs在口腔鳞癌发生发展中的作用。方法采用原代培养的方法,培养口腔鳞癌相关成纤维细胞,光学显微镜观察其形态,免疫组化和免疫印迹实验进行鉴定,CCK-8法检测细胞的增殖能力。 transwell迁移实验检测CAFs诱导口腔鳞癌细胞迁移的能力;萤火虫荧光素酶报告系统检测CAFs对肿瘤细胞增殖能力的影响;平板克隆形成实验评价 CAFs对肿瘤细胞克隆形成能力的影响。结果成功分离培养出口腔鳞癌来源的 CAFs。CAFs能促进口腔鳞细胞的迁移、增殖和克隆形成等生物学行为。结论 CAFs能显著影响口腔鳞癌的增殖和迁移,表明其在口腔鳞癌的发生发展中发挥着一定的作用。     

凝溶胶蛋白gelsolin对人牙髓细胞增殖及迁移的影响

目的研究凝溶胶蛋白gelsolin（GSN）对人牙髓细胞（hDPC）增殖及迁移的影响。 方法激光共聚焦检测gelsolin在hDPC中的表达。以小干扰RNA（siRNA）沉默hDPC中的gelsolin,CCK-8法检测细胞增殖;流式细胞仪检测细胞凋亡及细胞周期;Transwell实验观察细胞的迁移情况。采用单因素方差分析数据。 结果激光共聚焦显示gelsolin普遍表达于hDPC胞浆。沉默gelsolin,hDPC的增殖率在24、48、72 h时分别下降47.8%（F= 6.182,P= 0.035）、48.9%（F= 5.520,P= 0.044）、64.1%（F= 15.440,P= 0.004）;流式结果显示干扰gelsolin,hDPC的凋亡率增加约4.5%（F= 21.162,P= 0.002）,而细胞周期未发生明显阻滞,S期细胞比例升高约5%（F= 4.526,P>0.05）,G1期细胞比例下降约8%（F= 4.743,P>0.05）;Transwell结果显示,24 h内hDPC的迁移能力降低约60%（F= 12.781,P<0.001）。 结论gelsolin对hDPC的增殖及迁移具有调控作用。     

TRPM7离子通道在口腔鳞癌细胞增殖和迁移中的作用

目的:探讨TRPM7离子通道在口腔鳞癌细胞增殖和迁移中的作用.方法:Western blotting,RT-PCR和间接免疫荧光法检测口腔鳞癌细胞OC2中TRPM7的表达和细胞内定位.MTT法和Transwell试验分别检测抑制TRPM7通道(2-APB、siRNATRPM7)对OC2细胞增殖和迁移的影响.Western blotting检测2-APB或siRNA TRPM7阻断PI3K/AKT通路对OC2细胞中TR-PM7的表达影响.采用膜片钳技术初步探索干预TRPM7通道对OC2细胞阳离子通透的影响.结果:OC2细胞中TRPM7过表达,主要表达在细胞质中.阻断TRPM7通道后,OC2细胞增殖和迁移能力受抑制,且呈浓度依赖性和时间依赖性.阻断TR-PM7通道后,抑制OC2细胞TRPM7可下调磷酸化AKT以及磷酸化ERK的表达,而对组成性AKT以及ERK的表达无明显影响.膜片钳技术显示在TRPM7过表达的OC2细胞中TRPM7样电流激活,2-APB阻断TRPM7通道后电流明显减弱,而采用TRPM7通道活化剂Bradykinin后,电流明显增强.结论:TRPM7在口腔鳞癌细胞OC2中过表达,可能通过影响PI3 K/AKT和MAPK-ERK信号转导途径以及阳离子内流发挥对细胞增殖、迁移的调节作用.     

成纤维细胞上清提取物对表皮细胞增殖迁移的影响

目的:在人皮肤成纤维细胞培养上清液中寻找有效的生长因子样活性组分.方法:收集超滤成纤维细胞的培养上清液,采用MTT法、划痕试验检测对人皮肤表皮细胞增殖和诱导迁移的影响.结果:上清液中分子量5～10 KD组和5～30 KD组的试验组对表皮细胞有明显促增殖趋势,而5～30 KD组有较强的促进单层细胞伤口愈合的能力,二者与对照组比较有显著性差异(P＜0.01).结论:人成纤维细胞培养上清液的提取物中含有促进表皮细胞增殖及迁移能力的活性物质,可以尝试用其替代部分条件培养基进行对组织工程皮肤种子细胞的实验研究.     

Bioactive polyphenol antioxidants protect oral fibroblasts from ROS-inducing agents

Background

Oxidative damage to soft oral tissues may result from exposure to the chemicals or biochemicals found in teeth-whitening products, dental restorations, tobacco, and alcohol. Our working hypothesis is that oral tissues are susceptible to the toxic effects of stressors such as hydrogen peroxide (H₂O₂), ethanol (EtOH) and nicotine (Nic), which decrease cell viability/DNA synthesis and elevate reactive oxygen species (ROS). In this study, we investigated specific polyphenols and turmeric derivative antioxidants (AO) in combinations that counteracted the effects of these stressors on cultured oral fibroblast proliferation and ROS production.

Methods

Oral fibroblasts were exposed to stressors for 30 min and then treated with 10⁻⁵ M of bioactive AO mixtures [resveratrol, ferulic acid and tetrahydrocurcuminoid (RFT), phloretin, ferulic acid and resveratrol (PFR), phloretin, ferulic acid and tetrahydrocurcuminoid (PFT)] for 24 h. Cell viability and DNA synthesis were monitored using incorporated 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulphophenyl]-2H-tetrazolium (MTS) and 5-bromo-2-deoxyuridine (BrdU) assays, respectively. Total ROS was measured with dichlorodihydrofluorescein diacetate (H₂DCFDA).

Results

Incubation of oral fibroblasts in the stressors for 30 min resulted in a dose-dependent decrease of DNA synthesis and number of viable cells, and an increased total ROS activity. AO treatment counteracted the insults by restoring DNA synthesis levels and cell viability, and decreasing the total ROS activity.

Conclusion

The AO combinations of RFT, PFR and PFT protected the oral fibroblasts from the detrimental effects of H₂O₂, EtOH and Nic by decreasing total ROS and increasing cell viability and DNA synthesis.     

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。     

巨噬细胞来源的白细胞介素6对口腔鳞癌细胞增殖活性的影响

目的: 探讨巨噬细胞来源的白细胞介素6（interleukin 6,IL-6）对口腔鳞状细胞癌细胞系增殖活性的影响。方法: 通过MTT法检测不同浓度重组IL-6对口腔鳞状细胞癌细胞系SCC-15和CAL-27增殖活性的影响。建立佛波酯诱导的THP-1巨噬细胞分化模型,分别通过实时定量荧光PCR（RT-PCR）和酶联免疫吸附试验,分析脂多糖（lipopolysaccharide,LPS）刺激下的THP-1细胞中mRNA水平的变化以及THP-1细胞培养基中IL-6的含量。利用MTT法检测含THP-1条件培养基对SCC-15和CAL-27细胞系增殖活性的影响。采用GraphPad 7.0软件包对数据进行统计学分析。结果: 培养基中添加IL-6,可促进SCC-15和CAL-27细胞系的增殖活性。LPS可刺激活化的THP-1巨噬细胞分泌IL-6;活化的THP-1细胞培养基可促进SCC-15和CAL-27细胞的增殖活性。结论: LPS刺激巨噬细胞分泌的IL-6,可促进口腔鳞状细胞癌细胞系SCC-15和CAL-27的增殖活性。     

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。     

POLD1对口腔鳞癌细胞增殖、侵袭的影响及调控机制

目的:探讨人源DNA聚合酶 δ 催化亚基(POLD1)的表达对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、迁移、侵袭、细胞周期的影响及其相关机制.方法:使用生物信息数据分析POLD1在头颈部鳞癌及癌旁组织中的表达,利用免疫组织化学染色检测POLD1在40对口腔鳞癌及癌旁...     

釉原蛋白对牙周膜干细胞迁移、黏附及增殖的影响

目的：检测釉原蛋白（amelogenin,AML）对牙周膜干细胞（PDLSCs）迁移、黏附和增殖的影响。方法：用0．25、50和100μg／ml 的 AML 培养人 PDLSCs。用划痕实验和 transwell 法检测 AML 对 PDLSCs 迁移的影响。用黏附实验检测 AML 对PDLSCs 黏附的影响。用 MTT 法和计数法检测 AML 对 PDLSCs 增殖的影响。结果：AML 可促进 PDLSCs 的迁移,而且呈剂量效应关系（P ＜0．05）。AML 对 PDLSCs 迁移和黏附有促进作用（P ＜0．05）,且随培养时间延长而增加。AML 可促进 PDLSCs的增殖,且呈剂量效应关系。结论：AML 可促进 PDLSCs 的迁移、黏附和增殖。     

The downregulation of ANGPTL4 inhibits the migration and proliferation of tongue squamous cell carcinoma

ObjectiveTongue squamous cell carcinoma (TSCC) is the most common malignant cancer in the oral cavity, with a high rate of metastasis to the neck lymphoid node. Angiopoietin-like protein 4 (ANGPTL4) and microvessel density (MVD) may be novel indicators for tumor metastasis. The aim of the present study was to investigate the expression and function of ANGPTL4 in TSCC and the relationship between ANGPTL4 and MVD.MethodsThe expression levels of ANGPTL4 and MVD (CD34) were analyzed in 65 TSCC specimens and the adjacent non-cancerous tissues using immunohistochemistry (IHC). siRNA was delivered into TSCCA cells to downregulate ANGPTL4 expression. Subsequently, validation with real-time RT-PCR and western blot analyses was performed to analyze ANGPTL4 expression levels. In addition, a proliferation assay, migration and invasion assays were carried out.ResultsANGPTL4 expression was associated with tumor stage, lymph node metastasis and MVD expression. Cox regression analysis showed that high levels of ANGPTL4 expression were closely associated with poor survival time. In vitro analyses using qRT-PCR and western blot confirmed that ANGPTL4 was successfully inhibited in TSCCA cells. Suppressing ANGPTL4 resulted in the inhibition of cell proliferation and migration, but neither invasion nor cisplatin resistance was significantly affected.ConclusionHigh expression levels of ANGPTL4 are associated with the T stage, lymphatic metastasis, angiogenesis and poor overall survival in TSCC patients. The downregulation of ANGPTL4 inhibits the migration and proliferation of cells in TSCC. Taken together, ANGPTL4 may serve as a novel biomarker and therapeutic target for TSCC.     

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.