Adipose‐derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Chronic wounds represent a major socio‐economic problem in developed countries today. Wound healing is a complex biological process. It requires a well‐orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose‐derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell‐migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b‐FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases‐2 (MMP‐2) and MMP‐9 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b‐FGF, VEGF, MMP‐2 and MMP‐9 in ASC, and b‐FGF, VEGF and MMP‐9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.     

氧化低密度脂蛋白对巨噬细胞的作用及机制

目的 观察氧化低密度脂蛋白(oxLDL)对巨噬细胞增殖和分泌巨噬细胞迁移抑制因子(MMIF/MIF)的作用.方法 (1)制备人单核细胞来源巨噬细胞(MDMs),25、50和75 mg/L浓度oxLDL培养12、24、48 h后,细胞计数试剂盒(CCK-8)法检测细胞增殖,酶联免疫吸附法测定上清MIF浓度;不含oxLDL培养为正常对照(NC)组.(2)制备含稳转核因子-κB (NF-κB)-LUC质粒的MDMs,同法培养,荧光素酶底物(Luciferase)检测细胞NF-κB通路活性.(3)含NF-κB-LUC质粒的MDMs中加入终质量浓度为0、10 μmol/L的NF-κB通路抑制剂BAY 11-7082,同法培养并检测上清MIF浓度.结果 (1)与NC组比较,25、50、75 mg/L oxLDL组12 h时MDMs增殖显著升高,48 h时分别达到17.7％、27.8％和41.2％ (P ＜0.01);其上清液MIF也在12h起升高,48 h时分别是NC组的1.49、1.67和2.09倍(P＜0.01).(2)与NC组比较,oxLDL各组在12h即可检测到NF-κB通路明显激活,24h时MDMs内NF-κB通路活性分别增加38.1％、60.3％和61.1％ (P＜0.01).(3)加入BAY11-7082后,各组上清液MIF浓度在12 h即出现明显下降,48 h时分别下降58.6％、69.3％、69.7％和73.5％ (P＜0.01).结论 25 ～ 75 mg/L浓度的oxLDL可促进MDMs增殖和MIF的分泌,其作用随oxLDL作用时间的延长和浓度的增高而增强.oxLDL可能通过诱导激活NF-κB信号通路促进MDMs合成分泌MIF.     

小鼠脊髓背根神经节细胞与人微血管内皮细胞共培养对细胞增殖的影响

目的 观察小鼠脊髓背根神经节细胞(DRGC)与人微血管内皮细胞(HMVEC)共培养对细胞增殖的影响,并探讨其机制.方法 分离培养小鼠DRGC,并与HMVEC进行共培养,设为DRGC组、HMVEC组、DRGC+ HMVEC组.噻唑蓝(MTT)法检测细胞增殖,实时定量聚合酶链反应(Real-time PCR)及Western blot检测血管内皮生长因子(VEGF)、神经生长因子(NGF)、增殖细胞核抗原(PCNA)和细胞周期蛋白D1(Cyclin Dl) mRNA及蛋白的表达.结果 细胞培养24h后,DRGC+ HMVEC组相对于DRGC组和HMVEC组的细胞增殖率分别为136.29％和137.45％,细胞增殖能力显著提高(P＜0.05).VEGF、NGF、PCNA和Cyclin Dl mRNA和蛋白的表达在DRGC+HMVEC组中最高,与其他两组比较差异有统计学意义(P＜0.05).结论 共培养DRGC和HMVEC能够促进VEGF和NGF的表达,并可能通过调控PCNA和Cyclin D1的表达促进共培养体系细胞增殖能力.     

乳铁蛋白促进成骨细胞增殖的最佳浓度及促进分化的最佳时间

目的 探索乳铁蛋白促进大鼠成骨细胞增殖的最佳浓度及1000μg/mL乳铁蛋白对促进成骨细胞分化的最佳时间。方法 用混合酶消化法分离大鼠颅盖骨成骨细胞进行原代培养;不同浓度乳铁蛋白干预成骨细胞,浓度分别为0、0.1、1、10、100、200、400、600、800和1000μg/mL,在1、3、5、7d后分别采用CCK-8法测定细胞增殖;用1000μg/mL的乳铁蛋白干预成骨细胞7、14、21d后,采用碱性磷酸酶染色法检测细胞内碱性磷酸酶的活性。结果 1.CCK-8法测定细胞增殖结果显示,低于100μg/mL浓度乳铁蛋白组其OD值随着浓度的增高而增高,而高于100μg/mL浓度乳铁蛋白组其OD值随着乳铁蛋白浓度的增高而递减;2.碱性磷酸酶染色法结果显示,1000μg/mL的乳铁蛋白在干预第21天时碱性磷酸酶活性最高。结论 100μg/mL乳铁蛋白是促进大鼠成骨细胞增殖的最佳浓度;1000μg/mL的乳铁蛋白促进成骨细胞分化作用呈时间依赖性。     

釉基质蛋白对人牙龈上皮细胞增殖的影响

目的:研究釉基质蛋白(enamelmatrixproteins,EMPs)对体外培养的人牙龈上皮细胞增殖活性的影响。方法:乙酸提取法获取EMPs,取龈切术中切除的牙龈组织,采用酶消化法培养牙龈上皮细胞。将第1代牙龈上皮细胞按照22500个/孔接种,分为4组:对照组(不加EMPs)及EMPs分别为50、100、200μg/ml的实验组。采用MTT法检测各组细胞数量,对每个时间点的各组实验数据进行方差分析。结果:人牙龈上皮细胞能在EMPs覆盖的平皿上生长。统计学分析表明,不同浓度的EMPs在早期对牙龈上皮细胞的增殖无显著影响;从第3天开始,当培养液中EMPs浓度为200μg/ml时,牙龈上皮细胞增殖显著受抑。结论:EMPs影响牙龈上皮细胞的增殖,并存在剂量和时间依赖性。     

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.     

腮腺Warthin瘤及瘤旁腺泡细胞增殖与细胞凋亡的定量研究

目的：探讨细胞增殖与细胞凋亡在腮腺Warthin瘤及瘤旁腺泡中的作用。方法：取64例中部分Warthin瘤及瘤旁腺泡作流式细胞术（flow cqtometry,FCM)。以ModFit LT软件进行细胞增殖分析，以Sub-G1法检测凋亡率.结果：Warthin瘤的DNA指数（DNA index ,DI）、S期细胞比例（schizocqto%,S%)、PI增殖指数（proliferation index,PI）与凋亡率分别为1．13、8．83、9．95和8．24,属最高值，瘤旁腺泡的DI、S％、PI和凋亡率分别为1．08、8．94、8．66和6．67。正常腮腺为0．99、5．10、8．01和5．75。三者之间无显著差异（P＞0．05）。结论：腮腺Warthin瘤和瘤旁腺泡的细胞增殖与细胞凋亡水平稍高于正常腺泡，但差异不大，预示该肿瘤术后复发可能性较小。     

维甲酸对口腔黏膜上皮细胞增殖的影响

目的探讨不同浓度维甲酸对体外培养的口腔黏膜上皮增殖的影响。方法将第二代人口腔黏膜上皮细胞以4×104个/cm2接种于玻片上,在含钙1.2 mmol/L的无血清角朊细胞培养基中培养,将其按培养基中的维甲酸浓度分为0、10-8、10-7、10-6、2×10-6mmol/L共5组。对各组细胞进行硝酸银染色,观察核仁区相关嗜银蛋白(AgNOR s)的着色情况,并对细胞核AgNOR s面积进行图像分析,以评价维甲酸对口腔上皮细胞增殖能力的影响。结果高钙培养基中维甲酸浓度为10-7~10-6mmol/L时,体外培养的上皮细胞内AgNOR s面积与维甲酸浓度0、10-8mmol/L 2个实验组有显著差异(P<0.01)。结论维甲酸可有效地拮抗因钙离子诱导上皮细胞分化所致增殖抑制。