人牙周膜成纤维细胞的原代培养及鉴定

目的体外分离培养人牙周膜成纤维细胞,进一步研究细胞因子对牙周膜细胞功能的调控作用。方法利用组织块法原代培养牙周膜成纤维细胞并传代,通过形态学及免疫细胞化学方法对其进行鉴定。结果牙周膜成纤维细胞培养成功传代,5代后细胞生长旺盛,表现为长梭形细胞,免疫细胞化学鉴定细胞抗波形丝蛋白阳性,抗角蛋白阴性,为中胚层来源的成纤维细胞。结论组织块法培养出的细胞符合牙周膜成纤维细胞的形态学及免疫学特征,生长状态良好,可作为体外的细胞模型为研究细胞因子对其功能的调控作用奠定基础。     

小白菊内酯对TGF-β1诱导的大鼠肺成纤维细胞转分化的影响

目的探讨小白菊内酯(PNT)对转化生长因子-β1(TGF-β1)诱导的大鼠肺成纤维细胞转分化的影响。方法选择12周龄健康SD大鼠12只,组织块法体外培养大鼠肺成纤维细胞,免疫细胞化学染色,观察不同浓度PNT对TGF-β1诱导的肺成纤维细胞表达α-平滑肌肌动蛋白(α-SMA)的影响。结果 TGF-β1诱导肺成纤维细胞转分化为肌成纤维细胞,表达α-SMA增强(P<0.05),20μmol/L的PNT部分抑制TGF-β1的诱导作用(P<0.05);50μmol/L的PNT可完全抑制TGF-β1的诱导作用。结论 PNT有抑制TGF-β1诱导肺成纤维细胞转分化的作用。     

人表皮干细胞体外分离和培养的研究

目的建立人表皮干细胞体外分离和培养的技术方法。方法用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞,并以人成纤维细胞条件培养基为基础制备表皮干细胞培养液,用以人表皮干细胞(Ⅳ型胶原快速粘附法富集分离)的体外培养,通过检测β1整合素和角蛋白19的表达水平及其克隆形成率和克隆维持时间对其进行鉴定,角朊细胞作对照。结果表皮干细胞体外培养,可见细胞呈克隆样生长、β1整合素及角蛋白19免疫组织化学染色呈阳性,且其克隆形成率和克隆维持时间分别为(17.04±1.01%)和15～18d,明显高于对照组的(8.72±0.73%)和9～10d(P<0.01)。结论应用Ⅳ型胶原快速粘附法及人成纤维细胞条件培养基,对人表皮干细胞成功进行了体外分离和培养,为表皮干细胞体外的大量扩增奠定了基础。     

红景天苷对力达霉素诱导人二倍体成纤维细胞衰老的干预作用

目的:研究红景天苷对力达霉素导致DNA损伤后所诱导的细胞衰老的调节作用。方法:采用MTT法测定药物对人胚肺二倍体成纤维细胞2BS活力的影响;衰老相关的β-半乳糖苷酶染色法观察细胞衰老形态变化;流式细胞术检测细胞周期分布以及γ-H2AX蛋白表达变化;蛋白免疫印迹技术观察p53和p21蛋白表达水平变化。结果:较低浓度(低于0.1 nmol.L-1)力达霉素能够抑制2BS细胞增殖,诱导β-半乳糖苷酶染色率显著升高和G2/M期阻滞,并使DNA损伤相关的γ-H2AX蛋白以及p53和p21蛋白表达升高。红景天苷能够显著干预力达霉素诱导的上述细胞衰老样表型及相关分子信号变化。结论:红景天苷能够抑制基因毒药物所致DNA损伤及其诱导的细胞衰老。     

复制缺陷重组腺病毒有效转染兔心包组织的实验研究

目的观察腺病毒载体转染兔心包组织的有效性和外源性基因表达的时相性。方法用第五代腺病毒粘粒(Adv5-CAG)为对照组或含β-半乳糖苷酶基因的第五代腺病毒质粒(Adv5-CAG／LacZ)为实验组转染兔心包组织，转染后第3、7、14、28d取被转染组织行β-半乳糖苷酶(X-gal)染色及β-半乳糖苷酶活性检测。结果X-gal染色显示实验组转染后第3、7、14、28d注射部位心包组织均见蓝染，尤以第7d最明显，而对照组心包组织均无蓝染；β-半乳糖苷酶活性检查示实验组转染后第3d心包组织中即有β-半乳糖苷酶表达，7、14d达高峰，28d表达量明显减少。结论重组腺病毒载体可有效地转染兔心包组织，外源性基因在兔心包组织中能够表达并能维持1个月左右。     

人参皂苷Rg1对细胞光老化模型中p53信号转导途径的影响

目的观察人参皂苷Rg1对光老化p53信号转导途径中相关基因损伤及蛋白表达水平的影响。方法采用8-MOP/UVA(8-methoxypsoralen and subsequent ultraviolet A irradiation)联合处理人皮肤成纤维细胞建立光老化模型,用流式细胞周期分析、SA-β-半乳糖苷酶(senescence associatedβ-galactosidase)化学染色,免疫荧光及Western blot等方法检测人参皂苷Rg1对培养真皮成纤维细胞多项细胞衰老指标及p53信号途径中蛋白表达的影响。结果人参皂苷Rg1可明显抑制细胞和组织老化的指标表达(包括SA-β-Gal表达减少及细胞周期G1阻滞率降低);减少基因氧化应激损伤产物8-oxo-dG及老化相关蛋白p53,p21WAF-1及p16INK-4a的表达。结论人参皂苷Rg1可能通过缓解基因的氧化应激损伤,抑制相关信号转导,从而缓解细胞光老化进程。     

乳鼠心肌成纤维细胞培养方法的改进

目的建立乳鼠心肌成纤维细胞培养模型.方法通过对经典的差速贴壁分离法稍作改进,采用每次贴壁60min、两次差速贴壁分离法收集、培养心肌成纤维细胞.结果形态学观察和免疫细胞化学染色鉴定显示,成功培养心肌成纤维细胞,纯度达95%以上.结论该研究成功建立乳鼠心肌成纤维细胞培养模型,为高纯度心肌成纤维细胞的获得和对其病理生理学的进一步研究打下良好基础.     

藻红蛋白诱导人乳腺癌MCF-7细胞衰老研究

目的:研究藻红蛋白对人乳腺癌MCF-7细胞的诱导衰老作用。方法:采用MTT法检测藻红蛋白对MCF-7细胞的生长抑制作用,采用倒置显微镜观察形态学变化,酸性β-半乳糖苷酶染色法和衰老相关异染色质聚集检测藻红蛋白诱导人乳腺癌MCF-7细胞衰老作用。结果:MTT法结果显示,藻红蛋白对体外培养的人乳腺癌细胞MCF-7具有明显的生长抑制作用,IC50为134.87μg/mL。结果发现藻红蛋白作用72h后,细胞呈现变大变扁平,细胞内形成空泡、颗粒增多等明显衰老形态。在倒置显微镜下观察,酸性β-半乳糖苷酶染色呈阳性;在荧光显微镜下观察,DAPI染色出现衰老相关异染色质聚集现象。结论:藻红蛋白可通过诱导MCF-7细胞衰老达到抗肿瘤作用。     

黄精降低活性氧水平促进衰老内皮祖细胞功能的研究

目的观察黄精在老年大鼠内皮祖细胞(endothelial progenitor cells,EPCs)传代培养衰老进程中对细胞功能及活性氧(reactive oxygen species,ROS)水平的影响。方法分离培养大鼠骨髓EPCs并鉴定,将培养至第2代的EPCs分为4组,分别用黄精含药血清和空白对照血清继续培养至第4、6、8代。β-半乳糖苷酶染色法检测细胞衰老程度;MTT法检测细胞增殖功能;Transwell小室检测迁移功能;体外成血管试剂盒检测成血管功能;流式细胞仪检测ROS水平。结果 EPCs在体外传代培养中,其衰老程度逐渐加重,并伴有增殖、迁移和成血管功能的明显减退,ROS水平急剧升高(P<0.05);经黄精干预后,EPCs衰老程度有所减轻,其功能有所增强,ROS水平明显降低(P<0.05)。结论黄精可能通过降低ROS水平来延缓传代培养EPCs的衰老进程,保护EPCs的功能。     

银杏叶提取物EGB761对D-半乳糖诱导的心肌细胞老化的保护作用

目的：探讨银杏叶提取物EGB761对D-半乳糖诱导的心肌细胞老化的影响及其可能机制。方法：用5 g/L D-半乳糖处理培养心肌细胞建立衰老模型,并以不同浓度EGB761（5、10、20μg/mL）分别干预48 h。以β-半乳糖苷酶染色判断细胞衰老,ELISA法检测心肌细胞晚期糖基化终产物（advanced glycation end products,AGEs）的含量。显微镜下观察心肌细胞的形态和搏动情况。结果：与正常对照组相比,D-半乳糖诱导的心肌细胞内β-半乳糖苷酶染色阳性率显著提高（75.6%±4.9%vs 17.2%±2.9%,P〈0.01）;细胞内AGEs含量明显增高[（703±32）vs（93±26）pg/mL,P〈0.01];同时出现细胞形态学和搏动的异常。与D-半乳糖组比较,不同浓度（5、10、20μg/mL）EGB761干预组β-半乳糖苷酶染色阳性率显著下降（各组分别为57.7%±7.9%、49.7%±9.2%、34.0%±6.6%,P〈0.01）,同时AGEs含量显著降低[分别为（404±33）、（357±25）、（249±77）pg/mL,P〈0.01],心肌细胞的搏动明显增加。结论：EGB761可通过抑制心肌细胞的非酶糖基化,预防D-半乳糖诱导的心肌细胞老化。     

aFGF、bFGF和EGF对小鼠皮肤成纤维细胞生长及蛋白酶活化受体1的影响

目的比较酸性成纤维细胞生长因子(aFGF)、碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)对小鼠皮肤成纤维细胞增殖的作用以及对蛋白酶活化受体1(PAR1)的影响,探讨PAR1与促进成纤维细胞增殖的关系。方法取出生3周体质量为15~20 g的NIH小鼠皮肤进行成纤维细胞培养,采用MTT法测定细胞数和活度,免疫细胞学XTT法检测aFGF、bFGF和EGF对小鼠皮肤成纤维细胞生长的影响,RT-PCR法检测小鼠皮肤成纤维细胞中PAR1 mRNA的表达。结果小鼠皮肤成纤维细胞健康生长,aFGF、bFGF和EGF对小鼠皮肤成纤维细胞生长的促进作用在实验组与对照组间有显著性差异(P<0.05),其对成纤维细胞作用浓度梯度较为明显;小鼠皮肤成纤维细胞存在PAR1 mRNA的表达,且三种生长因子对其表达的影响有差异,以aFGF组表达最显著。结论aFGF、bFGF和EGF均能促进成纤维细胞增殖,以aFGF的促进作用最强,PAR1可能参与成纤维细胞的增殖。     

CD147siRNA对恶性黑色素瘤细胞VEGF及血管内皮细胞体外迁移活性的影响

目的研究CD147siRNA对恶性黑色素瘤细胞株A375中VEGF表达水平和血管内皮细胞体外迁移活性的影响。方法采用半定量RT-PCR法检测转染后A375细胞中VEGF mRNA表达的变化。采用ELISA法检测A375细胞与成纤维细胞共同培养后VEGF表达水平的变化。采用细胞迁移实验检测转染CD147siRNA前后的A375细胞对脐静脉内皮细胞株ECV-304在体外迁移活性的影响。结果转染CD147siRNA后,A375细胞中VEGF的mRNA和蛋白表达水平显著降低,其下调率分别为10.77%（P〈0.05）和38.5%（P〈0.01）。转染CD147siRNA前后的A375细胞与成纤维细胞共同培养后VEGF的表达水平均较单独培养时显著升高,其升高率分别为98.5%（P〈0.01）和159.9%（P〈0.01）。转染CD147siRNA后的A375细胞使ECV-304细胞的体外迁移活性下降44.77%（P〈0.01）。结论 CD147siRNA能有效抑制恶性黑色素瘤细胞株A375中VEGF的表达水平和脐静脉内皮细胞株ECV-304在体外的迁移活性。     

Colorimetric quantitation of in vitro cell density using carmine, a chromosome-specific stain.

Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 M NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 x 10(3) to 5 x 10(5) cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.     

An in vitro screening method for antitumor and/or antitumorigenic substances involving the transformation of chick embryo fibroblasts infected with Rous sarcoma virus

An efficient in vitro screening method for antitumor and/or antitumorigenic substances was established. The method is based on determining inhibitory effect of a compound on an RSV-induced tumorigenic process and the growth of the established tumor cells in a single assay system in the presence of normal cells. These effects were determined by inhibition of focus formation in a culture of chick embryo fibroblasts, while the nonspecific cytotoxic effect was determined by inhibition of the protein content of the same culture. The efficiency of this method in screening drugs was confirmed by testing various clinical and preclinical compounds.     

p53基因在病理性瘢痕成纤维细胞中的表达

目的　探讨 p5 3基因在病理性瘢痕成纤维细胞中的表达情况及其意义。方法　取手术切除的正常皮肤、增生性瘢痕和瘢痕疙瘩组织各 8例为标本 ,通过细胞培养 6～ 8代后 ,应用反转录 -聚合酶链反应 (RT-PCR)的方法检测 p5 3m RNA的表达。结果　 p5 3m RNA在正常皮肤高表达 ,而在增生性瘢痕和瘢痕疙瘩组明显减弱 ,差异具有显著性 (P<0 .0 5 )。结论　 p5 3基因介导了瘢痕疙瘩成纤维细胞的异常增生 ,是病理性瘢痕发生的分子机制之一。     

Regulation of interferon-beta activity by fibroblast cells

Exogenously administered interferons are rapidly cleared from the body. Several pharmacological mechanisms have been implicated in this clearance; however, they do not entirely explain the different clearance rates of the interferons. Cultured cells were studied for their ability to regulate interferon levels in vitro. Preparations of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma were exposed to cells in culture and monitored for any loss in titer. MuIFN-beta titers were found to be significantly reduced following exposure to mouse L-929 fibroblast cells. The reduction of MuIFN-beta activity appeared to be specific for fibroblasts, since the reduction occurred following exposure to L-cells and to mouse embryo fibroblasts, but not to mouse reticuloendothelial cells. Moreover, the ability of the mouse fibroblast cells to reduce MuIFN-beta titers was blocked if the cells were pre-treated with actinomycin D, suggesting that de novo RNA synthesis was required. The titers of IFN-alpha and IFN-gamma were not reduced following exposure to either fibroblast or reticuloendothelial cells. Thus, the reduction of interferon titer by fibroblasts was IFN-beta specific. Similarly, HuIFN-beta titers were reduced following exposure to human fibroblasts. The ability of fibroblast cells to reduce IFN-beta titers was also found to be species-specific, since human fibroblast cells reduced the titer of HuIFN-beta but not MuIFN-beta while murine fibroblasts reduced the titer of MuIFN-beta but not HuIFN-beta. These results suggest that IFN-beta-treated fibroblasts specifically regulate their response to IFN-beta by reducing the titer of the IFN-beta activity.     

Fibronectin promotes lung colony formation in the mouse by B16 melanoma cells spheroids.

By microscopical observation and using an original method of automatic image analysis, we studied on histological sections the rate of lung colony formation after intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as pure or mixed spheroids (B16 + 3T3 fibroblasts). The preincubation in vitro of pure spheroids with fibronectin significantly increased the percentages of lung section area occupied by tumors and the relative number of internal lung colonies. This effect of fibronectin was even more obvious when mixed spheroids were injected.     

Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro.