纳米钛酸钙对鼠成骨细胞增殖与分化的影响

背景：以不同修饰剂制备的纳米钛酸钙具有良好的骨传导性、生物相容性及生物活性。 目的：观察纳米钛酸钙材料对成骨细胞增殖、分化的影响。 方法：分别以聚乙二醇、十六烷基三甲基溴化铵、柠檬酸钠为修饰剂制备纳米钛酸钙,同时制备无修饰剂的纳米钛酸钙。将制备的4组纳米钛酸钙分别配制为0.1,1.0,10 g/L的浸提液,MTT方法检测材料浸提液对乳鼠成骨细胞增殖及碱性磷酸酶活性的影响,以正常培养的细胞为对照。 结果与结论：以聚乙二醇、十六烷基三甲基溴化铵、柠檬酸钠为修饰剂制备的纳米钛酸钙,无修饰剂的纳米钛酸钙在1.0,10 g/L质量浓度下可明显促进成骨细胞的增殖;上述4组纳米钛酸钙浸提液在1.0,10 g/L质量浓度下,培养3,6,9 d的细胞A值也高于对照组(P < 0.05,P < 0.01),培养6,9 d的细胞碱性磷酸酶活性值均高于对照组(P < 0.05,P < 0.01)。表明以聚乙二醇、十六烷基三甲基溴化铵、柠檬酸钠、无修饰剂制备的钛酸钙对成骨细胞无毒性作用,可促进成骨细胞的增殖和分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

基质细胞衍生因子1预处理抑制大鼠骨髓间充质干细胞的凋亡

背景：研究发现基质细胞衍生因子1除参与趋化干细胞定向迁移途径,还具有抗凋亡作用。 目的：观察基质细胞衍生因子1预处理后对骨髓间充质干细胞凋亡的影响。 方法：以不同浓度H₂O₂诱导大鼠骨髓间充质干细胞凋亡,取最适宜浓度100 μmol/L用于实验。不同质量浓度基质细胞衍生因子1干预100 μmol/L H₂O₂诱导后的大鼠骨髓间充质干细胞,选择0.2 mg/L最佳保护质量浓度用于实验。取第3代大鼠骨髓间充质干细胞,随机分组：正常对照组不进行任何处理;损伤组在培养液中加入H₂O₂作用24 h;基质细胞衍生因子1预处理组于H₂O₂损伤细胞前6 h加入基质细胞衍生因子1;基质细胞衍生因子1+AMD3100(基质细胞衍生因子1受体CXCR4的阻断剂)组于H₂O₂细胞损伤前6 h加入基质细胞衍生因子1与AMD3100共孵。 结果与结论：H2O2能体外模拟缺血缺氧环境诱导骨髓间充质干细胞凋亡,且作用呈剂量依赖性。与损伤组比较,加入基质细胞衍生因子1预处理后细胞凋亡明显减轻(P < 0.01),细胞E2F6基因表达增强(P < 0.05),E2F1基因表达减少(P < 0.05),线粒体细胞色素C转位减少(P < 0.05),Caspase-3活性降低(P < 0.05),AMD3100可阻断基质细胞衍生因子1对骨髓间充质干细胞的保护作用。提示基质细胞衍生因子1可能通过增强E2F6基因,负性调控E2F1基因抑制线粒体损伤导致的骨髓间充质干细胞凋亡。     

新型输尿管支架材料的生物相容性

背景：有研究表明,聚乙烯高分子合成的输尿管支架在体内实验中具有良好的生物相容性,但实验发现其毒副作用,诱导机体产生的炎症、热原、过敏反应均较明显,故对于新研发的L-乳酸、乙交酯、硫酸钡合成的新型输尿管支架在生物相容性、毒副作用、机体炎症反应等方面的探索是实验研究的重点内容。 目的：通过对临床上使用的聚乙烯高分子合成输尿管支架和L-乳酸、乙交酯、硫酸钡合成的新型输尿管支架在细胞毒性试验、动物体植入后引起的炎症反应以及过敏反应、热原反应、致死率等生物相容性方面的比较,为临床上引入新型输尿管支架材料提供一定的参考依据。 方法：实验分为3组,空白对照组不作任何处理,使用正常培养基培养;新型材料组细胞采用L-乳酸、乙交酯、硫酸钡合成材料浸提液进行培养;聚乙烯组细胞采用聚乙烯浸提液进行培养。 结果与结论：活细胞数目检测结果显示,在尿道上皮细胞培养皿中加入两种输尿管支架浸提液后,与空白对照组相比,毒性反应均明显增强(P < 0.05),但两组毒性差异无显著性意义。苏木精-伊红染色结果显示,将两种材料植入大鼠体内2,6周后,肌肉组织均有明显的炎性浸润、中性粒细胞和嗜酸性粒细胞增加,但植入新型材料组的炎性浸润和组织损伤明显少于植入聚乙烯组(P < 0.05)。酶联免疫吸附试验结果显示,植入两种材料6周后,大鼠血清中炎性因子白细胞介素10,23的含量均明显增高(P < 0.05),但植入新型材料组的炎性因子表达量明显低于植入聚乙烯组(P < 0.05)。支架植入6周后,新型材料组的大鼠在实验过程中出现过敏反应和热原反应的数量均少于聚乙烯组。结果证实,L-乳酸、乙交酯、硫酸钡合成的新型输尿管支架材料能减少组织对于材料的炎症反应、过敏反应、热原反应,具有比聚乙烯合成材料更好的生物相容性。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

TiAl6Vi4表面超疏水修饰后的体外抑菌实验

背景：研究表明,材料表面亲、疏水性(即表面浸润性)是影响细菌黏附的重要原因。 目的：探讨钛金属TiAl₆Vi₄表面超疏水改性后对金黄色葡萄球菌的抑菌作用。 方法：将TiAl6Vi4板块经砂纸、酸溶液抛光和超声清洗后,随机分组：超疏水表面组采用电化学阳极氧化法在TiAl₆Vi₄表面制备TiO₂纳米管薄膜,并通过氟硅烷自组装修饰;亲水表面组采用电化学阳极氧化法在TiAl₆Vi₄表面制备TiO₂纳米管薄膜;疏水表面组对TiAl₆Vi₄表面行氟硅烷自组装修饰,分别测量3组表面的接触角。将3组样品浸泡于金黄色葡萄球菌菌液中2 h,观察样品表面细菌黏附和分布状态,以及浸泡过样品剩余菌液的A值。 结果与结论：亲水表面组表面多数金葡菌彼此聚集、重叠,呈葡萄串形态;疏水表面组表面细菌有聚在一起的趋势,但没有彼此重叠、覆盖,只是单层排列,没有形成葡萄串表面;超疏水表面组表面细菌分散排布,一般只有两三个细菌在一起,不成串,不重叠。3组表面随着亲水性的降低细菌数量逐渐减少,以超疏水表面组为最少,而且细菌相互分离的也更加明显。超疏水表面组剩余菌液的A值明显高于亲水表面组和疏水表面组(P < 0.05)。表明钛金属表面超疏水修饰能有效抑制金黄色葡萄球菌贴附。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

葛根素抑制1，25-二羟基维生素D3促破骨细胞骨吸收功能的最佳剂量

背景：近年来国内外学者对葛根素的骨形成研究较多,但对于葛根素抑制1,25-(OH)₂D₃促破骨细胞骨吸收功能尚未见报道。 目的：观察不同浓度葛根素在体外对1,25-(OH)₂D₃促破骨细胞骨吸收功能的抑制作用。 方法：收集3周龄小鼠骨髓细胞并接种到24孔培养板中。实验组培养液为1,25-(OH)₂D₃ +α-MEM完全培养基+不同质量浓度的葛根素,对照组不加葛根素。 结果与结论：不同浓度组葛根素处理的培养体系均诱导出破骨样细胞且形态差异不明显;除100 μg/L葛根素组在第3天时碱性磷酸酶表达显著增高外,各组之间培养液上清中碱性磷酸酶水平无显著性差异(P > 0.05);10 μg/L葛根素组培养液上清Ca2+水平在第3,5,12天时较对照组显著降低(P < 0.05~0.01),50 μg/L葛根素组培养液上清Ca²⁺水平在第12天时较对照组显著降低(P < 0.01),100 μg/L葛根素组培养液上清Ca²⁺水平在所有检测点较10 μg/L葛根素组显著上升(P < 0.05)。提示葛根素最佳抑制剂量在10~50 μg/L之间。     

三氧化二砷/锰锌铁氧体复合纳米粒体外治疗食管癌

背景：与传统的热疗方法相比,As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒可以同时发挥As₂O₃的细胞毒性作用和磁感应加热的联合定向治疗作用,效果优于单一治疗。 目的：制备As₂O₃/Mn_0.5Zn₀.₅Fe₂O₄复合纳米粒,观察其对食管癌Eca109细胞增殖的抑制作用。 方法：采用浸渍法制备As₂O₃/Mn₀.₅Zn_0.5Fe2O4复合纳米粒,含砷量0.012%,以透射电镜、能谱仪、原子分光光度仪对其进行表征。向两块培养板的食管癌Eca109细胞中分别加入DMEM培养液(阴性对照)、Mn_0.5Zn_0.5Fe₂O₄纳米材料、含As₂O₃终浓度5 μmol/L的As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒、游离As₂O₃(终浓度5 μmol/L),其中一块培养板进行磁流体热疗,另一块培养板正常培养。 结果与结论：As2O3/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒近似球形,As₂O₃成功浸渍在Mn_0.5Zn_0.5Fe₂O₄纳米材料表面,砷含量在0.012%-0.066%之间。当As₂O₃浓度为5 μmol/L时,As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒组细胞增殖率明显低于阴性对照组和Mn_0.5Zn_0.5Fe₂O₄纳米材料组(P < 0.05);而在磁流体热疗中,As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒组细胞增殖率明显低于游离As₂O₃组或Mn_0.5Zn_0.5Fe₂O₄组(P < 0.05);在凋亡率检测中,As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合磁流体热疗组细胞凋亡率明显高于Mn_0.5Zn_0.5Fe₂O₄纳米材料联合磁流体热疗组或游离As₂O₃组(P < 0.05)。表明As₂O₃/Mn_0.5Zn_0.5Fe₂O₄复合纳米粒联合磁流体热疗可显著抑制食管癌细胞增殖。     

胶原蛋白-硫酸肝素生物支架在猪脑内的生物相容性分析

背景：胶原蛋白-硫酸肝素支架与人体神经内部结构十分相似。 目的：探讨胶原蛋白-硫酸肝素支架的体内生物相容性。 方法：将40只小猪随机均分为两组,以距离前囟中线旁开1.0 cm为穿刺点,利用骨穿针进行纵向穿刺,至脑蛛网膜下腔,感到落空感后,将骨穿针缓慢拔除,观察组植入胶原蛋白-硫酸肝素支架,对照组不予处置。术后1,3,7,14,30 d对两组脑组织进行透射电镜观察、细胞凋亡及Caspase-3蛋白表达检测。 结果与结论：早期电镜观察发现,观察组支架周边有个别脑组织神经元受损情况,并出现髓神经纤维脱髓鞘变化,支架与脑组织交界处及支架内部存在阳性Caspase-3蛋白,但在远离支架周边脑组织则无阳性蛋白表达。从术后1 d开始,观察组支架周边脑组织出现个别脑细胞凋亡情况,至术后30 d无明显凋亡细胞,对照组也表现出相似的变化,观察组术后1,3,7,14 d细胞凋亡数量高于对照组(P < 0.05),两组术后 30 d细胞凋亡数量比较差异无显著性意义(P > 0.05)。两组Caspase-3蛋白表达均处于较低状态,且观察组术后3,7 d的Caspase-3蛋白表达高于对照组(P < 0.05),其余时间点比较差异无显著性意义。表明胶原蛋白-硫酸肝素支架在猪脑内具有良好的生物相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

过氧化氢对胚胎干细胞移植治疗心肌梗死的影响

背景：干细胞移植治疗心肌梗死的具体机制一直不十分清楚。 目的：探讨H₂O₂体外预处理小鼠胚胎干细胞对治疗心肌梗死疗效的影响。 方法：选取24只9周龄C57BL/6小鼠随机分为心肌梗死组、胚胎干细胞组、H₂O₂预处理胚胎干细胞组,每组8只,所有小鼠制备心肌梗死模型1周后,分别于尾静脉分别注入150 μL的PBS、150 μL的胚胎干细胞(1×10⁶)、150 μL的H₂O₂预处理胚胎干细胞(1×10⁶)。移植后第3周超声测量各组小鼠左心室舒张末内径、左心室收缩末内径和左心室射血分数。超声心动图检查后取心肌组织行天狼星红染色检测心肌胶原含量,计算平均胶原容积分数。 结果与结论：与心肌梗死组比较,胚胎干细胞组和H₂O₂预处理胚胎干细胞组左心室舒张末内径和左心室收缩末内径明显下降,左心室射血分数明显升高(P < 0.05),全心质量/体质量、左心室质量/体质量、平均胶原容积分数明显降低(P < 0.05);与胚胎干细胞组比较,H₂O₂预处理胚胎干细胞组小鼠左心室舒张末内径和左心室收缩末内径明显下降,左心室射血分数明显升高(P < 0.05),全心质量/体质量、左心室质量/体质量、平均胶原容积分数明显降低(P < 0.05)。所以体外H₂O₂预处理胚胎干细胞减轻了心肌梗死后心肌纤维化,心功能改善更为明显。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

渗透树脂在早期釉质龋修复中的应用：颜色稳定性评价

 背景：采用渗透树脂微创技术治疗釉质脱矿的修复效果优于传统的再矿化治疗,但目前研究渗透树脂修复非实质性缺损龋病后颜色变化的文献很少。目的：通过体外研究评估渗透树脂修复早期釉质龋的颜色稳定性。方法：将具有完整釉质面的48颗人离体牙浸泡在脱矿液中4周,制作成人工龋模型,之后进行渗透树脂修复,再随机均分成4组,分别置于红酒、咖啡、茶和人工唾液中浸泡2,4周。采用分光光度仪对病损区的颜色进行检测,以渗透树脂治疗后的检测作为基准色,计算出色差数值。结果与结论：浸泡后,4组样本都有不同程度的颜色变化。浸泡2周后,颜色变化大小顺序为：红酒>咖啡>茶>人工唾液,4组间比较差异有显著性意义(P < 0.05);浸泡4周后,颜色变化大小顺序为：红酒≈咖啡>茶>人工唾液,红酒组与咖啡组比较差异无显著性意义(P > 0.05),其他组间比较差异有显著性意义(P < 0.05)。红酒组与咖啡组浸泡不同时间点的颜色变化比较差异有显著性意义(P < 0.05)。浸泡在红酒、咖啡和茶一定的时间后,均显示在临床上不可以接受的色彩改变(?E >3.7)。说明渗透树脂颜色的稳定性受浸泡时间长短和浸泡溶液种类的影响,其中红酒、咖啡对其颜色稳定性影响较大,在人工唾液中颜色稳定性最好。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

软骨下钻孔联合软骨源性形态发生蛋白缓释系统修复膝关节软骨缺损

背景：有研究表明软骨源性形态发生蛋白等因子在诱导细胞分化、促进软骨修复过程中起到重要调节作用;软骨下钻孔治疗软骨缺损在临床已广为应用,但其与软骨源性形态发生蛋白等因子联合应用的相关研究至今少有报道。 目的：将软骨下钻孔技术与关节内注射透明质酸/软骨源性形态发生蛋白1缓释载药微球(hyaluronic acid- coated cartilage-derived morphogenetic protein-1,HA/CDMP-1)相结合,观察其对软骨缺损修复的效果,并通过与单纯钻孔组及HA/CDMP-1微球组治疗结果比较,观察两者有无协同效应。 方法：用透明质酸包被软骨源性形态发生蛋白制备缓释微球冻干保存,制备兔实验膝关节全层关节软骨缺损模型,随后将兔随机分为模型组、钻孔组、HA/CDMP-1微球组和钻孔联合HA/CDMP-1微球组,分别采用生理盐水关节腔注射,软骨缺损区钻孔,HA/CDMP-1微球关节腔注射,软骨下钻孔并HA/CDMP-1微球注射。 结果与结论：建模后8,12,16周,组织学观察结果提示,钻孔联合HA/CDMP-1微球组软骨缺损区修复组织覆盖面积明显高于其他3组(P < 0.05);甲苯胺蓝染色和荧光定量PCR检测显示,钻孔联合HA/CDMP-1微球组修复的软骨组织中蛋白多糖和Ⅱ型胶原mRNA的表达明显高于其他3组(P < 0.01或P < 0.05)。结果证实,软骨下钻孔与关节腔内注射HA/CDMP-1联合应用修复兔膝关节软骨缺损近期疗效满意,提示两者可能发生协同作用。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Regions of alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.     

Novel fabrication of fluorescent silk utilized in biotechnological and medical applications

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine.     

Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

ABSTRACT. Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760?nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.     

NFкB活化诱导剂筛选细胞的建立

目的：了解不同刺激剂作用ＨＬ－６０细胞后对ＮＦ－ｋＢ活化的特点，为人类功能基因的筛选提供新的方法。方法：采用基因转染技术获得能稳定表达ＩｋＢａ－ＥＧＦＰ融合蛋白的ＨＬ－６０细胞系，定性和定量检测８种刺激剂作用于该细胞系后荧光强度的变化。结果：定性结果发现，在所用的８种刺激剂中，除ＩＬ－１ｒａ作用后的细胞荧光强度没有明显的变化外，其它７种刺激剂作用后都有不同程度的细胞荧光强度的降低，尤以ＰＨＡ作用后的变化更为典型。定量检测结果发现，稳定表达的ＨＬ－６０细胞自身的荧光强度，随时间延长即有降低，ＩＬ－１ｒａ作用后细胞荧光强度的降低同没有刺激时相比没有明显区别；其它７种刺激剂作用后均有明显的荧光强度降低，但它们作用后的特点并不相同。结论：该真核转染的细胞可用于ＮＫ－ｋＢ活化刺激剂的初步筛选，可以作为功能基因组研究中新基因功能初筛的一个平台技术。     

Photoconversion of fluorescent retrograde tracers.

Photoconversion of fluorescent dyes, retrogradely transported through axons to their parent cell bodies, into a stable diaminobenzidine (DAB) reaction product was tested in the nigrostriatal and thalamocortical systems of rats. Satisfactory results were obtained with Propidium Iodide (PI), Fluoro-Gold (FG), Fast Blue (FB), Diamidino Yellow (DY), and rhodamine-labeled latex microspheres (RLM); some photoconversion was also observed in Evans Blue (EB)-labeled neurons. The red fluorescent tracers PI, EB and RLM were photoconverted under the excitation wavelength appropriate for eliciting their fluorescent emission. With the yellow or blue fluorescent tracers FG, FB, and DY satisfactory results could instead be obtained using an excitation wavelength which did not elicit visible fluorescent emission. This finding indicates that the latter is not a critical factor for obtaining photoconversion. The features and subcellular localization of photoconverted DAB were different from those of the fluorescent labeling: photoconversion resulted in the appearance of brown granules of DAB reaction products in the cytoplasm, independently from the occurrence of fluorescent labeling in the neuronal cytoplasm or nucleus. Photoconversion may enable new applications of fluorescent retrograde tracing and, in particular, its electron microscopic visualization.     

Foot-and-mouth disease virus: Rapid assay using the fluorescent antibody technique

Summary A fluorescent focus assay was used to enumerate infective units of foot-and-mouth disease virus (FMDV) in primary swine kidney cell monolayers. Fluorescent focus assay was rapid and compared well with the titers obtained by cytopathic effect and plaque assays for readily cytopathic strains of FMDV (A₂₄, A-4691 and Asia-1/1, Pakistan-'54). The fluorescent focus assay was superior to CPE or plaque assays for the less cytopathogenic strains, O₆ (O-VI) and C₁ (GC) and was more sensitive than suckling mice for titration of all 4 FMDV strains used. The fluorescent antibody technique has the advantage that FMDV titers can be obtained within 3 to 4 hours.A linear relationship between two-fold virus dilutions and the number of fluorescent foci indicated that each fluorescent cell at 3 hours after inoculation or each fluorescent focus after 6 and 18 hours probably resulted from infection with one infectious FMDV particle or aggregate not separable by dilution. The number of fluorescent cells per fluorescent focus at 6 hours after inoculation was inversely proportional to the concentration of homologous antisera added to the overlaying media.     

人eNOS启动子不同序列驱动的红色荧光蛋白载体的构建与表达

目的:为了研究人血管内皮细胞一氧化氮合酶(eNOS)启动子的功能,构建在哺乳动物细胞中表达的人eNOS启动子不同区段驱动的红色荧光蛋白报告基因载体。方法:从重组pDseNOSRed载体上将eNOS启动子的不同长度DNA序列亚克隆至红色荧光蛋白载体pDsRed1-1上,经PCR、酶切和DNA测序鉴定,将重组载体pDsF1033Red,pDsF494Red和pDsF166Red转染NIH3T3细胞,在倒置荧光显微镜下观察它们在细胞内的表达情况。结果:PCR、酶切和DNA测序结果均表明重组载体pDsF1033Red,pDsF494Red和pDsF166Red的构建正确,这些载体能在NIH3T3细胞中有效表达。由eNOS启动子不同区段驱动表达的95％以上的红色荧光蛋白均匀分布于整个细胞,转染后48-60h开始出现,96-144h为红色荧光蛋白(RFP)的表达高峰,RFP的荧光在144h最强,168h后红色荧光逐渐消退,21d后仍有很少量红色荧光残留。RFP的表达量和荧光强度明显低于强启动子p_CMVIE驱动的RFP。结论:成功构建了eNOS启动子不同区段驱动的红色荧光蛋白报告基因载体,它们能在哺乳动物细胞中有效表达,静息状态下呈现弱转录活性,为研究人eNOS启动子不同区及其顺式调控元件的作用提供了实用而又方便的工具。     

Imaging of chromosomes at nano-meter scale resolution using scanning near-field optical/atomic force microscopy

Topographic and fluorescent images of whole barley chromosomes stained with YOYO-1 were observed simultaneously by scanning near-field optical/ atomic force microscopy (SNOM/AFM). The chromosome was relatively smooth and flat in the topographic images and no significant difference in height was present between regions of high fluorescent and low fluorescent intensity in the chromosomes. The telomeric region, labeled by fluorescence in situ hybridization (FISH) method, was also observed by SNOM/AFM at high resolution, and fluorescent signals of the telomeric region were clearly defined on the topographic image of chromatin fibers on the chromosome at the nano-meter scale level. Although the telomeric signals were usually visualized as a single fluorescent region at the end of sister chromatids by conventional light microscopy, they were observed separately as two fluorescent regions, less than 100-200 nm distance, using the SNOM/AFM. The SNOM/AFM offers great potential in identifying particular single gene location on chromosomes in the near future.     

Use of the fluorescent retrograde tracer, Granular blue, in crustacean motor system studies

Retrograde axonal transport and fluorescent retrograde labelling of neurons were investigated in a crustacean motor system. A fluorescent tracer (Granular blue) was injected into an antennal muscle of rock lobster in order to identify the motor neurons innervating it. After an appropriate post-injection survival time, the fluorescent tracer was found to have labelled motor neurons in the central ganglion. This result indicates that after uptake by the axon terminals, the fluorescent tracer was retrogradely transported through axons of motor neurons to the parent cell bodies.     

Demonstration of succinate dehydrogenase activity in ascites tumor cells using fluorescent tetrazolium salts

In Ascites tumor cells a fluorescent tetrazolium salt, new synthesized (Stellmach 1984) was tested on its suitability for the histochemical enzyme demonstration. Optimal incubation conditions for the demonstration of the succinate dehydrogenase were found out. The red coloured and red fluorescent formazane formed by enzymatic reduction can be localized as well under the light microscope as under the fluorescent microscope. A quantification of the formed formazane as a measure for the enzyme activity was possible by measuring the absorption and fluorescent intensity.