A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles

Poly(MePEG₂₀₀₀cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems.Received 4 March 2005; accepted 14 April 2005     

Cellular responses to mild heat stress

Since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock proteins that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1–2°C during febrile diseases. Here we provide information that mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells.Received 14 May 2004; received after revision 2 August 2004; accepted 16 August 2004     

Human peripheral blood monuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro

The efficiency of test vaccines needs to be evaluated by quantification of the triggered cellular immune response. Usually, for these assays, autologous target cells expressing the vaccine antigen are required. In the context of messenger RNA (mRNA)-based vaccinations, the target cells used for the read-out are mRNA-transfected monocyte-derived dendritic cells (Mo-DCs). Their production typically requires samples of 100 ml blood from the patients, and limits the number of assays that can be performed. We show here that fresh peripheral blood mononuclear cells (PBMCs) can be transfected with mRNA by electroporation. Such cells are as efficient as mRNA-transfected Mo-DCs for their ability to activate memory T cells in vitro. Thus, mRNA-transfected PBMCs are a convenient replacement of mRNA-transfected Mo-DCs for the in vitro monitoring of natural or vaccine-induced immune responses.Received 17 February 2005; received after revision 1 May 2005; accepted 7 Juni 2005     

Marrow mesenchymal stem cells transduced with TPO/FL genes as support for ex vivo expansion of hematopoietic stem/progenitor cells

A new marrow-derived mesenchymal stem cell (hMSC) line that could support expansion of hematopoietic stem/progenitor cells (HSPCs) was developed. Primary hMSCs were infected with retrovirus containing Flt-3 ligand and thrombopoietin genes. CD34+ cells from cord blood were expanded with primary hMSCs or transduced hMSCs. The expansion of total nucleated cells, CD34+ cells and mixed colonies containing erythroid and myeloid cells and megakaryocytes for 2 weeks coculture with transduced hMSCs was remarkably increased. The outputs of long-term culture-initiating cells for 2 and 4 weeks coculture with transduced hMSCs were also largely increased. The expansion rates of HSPCs with transduced hMSCs were unchanged for 6 weeks. In contrast, the expansion rates of HSPCs with primary hMSCs declined drastically through 6 weeks. SCID-repopulating cell expansion with transduced hMSCs for 4 weeks was significantly higher than that of uncultured CD34+ cells and HSPCs expanded with primary hMSCs. Received 21 June 2005; received after revision 30 July 2005; accepted 24 August 2005     

A model of calcium signaling and degranulation dynamics induced by laser irradiation in mast cells

Recent experiments show that calcium signaling and degranulation dynamics induced by low power laser irradiation in mast cells must rely on extracellular Ca^2＋ influx. An analytical expression of Ca^2＋ flux through TRPV4 cation channel in response to interaction of laser photon energy and extracellular Ca^2＋ is deduced, and a model characterizing dynamics of calcium signaling and degranulation activated by laser irradiation in mast cells is established. The model indicates that the characteristics of calcium signaling and degranulation dynamics are determined by interaction between laser photon energy and Ca^2＋ influx. Extracellular Ca^2＋ concentration is so high that even small photon energy can activate mast cells, thus avoiding the possible injury caused by laser irradiation with shorter wavelengths. The model predicts that there exists a narrow parameter domain of photon energy and extracellular Ca^2＋ concentration of which results in cytosolic Ca^2＋ limit cycle oscillations, and shows that PKC activity is in direct proportion to the frequency of Ca^2＋ oscillations. With the model it is found that sustained and stable maximum plateau of cytosolic Ca^2＋ concentration can get optimal degranulation rate. Furthermore, the idea of introducing the realistic physical energy into model is applicable to modeling other physical signal transduction systems.     

考虑主路左转延误的主路优先交叉口信号设置流量阈值

为了进一步完善主路优先交叉口信号灯设置流量条件,考虑了主路左转车流延误对交叉口信号灯设置条件的影响.通过分析主路左转车流在信号灯设置前后的交通特性,利用控制延误方法和Webster方法的研究思路,分别建立主路左转车流在信号灯设置前后的车均延误改进模型.从交叉口交通运行效率的角度出发,以交叉口车均延误作为判别指标,建立信号灯设置前后交叉口等车均延误曲线,以此曲线作为判断交叉口是否需要设置信号灯的阈值条件,同时还给出了不同主路左转车流比例下的流量阈值.与美国MUTCD提出的阈值曲线相比,考虑左转车流延误影响的流量阈值曲线更符合我国主路优先交叉口交通特性,具有更强的实用性.     

枯草芽孢杆菌高效转化及其转化子验证方法

用构建好的重组质粒转化枯草芽孢杆菌（Bacillus subtilis）WB600感受态细胞,进行不同时间预培养后,涂布于抗生素平板培养,研究一种更高效的转化方法。用载体与外源片段的连接产物转化WB600感受态细胞,进行最佳预培养时间培养后,涂布于抗生素平板培养,培养得到的转化子用PCR快速验证和双酶切鉴定,研究一种转化子的快速验证方法。     

克服昆明小鼠早胚2-细胞阻滞的研究

目的：评价两种培养液对昆明小鼠胚胎２－细胞阻滞的克服效果。方法：用添加１５％ＦＣＳ或ＢＳＡ的Ｍ１６和ＣＺＢ培养液培养昆明小鼠２－细胞胚胎和体外受精卵,以观察这两种培养液对昆明小鼠早胚发育的影响及在克服昆明小鼠２－细胞阻滞中的作用,并通过ＣＺＢ培养液中葡萄糖成份的增减,了解其作用及效应的时间。结果：添加ＦＣＳ的Ｍ１６和ＣＺＢ培养注均能较好地支持２－细胞胚胎发育到囊胚（７８．９％,７２．７％）,当进行     

碱性成纤维细胞生长因子对骨髓基质细胞的生物学效应

目的探讨碱性成纤维细胞生长因子(bFGF)对骨髓基质细胞(BMSC)粘附、增殖和分化等生物学效应的影响.方法利用体视学计数、MTT法及ALP试剂盒分别测定不同浓度bFGF诱导一定时间后BMSG的粘附特性、增殖和分化情况的变化.结果10n/mLbFGF明显促进BMSG的粘附,但是200ng/mLbFGF反而不利于细胞粘附;在细胞增殖和分化测定中,100ng/mLbFGF明显促进细胞增殖,但细胞碱性磷酸酶含量也最低.结论碱性成纤维细胞生长因子对骨髓基质细胞的生物效应是复杂和多方面的,可以作用于骨髓基质细胞的粘附、增殖和分化等多个环节,这种影响与碱性成纤维细胞生长因子的剂量有关.