Chemically distinct rat olivocochlear neurons.

We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203-214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD-positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP-positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP-positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems.     

Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.     

Primary Alloproliferative TH1 Response Induced by Immature Plasmacytoid Dendritic Cells in Collaboration with Myeloid DCs

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.     

骨髓基质干细胞修复兔关节软骨缺损的实验研究

目的研究以多聚乙醇酸(PGA)为支架的骨髓基质干细胞(BMSCs)复合物修复兔膝关节软骨缺损的情况。方法体外培养扩增的自体BMSCs种植于PGA支架并培养72h,然后将支架-细胞复合物植入兔关节软骨缺损模型。术后12周处死动物,标本行大体观察、组织学检查及Ⅱ型胶原免疫组化染色。结果BMSCs-PGA复合物植入后形成丰富的透明软骨样修复组织,新生软骨无明显退变。对照组主要为纤维组织及软骨下骨修复。结论BMSCs-PGA复合物可修复关节软骨缺损。     

AdEasy1／Cbfa1诱导间充质干细胞向成骨细胞分化的实验研究

目的：观察核心结合因子a1（Cbfa1）对兔骨髓间充质干细胞（MSCs）向成骨细胞分化的诱导作用。方法：体外分离培养兔骨髓MSCs，用AdEasy1／Cbfa1。转染MSCs，在转染后3d,1、2、3和4周时，组织化学和免疫组化等方法检测成骨标志碱性磷酸酶和骨钙素的表达。结果：AdEasy1／Cbfa1转染后的兔骨髓MSCs表现出与成骨细胞相似的形态，并且表达碱性磷酸酶和骨钙素。结论：Cbfa1可诱导兔骨髓MSCs向成骨细胞分化。     

种子细胞双相接种技术促进组织工程骨体外成熟的研究

目的 比较观察双相接种法和静置接种法构建组织工程骨对种子细胞增殖、分化和成骨表达的影响。方法 取健康志愿者骨髓，密度梯度离心法分离骨髓间充质干细胞并进行体外扩增、诱导分化，然后应用双相接种法和静置接种法与脱钙骨基质（DBM）复合构建组织工程骨，体外培养3周，用倒置显微镜、扫描电镜观察细胞密度、形态及基质分泌情况，并在不同时相点测定组织块中DNA含量、碱性磷酸酶（ALP）活性及钙含量变化。结果 倒置显微镜下双相接种法构建的组织块可见大量具有一定折光性的梭形细胞存在于胶原基质中。扫描电镜见双相接种法构建的组织块切面较平坦，孔隙较小，细胞包埋在胶原中，而静置接种法构建的组织块可见细胞、细胞外基质较少。在体外培养过程中，双相接种法组织块中细胞的DNA含量、ALP活性均高于静置接种法，钙含量在培养2周后高于静置接种法。结论 双相接种法是一种高效的组织工程骨构建方法，有利于提高接种效率和促进组织工程骨的体外成熟。     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的 探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料--多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性.方法 以直径3 mm、长5 mm的圆柱形HDPE,外裹 1 mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7∶3混合,以10×10 7/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组).经体外培养2周及在裸鼠皮下移植4、8周后取材 ,行大体观察、组织学、组织化学及免疫组化检测.结果 各组细胞均与材料黏附良好.实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密.组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达.结论 以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体.     

蛋白酶激活受体1介导人肺上皮细胞分泌白细胞介素-8

目的探讨蛋白酶激活受体1（PAR1）激动肽和凝血酶对人肺上皮细胞白细胞介索-8（IL-8）分泌的影响。方法人肺上皮细胞系A549细胞分别接种于12孔培养板各孔内，并分别用不同浓度的PAR1激动肽SFLLR和反PAR1激动肽RLLFS以及不同浓度的凝血酶和／或凝血酶抑制剂水蛭索进行刺激，刺激时间为2和16h。用ELISA方法检测上清液中的IL-8水平。结果经过16h的培养，SFLLR可引起浓度相关性IL-8的释放增加，增加到300μmol/L时诱导IL-8的释放量比基础分泌量增加了近16倍，RLLFS不能引起IL-8的释放增加。凝血酶也可引起浓度相关性IL-8释放．凝血酶在浓度1kU／L时就可引起IL-8释放量增加，10kU/L时诱导IL-8释放量达高峰，为基础分泌量的7．5倍。水蛭索可以抑制凝血酶对IL-8的释放作用。时间相关曲线表明，PAR1介导的IL-8释放从2h起即可引起增加，16h达高峰。结论PAR1激动肽和凝血酶可促进人肺上皮细胞分泌IL-8，PAR1拮抗剂和凝血酶抑制剂可能具有抗炎作用。     

Influence of tumours on normal cells and optimal chemotherapy regimens: the case of several drugs and toxicity constraints.

Cancer chemotherapy with the application of several drugs is studied. The negative and inhibiting effect of the tumour on normal cells is taken into account. Under certain hypotheses, we determine the optimal regimen that minimizes the tumour burden at the end of a fixed period of therapy while maintaining several normal cell populations above prescribed levels. More precisely, it is demonstrated that the optimal drug administration corresponds to the strategy of intensive chemotherapy.     

转化生长因子β1对人牙髓细胞微丝骨架的重组作用

目的:了解转化生长因子β1(TGF-β1)对人牙髓细胞微丝和微管骨架的作用。方法:I型胶原酶消化组织块法体外培养人牙髓细胞,以20ng/ml的TGF-β1处理细胞,分别在第30min、1、6和24h收集细胞爬片,BODYPY-Phalloidin对微丝作直接荧光染色、Rhodamine RedTM对微管蛋白α(tubulin-α)作间接免疫荧光染色,采用激光扫描共聚焦显微镜观察TGF-β1作用于牙髓细胞后不同时间点微丝和微管的变化情况。结果:TGF-β1作用于人牙髓细胞后,微丝出现解聚重组现象,在30min时间点,肌动蛋白(actin)在细胞膜下聚合成纤维形肌动蛋白F-actin,同时胞质内的F-actin解聚,6h解聚最明显,24h后可见胞质内微丝重组。观察的各时间点,微管结构未见明显解聚重组现象。结论:TGF-β1能够使牙髓细胞微丝骨架重组。