C-myc寡脱氧核苷酸与叶酸结合的生物学特性

目的：寡脱氧核苷酸为多聚阴离子分子，不能透过细胞膜，而叶酸受体是转运叶酸及与叶酸连接药物进入高表达叶酸受体肿瘤细胞的关键通道，因此将C-myc寡脱氧核苷酸与叶酸结合，观察C-myc寡脱氧核苷酸与叶酸连接药物的生物学特性。 方法：实验于2005-07/2006-07在中南大学湘雅医院中心实验室进行。①将C-myc反义、正义和无义寡脱氧核苷酸分别与叶酸连接，并标记放射性核素99Tcm，以酒石酸钠为转螯合剂。②将99Tcm标记的C-myc反义、正义和无义寡脱氧核苷酸－叶酸连接药物分别与新鲜人血浆孵育1.5，4和6 h，测定其与血浆白蛋白结合率；并在孵育0.5，2，4和6 h测定放射性化学纯度，以了解其血清稳定性。 结果：①随着时间增加，反义、正义和无义寡脱氧核苷酸的血清蛋白结合率增加，6 h为26%，高于1.5 h，差异有显著性意义（P < 0.05）。②99Tcm标记的C-myc反义、正义和无义寡脱氧核苷酸-叶酸连接药物的放射性化学纯度随着时间推移略降低，6 h略有下降，但仍> 90%,且各个时间段比较差异无显著性意义(P > 0.05)。 结论： C-myc寡脱氧核苷酸叶酸药物血浆蛋白结合率在6 h为26%，在血清中6 h内稳定，可满足体内外分析的要求。     

壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架制备及其蛋白缓释效果：与单纯纳米羟基磷灰石/聚乳酸-羟基乙酸支架、壳聚糖微球的比较

背景：骨组织工程骨构建中如何使生长因子持续高效发挥作用是影响成骨速度和质量的关键,现多以各种材料的微球或支架作为缓释载体,但缓释作用有待提高。 目的：实验拟制备壳聚糖微球,然后复合到纳米羟基磷灰石/聚乳酸-羟基乙酸支架上,形成双重缓释作用,并测量对牛血清白蛋白的释放效果。 方法：以牛血清白蛋白为模型药物,采用乳化交联法制备壳聚糖微球。将微球与纳米羟基磷灰石、聚乳酸-羟基乙酸按一定比例混合,以冰粒子为致孔剂,采用冷冻干燥法制备壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架。利用扫描电镜、激光粒度分析仪、压泵仪和力学性能测试仪检测复合支架的形态性能,考察药物在缓释支架上的体外释放规律。 结果与结论：所制备的壳聚糖微球形态良好,呈规则圆球形,粒径集中分布在20~40 μm,微球药物包封率为86.5%,载药量为0.8%,随牛血清白蛋白初始用量的增加,载药量可升高至2.6%,但包封率下降至74.1%。壳聚糖微球能均匀分布在聚乳酸-羟基乙酸支架上,形成壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架,孔径为100~400 μm,孔隙率> 80%,压缩强度为1.1~2.3 MPa,10周降解率为26.5%。单纯纳米羟基磷灰石/聚乳酸-羟基乙酸支架其牛血清白蛋白在36 h累积释放量达85%以上,壳聚糖微球其牛血清白蛋白10 d累积释放量为33.6%,复合支架其牛血清白蛋白40 d累积释放量为81.5%。结果证实包埋壳聚糖微球的纳米羟基磷灰石/聚乳酸-羟基乙酸支架其压缩强度和降解速率合适,对蛋白类药物具有良好的缓释作用,有望作为组织工程的支架材料和生长因子的缓释载体。 关键词：聚乳酸-羟基乙酸;支架;壳聚糖;缓释载体;骨修复材料,组织工程;生物材料 doi:10.3969/j.issn.1673-8225.2010.03.017     

人反义血管内皮生长因子基因载体构建及对肾细胞癌血管内皮生长因子表达的影响☆

目的： 构建反义血管内皮生长因子基因表达载体，观察其对肾癌细胞血管内皮生长因子表达的影响。 方法：实验于2006-07/2007-03在郑州大学第三附属医院实验中心完成。①实验材料：质粒 pcDNA3.1(－)，宿主菌DH5α，肾癌细胞株786-0。②实验过程：克隆人血管内皮生长因子基因，将反义血管内皮生长因子基因定向克隆于质粒pcDNA3.1(－)表达载体，酶切鉴定；转染人肾癌细胞，并分别命名为反义血管内皮生长因子组和空载体组，未转染细胞命名为对照组；G418筛选阳性克隆。③实验评估：反转录-聚合酶链反应方法检测血管内皮生长因子mRNA的表达，免疫组织化学法检测血管内皮生长因子基因的蛋白表达，流式细胞术检测细胞周期，四甲基偶氮唑盐法检测细胞生长情况。 结果：①成功构建反义血管内皮生长因子基因表达载体。②反义血管内皮生长因子组血管内皮生长因子mRNA的表达受到抑制，明显低于空载体组和对照组血管内皮生长因子mRNA的表达(P < 0.01),空载体组血管内皮生长因子mRNA的表达没有受到影响。③反义血管内皮生长因子组的血管内皮生长因子蛋白表达明显降低，明显低于空载体组和对照组(P < 0.01)，空载体组和对照组血管内皮生长因子蛋白的表达差异无显著性。④反义血管内皮生长因子组细胞生长减慢，G1期细胞比例增加，S期细胞比例减少,反义血管内皮生长因子组细胞生长明显减慢。 结论：成功构建血管内皮生长因子的pcDNA3.1(－)反义基因表达载体，人反义血管内皮生长因子基因可明显降低肾癌细胞血管内皮生长因子基因在转录和翻译水平的表达，抑制肾癌细胞生长，为肾癌基因治疗提供一定的实验依据。     

生物素化聚乙二醇/聚乳酸纳米粒子的体外主动靶向行为

背景：目前研究的大部分高分子药物载体没有靶向性,在应用上有局限性,只有几个国外课题组报道生物素化聚乙二醇/聚乳酸(Biotin-PEO-PLA)纳米粒子的体外靶向行为,国内没有这方面的研究报道。 目的：分析Biotin-PEO-PLA纳米粒子作为靶向药物载体的可行性。 方法：透析法制备包埋紫杉醇的Biotin-PEO-PLA纳米粒子并表征;通过高效液相色谱研究包埋紫杉醇的Biotin-PEO-PLA纳米粒子的体外释放行为;利用细胞毒性法比较研究生物素-亲和素三步法实施的包埋紫杉醇的Biotin-PEO-PLA纳米粒子对OVCAR-3(表面表达CA-125抗体)和SKOV-3(表面不表达CA-125抗体)细胞的体外靶向行为。 结果与结论：包埋在Biotin-PEO-PLA纳米粒子中的紫杉醇的释放呈现初期的快速释放以及随后的缓慢释放。利用三步法处理的OVCAR-3细胞存活率明显低于SKOV-3细胞,表明通过Biotin-PEO-PLA/avidin/biotinylated MAB X306与OVCAR-3细胞表面CA-125抗原的特异性相互作用,包埋紫杉醇的Biotin-PEO-PLA纳米粒子被更为有效地传递进了OVCAR-3细胞。     

不同浓度肝细胞生长因子及表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化******

背景：研究证实在多种微环境下可以将骨髓间充质干细胞诱导分化为成熟肝细胞,但诱导条件及诱导分化率尚无定论,选择合适的诱导剂及诱导剂浓度尤为重要。 目的：观察肝细胞生长因子和表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化的最适浓度。 方法：不同浓度肝细胞生长因子、表皮细胞生长因子联合诱导培养原代兔骨髓间充质干细胞,观察细胞形态学变化,并检测肝细胞表面标志物甲胎蛋白、白蛋白表达及肝细胞合成功能。 结果与结论：随诱导时间延长,可观察到多极性的肝细胞样细胞。7 d时甲胎蛋白表达阳性,14 d达最大值后即开始下降(P < 0.05),此后各浓度组间表达无差别(P > 0.05)。14 d时白蛋白表达阳性,随时间延长,阳性细胞数递增(P < 0.05),且细胞生长因子浓度越高,阳性细胞数越高(P < 0.05)。诱导早期(9~15 d) 白蛋白上清液中白蛋白水平与诱导剂浓度成正比(P < 0.05)。18 d或21 d达高峰后下降,此时各浓度组间含量无差别(P > 0.05)。结果显示高浓度的肝细胞生长因子及表皮细胞生长因子可提高骨髓间充质干细胞向肝细胞的分化率,诱导剂最适浓度为肝细胞生长因子60 μg/L、表皮细胞生长因子4.5 mg/L。     

体外诱导人骨髓间充质干细胞向肝样细胞分化：肝细胞生长因子和表皮细胞生长因子的作用*

背景：研究证实人骨髓间充质干细胞能够诱导分化为肝样细胞,但其具体生物学特性及分化机制尚不清楚,且诱导分化培养体系尚不成熟。 目的：探讨肝细胞生长因子和表皮细胞生长因子体外诱导人骨髓间充质干细胞向肝样细胞分化的可行性。 方法：取食管癌手术患者肋骨,采用密度梯度离心联合贴壁筛选法获取人骨髓间充质干细胞,流式细胞仪检测细胞表面分子的表达。取第3代人骨髓间充质干细胞,分为4组,肝细胞生长因子组加入20 μg/L肝细胞生长因子,表皮细胞生长因子组加入20 μg/L表皮细胞生长因子,联合组同时加入上述两种生长因子,空白对照组不加任何生长因子。倒置显微镜下观察细胞形态的变化,诱导7,14 d RT-PCR检测甲胎蛋白与白蛋白mRNA的表达。 结果与结论：人骨髓间质干细胞不表达造血细胞标志CD34,CD45,强表达β1-整合素CD29和基质受体CD44。肝细胞生长因子组、表皮细胞生长因子组、联合组诱导后,细胞形态由长梭形变为类圆形或多角形,诱导第7,14天甲胎蛋白、白蛋白 mRNA均呈阳性表达;空白对照组未见多角形细胞,甲胎蛋白、白蛋白 mRNA均呈阴性表达。证明肝细胞生长因子、表皮细胞生长因子以及二者联合均具有诱导人骨髓间充质干细胞向肝样细胞分化的能力,至于二者联合是否能增强其分化能力尚待进一步免疫细胞化学染色定量分析予以验证。     

CD40配体-受体载体介导的CpG ODN靶向脐血B淋巴细胞的特异传递及其免疫效应

背景：CpG寡核苷酸是一种高效低毒的免疫佐剂,在许多疾病的基因治疗中具有广阔的应用价值,但存在种属和细胞特异性,细胞摄取率低,易被酶降解,成为临床应用的障碍。 目的：拟验证CpG寡核苷酸通过CD40配体-受体载体系统对脐血B淋巴细胞特异靶向传递及其免疫效应的增强作用。 设计、时间及地点：观察对比实验,于2004-04/2007-10在广州暨南大学附属第一医院血液科、儿科完成。 材料：取健康新生儿的肝素抗凝新鲜脐血,事先征得父母知情同意并获医院伦理委员会审核批准。 方法：制备CD40配体-碳二亚胺-多聚左旋赖氨酸-CpG寡核苷酸交联复合物,与脐血单个核细胞共培养。分别于24,48,72,96 h应用流式细胞仪检测单个核细胞对FAM标记CpG寡核苷酸的摄取率及平均摄入强度;培养96 h后用直接免疫荧光标记法标记CD19,CD20,CD22单抗,以流式细胞仪检测阳性表达率;细胞悬液加入96孔板,3组分别加入CD40配体-碳二亚胺-多聚左旋赖氨酸-CpG寡核苷酸、单纯CpG寡核苷酸及ddH2O,培养92 h,以四甲基偶氮唑盐比色法测细胞增殖能力;以酶联免疫吸附法测以上3组细胞培养上清IgG水平。 主要观察指标：单个核细胞对CpG寡核苷酸的摄取率及平均摄入强度,淋巴细胞亚群的表达,细胞增殖能力,培养上清IgG水平。 结果：与单纯CpG寡核苷酸未交联组相比,单个核细胞对交联复合物摄取率更高（> 98%）,达摄取峰值时间更早。共培养后CD19+,CD22+,CD20+细胞表达升高,细胞增殖量及上清IgG水平均高于对照组（P < 0.05）。 结论：CD40配体-受体载体系统有助于CpG寡核苷酸特异高效的B细胞靶向投递,并增强其免疫效应。     

三种因子联合诱导大鼠骨髓间充质干细胞向肝细胞的分化**

背景：间充质干细胞的生物学特性及影响分化调控因子的研究认为,体外原代培养的间充质干细胞自然分化为肝细胞的比例较低,选择一种合适的诱导剂提高其分化为肝细胞的比例尤为必要。 目的：以肝细胞生长因子、表皮生长因子及成纤维生长因子体外联合,验证其诱导大鼠骨髓间充质干细胞向肝细胞分化的可行性。 设计、时间及地点：细胞学体外观察,于2007-08在潍坊医学院实验中心完成。 材料：Sprague-Dawley大鼠40只,由潍坊医学院实验动物中心提供。 方法：采用贴壁法分离培养大鼠骨髓间充质干细胞,取传至第3代细胞,设立2组：空白对照组加入含体积分数为10%胎牛血清的L-DMEM培养基;联合诱导组在此基础上,另加入10 µg/L成纤维生长因子、8 µg/L肝细胞生长因子、8 µg/L表皮生长因子。 主要观察指标：倒置显微镜观察诱导后细胞形态变化,免疫荧光染色观察甲胎蛋白及白蛋白的表达,PAS检测糖原的表达,靛青绿摄入情况,酶学检测谷丙转氨酶、谷草转氨酶、碱性磷酸酶的水平。 结果：联合诱导组细胞呈多角形、卵圆形或圆形细胞的特征性改变,空白对照组骨髓间充质干细胞仍保持梭形或纺锤形。联合诱导组培养14 d可见白蛋白和甲胎蛋白免疫反应阳性细胞;诱导7 d时偶见PAS阳性细胞与靛青绿阳性细胞,随诱导时间延长,阳性细胞逐渐增多;诱导14 d时开始检测到谷丙转氨酶、谷草转氨酶、碱性磷酸酶的合成,21 d达高峰,之后下降。上述各指标空白对照组细胞均呈阴性。 结论：成纤维生长因子、肝细胞生长因子与表皮生长因子体外联合应用,能够成功诱导大鼠骨髓间充质干细胞向肝样细胞的分化。     

小鼠肝细胞胰岛素与表皮生长因子信号转导磷蛋白质组的对比分析*

背景：表皮生长因子和胰岛素都主要通过PI3K通路和MAPK通路进行信号转导,但各自的生理功能不同。 目的：从细胞整体水平对比分析小鼠肝细胞内胰岛素与表皮生长因子信号转导磷蛋白质组的动力学行为差别，以此找出二者关键性的信号蛋白。 设计、时间及地点：随机分组设计的对比观察实验，于2005-07/2006-04在泸州医学院分子生物学实验室完成。 材料：实验用肝细胞来源于昆明种封闭群乳小鼠。 方法：选取细胞长势相当的肝细胞，采用放射性同位素(32P)对其进行标记，标记后按随机数字表法分为3组，空白对照组、表皮生长因子刺激组(给予10 μg/L 表皮生长因子)及胰岛素刺激组(给予100 nmol/L胰岛素)。 主要观察指标：表皮生长因子、胰岛素分别作用0，5，20，60，120 min，采用双向电泳技术分析比较表皮生长因子与胰岛素分别介导的磷酸化蛋白质组的动力学行为，即磷酸化水平。 结果：参与两者信号转导的磷蛋白质种类没有多大差异，大多数信号磷蛋白磷酸化水平的动力学行为也表现为一致性，但有4个蛋白点其磷酸化水平随时间变化趋势明显不同。 结论：胰岛素与表皮生长因子在同一细胞内参与信号转导的个别信号蛋白其磷酸化水平的动力学行为差别较大，估计这些蛋白就是导致表皮生长因子与胰岛素最终生物学活性的关键蛋白。     

重组人表皮生长因子纳米微球修复糖尿病大鼠溃疡创面

背景：在糖尿病合并有溃疡创面的动物实验中,各种生长因子单药或联合治疗都有报道。但是以纳米微球作为表皮生长因子载体进行糖尿病溃疡愈合的研究作者未检索到。 目的：比较重组人表皮生长因子(recombinant human epidermal growth factor,rhEGF)纳米微球和rhEGF原液2种不同剂型修复糖尿病大鼠溃疡效果的差异。 设计、时间及地点：随机对照动物实验,于2006-06/2007-02在卫生部及天津市激素与发育重点实验室完成。 材料：rhEGF原液由深圳华生元基因工程有限公司提供[rhEGF含量为1μg(500 U)],rhEGF纳米微球[rhEGF含量为1μg (500 U)]、空纳米微球由天津医科大学代谢病医院与天津大学材料学院纳米生物技术研究所联合制备,并完成鉴定。 方法：88只SD大鼠尾静脉注射链脲佐菌素制备糖尿病大鼠模型,用打孔器制备糖尿病大鼠溃疡模型。随机分为4组：rhEGF纳米微球组24只给予rhEGF纳米微球,rhEGF原液组24只给予rhEGF原液,空微球组24只给予空微球(与rhEGF纳米微球质量相同),PBS溶媒对照组16只给予PBS。各组均于造模后第1天开始用注射器将溶解好的药物均匀的喷洒在创面,待作用5 min后用无菌纱布包扎。第1天给液量为0.12 mL,以后根据创面愈合情况逐渐减少给液量。 主要观察指标：于3,7,14,21 d计算创面愈合率。同时取创面肉芽组织,免疫组织化学染色测增殖核细胞抗原阳性表达情况,透射电镜观察成纤维细胞超微结构改变。 结果：从第14天开始,rhEGF纳米微球组创面愈合率、增殖核细胞抗原阳性细胞率高于rhEGF原液组(P < 0.05,P < 0.01)。透射电镜下可见rhEGF纳米微球组成纤维细胞胞质内有大量的内质网,扩张成池状,腔内充满了合成的胶原蛋白。 结论：与rhEGF原液比较,rhEGF纳米微球促进溃疡愈合更快。     

EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture.

Epidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia. In this study, we investigated the effect of EGF on cells cultured from 16-day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo. EGF receptors were detected in untreated cultures by [125I]-EGF binding. Treatment of the cultures with EGF resulted in up to 50-fold increases in neuronal high-affinity dopamine uptake. Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase-immunoreactive cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons. By contrast, the high-affinity uptake for serotonin was increased only threefold over its controls. There was no significant effect on high-affinity gamma-aminobutyric acid (GABA) uptake. These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures. Immunocytochemistry for glial fibrillary acidic protein demonstrated an increase in astroglia with EGF treatment. Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.     

Transport of CSF antibodies to Galpha subunits across neural membranes requires binding to the target protein and protein kinase C activity

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.     

纳米载体在肿瘤靶向治疗中的应用

摘要：近年来纳米材料的研究正成为医药学者研究的焦点,纳米载体作为控释系统所具有的特殊性质,使其在药物输送方面具有高效,稳定,特异性强等优点,因其粒径呈纳米态,可透过细胞膜和穿透血脑屏障等,能更好的向靶器官,靶细胞,靶分子输送药物,起到更好的治疗效果。文章针对纳米载体的结构和特点,综述了纳米载体在肿瘤靶向治疗方面的国内外最新研究进展。     

Effects of an oncologist’s recommendation to exercise on self-reported exercise behavior in newly diagnosed breast cancer survivors: a single-blind,randomized controlled trial

Background: Increased attention has focused on exercise as a quality of life intervention for breast cancer survivors during and after adjuvant therapy.Purpose: Our objective was to examine the effects of an oncologist’s recommendation to exercise on self-reported exercise behavior in newly diagnosed breast cancer survivors attending their first adjuvant therapy consultation.Methods: Using a single-blind, 3-armed, randomized controlled trial, 450 breast cancer survivors were randomly assigned to receive an oncologist exercise recommendation only, an oncologist exercise recommendation plus referral to an exercise specialist, or usual care. The primary outcome was self-reported total exercise (in metabolic equivalent [MET] hours per week) at 5 weeks postconsultation.Results: The follow-up assessment rate was 73% (329 of 450). Intention-to-treat analysis based on participants with follow-up data indicated a significant difference in total exercise in favor of the recommendation-only group over the usual care group (mean difference, 3.4 MET hr per week; 95% confidence interval [CI], 0.7–6.1 MET hr per week; p = .011). There was no significant difference between the recommendation-plus-referral group and the usual care group (mean difference, 1.5 MET hr per week; 95% CI, −1.0 to 4.0 MET hr per week; p = .244). Ancillary on-treatment analyses showed that participants who recalled an exercise recommendation reported significantly more total exercise than participants who did not recall an exercise recommendation (mean difference, 4.1 MET hr per week; 95% CI, 1.9–6.4 MET hr per week; p < .001).Conclusions: Our findings suggest that an oncologist recommendation may increase exercise behavior in newly diagnosed breast cancer survivors, particularly if it is recalled 1 week after the recommendation. At the time of this study, Lee W. Jones was supported by an Alberta Heritage Foundation for Medical Research studentship. Kerry S. Courneya is supported by an Investigator Award from the Canadian Institutes of Health Research. Adrian S. Fairey was supported by an Izaak Walton Killiam Memorial Scholarship. This study was funded by a Research Team Grant from the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society (CCS) and the CCS/NCIC Sociobehavioral Cancer Research Network. We gratefully acknowledge the Northern Alberta Breast Cancer Program at the Cross Cancer Institute; Blair St. Martin, B.S., Neil Eves, M.S., and BenWilson for their assistance in assessment and data management. We also acknowledge doctoral dissertation committee members Ron Plontikoff, Ph.D., John Spence, Ph.D., and Cameron Wild, Ph.D. for feedback on an earlier version of this article. Papers were presented in part at the 38th annual meeting of the American Society of Clinical Oncology, Orlando, Florida (May 2002) and the 24th annual meeting of the Society of Behavioral Medicine, Salt Lake City, Utah (2003).     

Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures

The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15-30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5,6-3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37 degrees C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr. The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4-10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied. Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures.     

Role of LRP in TGFbeta2-mediated neuronal uptake of Abeta and effects on memory

There is increasing evidence that soluble amyloid-beta peptide (Abeta) uptake into neurons is an early event in the pathogenesis of Alzheimer's disease (AD). Identification of the early events leading to neuronal dysfunction is key to developing therapeutic strategies, but relative roles of receptors and factors modulating uptake are poorly understood. Studies have shown that transforming growth factor beta (TGFbeta), particularly TGFbeta2, can influence the targeting of Abeta to cells in vitro. TGFbeta2 can target Abeta to neurons in organotypic hippocampal slice cultures (OHSC). We examine a specific mechanism for TGFbeta2-mediated targeting of Abeta to neurons. The receptor-associated protein (RAP), a low-density lipoprotein receptor-related protein (LRP) antagonist, can attenuate the cellular targeting of Abeta both in vitro and in vivo and prevent Abeta/TGFbeta2-induced memory retention deficits. Using both in vitro and in vivo methods, we identify LRP as playing a role in TGFbeta2-mediated Abeta uptake, neurodegeneration, and spatial memory impairment.     

Visualization of growth factor receptor sites in rat forebrain

It is now known that various growth factors may also act in the central nervous system. Among them, it has recently been shown that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) may possess trophic effects in the mammalian brain. We report here on the respective autoradiographic distribution of [125I]EGF and [125I]IGF-I receptor binding sites in the rat brain, both during ontogeny and in adulthood. It appears that [125I]EGF sites are mostly found in the rat forebrain during brain development. On the other hand, [125I]IGF-I sites are more widely distributed both during ontogeny and in adulthood. These results reveal the plasticity of the expression of EGF and IGF-I receptor sites in the mammalian brain. This could be relevant for the respective role of these two growth factors in the development and maintenance of neuronal function.     

复合壳聚糖纳米微球聚乳酸-羟基乙酸/纳米羟基磷灰石缓释载体系统对蛋白的缓释作用

背景：聚乳酸-羟基乙酸支架材料具有良好的生物相容性、无毒、可以良好的塑性,并具有一定的强度和韧性。但其降解产物为酸性,会影响局部pH值变化,不利组织生长。 目的：制备能够良好缓释蛋白类药物的复合支架。 方法：以牛血清蛋白为模型药物,以离子凝胶法制备壳聚糖微球。将微球与纳米羟基磷灰石和聚乳酸-羟基乙酸按一定比例混合,以冰粒子为致孔剂,采用粒子沥虑-冷冻干燥复合工艺制备CMs/nHA/PLGA复合缓释支架。利用扫描电镜、透射电镜、压泵仪和力学性能测试仪检测复合支架的形态和性能,并考察其在体外对蛋白类药物释放的规律。 结果与结论：制备的壳聚糖纳米微球形态良好,呈规则球形或类球形,粒径分布在220~770 nm,以380~650 nm为多。微球对药物的载药量为39.2%,包封率为68.3%,两者均与牛血清蛋白的初始量相关,载药量随牛血清蛋白初始量的增加而增加,包封率则反之。复合支架呈白色多孔状,孔径为125~355 mm,孔与孔之间联通良好,孔隙率达83.4%,压缩强度为1.4~ 2.1 MPa,10周降解率为28.6%。PLGA/nHA支架对牛血清蛋白的2 d累积释放量为85%,而壳聚糖和CMs/nHA/PLGA复合支架对牛血清蛋白的9 d累积释放量分别是为48.9%和35.7%。提示制作的壳聚糖纳米微球和CMs/nHA/PLGA支架材料对牛血清蛋白有良好的缓释作用,复合支架材料形态好,强度和降解速率合适。