一氧化氮介导的神经胶质瘤细胞对重离子辐射抗性及动物实验降能装置设计

目的：１、研究一氧化氮(NO)介导的神经胶质瘤细胞对重离子辐射抗性；２、研究小鼠大脑受重离子辐照后的损伤修复；３、为了更好的研究小鼠大脑受重离子布拉格（Bragg）峰区辐照后的损伤修复，设计并制造了旋转轮状降能装置。材料与方法：１、采用兰州重离子研究装置（HIRFL）加速的碳离子束辐照人类神经胶质瘤三种基因型细胞株：野生型神经胶质瘤细胞（A172），带绿色荧光蛋白基因的神经胶质瘤细胞（EA172）和带诱导型一氧化氮合酶基因（iNOS）的神经胶质瘤细胞（iA172），以及一种加了一氧化氮合酶抑制剂（L-NAME）的A172细胞（L-NAME-A172）。分别利用化学法、流式细胞术和MTT法检测三种神经胶质瘤细胞中NO、谷胱甘肽（GSH）的含量，以及它们的细胞周期变化和辐照后存活状况。２、利用不同剂量的碳离子束辐照昆明小鼠全脑，采用八臂迷宫检测随时间推移及训练次数的增加小鼠记忆损伤恢复情况。３、采用蒙特卡罗法和模拟退火法设计制造了一个有机玻璃材料的旋转降能装置。结果：1、NO和GSH在iA172细胞中的含量比A172和EA172中显著要高；辐照后24小时，观察到A172和EA172细胞发生周期阻滞即细胞阻滞于G2／M期，而这种现象没有在iA172细胞中观察到，并且iA172在接受同样辐射刺激后，细胞存活率显著高于其它三个细胞株。2、动物实验表明，在0－2Gy的碳离子辐照在短期内影响小鼠的记忆，经过一定时间后，这种影响得到恢复。3、物理实验显示，降能装置的实验数据与理论计算相符。结论：在低剂量的重离子坪区辐射条件下，一定浓度的NO可以使神经胶质瘤细胞产生辐射抗性。低剂量的重离子辐射致脑损伤可以得到很快的修复

超抗原SEA与抗黑色素瘤ScFv融合基因的构建及表达

采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肽的金黄色葡萄球菌肠毒素A基因进行融合 ,并将融合基因克隆于pET2 8 a表达载体上 ,转化大肠杆菌BL2 1(DE3)。用Ni NTA系统对表达产物进行分离、纯化。MTT法检测融合蛋白对黑色素瘤细胞的体外抑制率。结果表明 6His ScFv SEA融合蛋白可在E .coliBL2 1(DE3)中稳定表达 ,表达量占菌体蛋白的 30 % ,主要以包涵体的形式存在。融合蛋白可通过激活效应细胞对表达相关抗原的黑色素瘤细胞发挥抑制作用。

抗HER2/neu单链抗体增强TNF-α对卵巢癌细胞的抗肿瘤活性

为了探索抗HER2/neu单链抗体与TNF-α联合应用对表面过度表达HER2/neu卵巢癌细胞的生物效应,同时构建了抗HER2/neu单链抗体scFvC6.5的原核表达载体和人TNF-α的原核表达载体,将上述两种重组子分别转化入感受态宿主菌BL21(DE3),得到稳定表达.表达产物主要以包含体形式存在;包含体经过溶解、变性、复性和纯化,得到了分纯的产物.SDS-PAGE和Western-blot检测结果证实,蛋白表达正确.ELISA法验证了scFvC6.5和人TNF-α具有与卵巢癌细胞SKOV-3的结合活性.MTT细胞毒活性试验进一步表明,相对于单独使用TNF-α,联合应用上述两个重组蛋白细胞毒效应明显提高,SKOV-3细胞对TNF-α的敏感性增强.这将为抗HER2/neu抗体的联合抗肿瘤疗法提供一种新的途径,具有潜在的临床应用价值.图5参23

抗HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子.为了增加过度表达HER2/neu的肿瘤细胞对肿瘤坏死因子(TNF)的敏感性和提高HER2/neu抗体的肿瘤杀伤效应,将抗HER2/neu单链抗体C6.5与人肿瘤坏死因子hTNF-α融合,构建了scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为400μg/L菌液.经过亲和层析和柱复性,融合蛋白的纯度达95%以上.ELISA试验表明,scFvC6.5-hTNF-α能够特异结合HER2/neu阳性卵巢癌细胞SKOV-3和乳腺癌细胞MCF-7,而不结合HER2/neu阴性的黑色素瘤细胞A375.MTT试验表明,scFvC6.5-hTNF-α能够选择性地杀伤SKOV-3和MCF-7细胞,而不影响A375细胞的生长.这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值.

抗Her-2-scFv-SEC2融合免疫毒素的构建和功能研究(英文)

用基因工程方法,将金黄色葡萄球菌肠毒素 C2 与抗人表皮生长因子受体 HER-2 单链抗体 scFv-B1,以一连接短肽连接,构建融合免疫毒素 B-L-SEC2,并用改进的新型表达载体 pASK75-EX,在大肠杆菌 BL21(ED3)中表达. 以不溶性包涵体形式表达的目的蛋白经变性后以镍离子螯和层析纯化,并以透析法进行复性. 流式细胞术和 MTT 实验结果表明,纯化复性的融合免疫毒素 B-L-SEC2,在体外具有与 HER-2 过表达的靶细胞 SK-Br-3 特异性结合的活性,并对该细胞产生显著的特异性生长抑制作用.

鸭瘟病毒gB蛋白N端抗原域的高效表达及其免疫特性

在分析鸭瘟病毒(Duck plague virus,DPV)gB蛋白抗原性的基础上,设计1对引物克隆gB蛋白N端抗原性较好的抗原域编码基因,克隆到表达载体pET32a中,构建了原核表达质粒pET-gB1。将pET-gB1转化到感受态大肠杆菌(Escherichia coli)BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约42.4kD的目的蛋白以包涵体形式表达。Western blot分析发现,表达产物与抗鸭瘟的鼠阳性血清发生特异性反应。将包涵体溶解于8mol/L的尿素中,利用His·Bind试剂盒获得纯化的蛋白,将纯化的蛋白皮下注射免疫小鼠,间接ELISA法测得抗体的效价,MTT法检测免疫小鼠的T淋巴细胞增殖反应能力。结果说明,该融合蛋白能够诱导机体产生较强的体液免疫和细胞免疫。

大鼠脑胶质瘤干细胞分离及作为树突状细胞治疗抗原来源的研究

脑胶质瘤是原发性恶性脑肿瘤中最常见的一类肿瘤，其具有较差的预后性和高致死率。尽管包括手术、化疗及放疗在内的综合治疗水平在不断提高，但由于胶质瘤细胞的高度侵袭性导致治疗结果仍未得到明显改善，病人常常在治疗后很短时间内发生复发。近年来在肿瘤发生的领域提出了一个新的理论，即肿瘤干细胞理论。这个理论认为，肿瘤干细胞是肿瘤中具有自我更新、无限增殖及分化潜能的细胞，这部分细胞虽然只占少部分，但却是肿瘤发生、发展的关键。本研究主要探讨了利用化疗药物秋水仙素分离大鼠胶质瘤细胞中的干细胞样肿瘤细胞（Stem-like Cancer Cell, SLCC）的可行性，负载胶质瘤SLCC的树突状细胞疫苗在体外对肿瘤细胞的杀伤能力，以及对秋水仙素处理胶质瘤细胞的基因组不稳定性的初步探索。 C6大鼠胶质瘤细胞经不同剂量水平（0、0.5、1.0、2.0μg/ml）的秋水仙素处理后，大部分细胞发生凋亡，剩下存活的细胞经过免疫荧光染色发现其表达干细胞标志分子Nestin，提示可能是SLCC。流式和RT-PCR实验表明，经过秋水仙素处理后的存活的C6细胞表达干细胞标志分子nestin，sox2和bmi1，并且其数量随着秋水仙素浓度的增加而增加。 树突状疫苗是一种脑肿瘤的生物治疗方法。本实验通过分离大鼠的树突状细胞和淋巴细胞，体外培养并用经不同剂量组处理分离的SLCC全蛋白刺激一周后，使用MTT法检测激活的细胞毒T淋巴细胞（cytotoxic T lymphocyte，CTL）体外对肿瘤细胞的杀伤效果。结果表明，经药物分离的SLCC组诱导的CTL效果明显大于对照组，提示脑胶质瘤SLCC可以作为树突状疫苗免疫治疗的新靶点。 此外RT-PCR证明经秋水仙素处理后的胶质瘤细胞中mad2水平明显升高，表明化疗药物可能增加了肿瘤细胞中基因组不稳定性，从而导致SLCC的聚集。 综上，本实验证明了利用化疗药物秋水仙素可以分离大鼠胶质瘤细胞C6中的SLCC细胞，且该分离可能与肿瘤细胞基因组不稳定性提高有关。同时，利用负载SLCC全蛋白的树突状细胞激活的T淋巴细胞在体外具备比对照组更强的细胞杀伤能力。这些结果为以脑胶质瘤SLCC最为靶点的免疫治疗方法开发提供了基础依据。

聚乳酸接枝改性纳米生物玻璃/PLGA复合材料的制备、表面性质及生物活性

以丙交酯开环聚合原位接枝改性的纳米生物玻璃(PLLA-g-BG)与聚丙交酯-乙交酯(PLGA)复合材料为研究对象,采用TGA,ESEM和EDX分析其接枝率,粒子分散性和表面元素分布,通过将兔成骨细胞种植于材料膜表面进行体外培养,采用荧光染色法、NIH Image J图像分析软件、MTT法和流式细胞术等手段检测细胞在材料表面的平均黏附数量、扩展面积比、增殖能力和细胞周期的变化,综合评价新型改性纳米复合材料的生物相容性和生物活性.结果表明,聚乳酸表面接枝改性可明显改善纳米生物玻璃粒子的团聚;PLGA中掺入一定比例的改性PLLA-g-BG可明显促进兔成骨细胞的黏附、扩展与增殖;改性纳米生物玻璃的应用可提高生物可降解聚酯材料的生物相容性和生物活性.

交联型聚乙烯亚胺智能基因载体的制备及PEG化影响

使用胱胺双丙烯酰胺(CBA)对低分子量聚乙烯亚胺(PEI)进行交联反应制备智能降解型聚阳离子基因载体.通过与聚乙二醇(PEG)反应得到不同程度PEG化的聚阳离子载体.利用核磁、黏度测试、粒度仪、zeta电位仪和凝胶电泳对聚阳离子载体及其与DNA的复合物进行了表征.研究表明随着PEG含量的增加,聚阳离子载体/DNA复合物颗粒粒径变小、表面正电荷降低,PEG具有明显的屏蔽作用,但过多的PEG也使载体与DNA复合能力下降.通过MTT细胞毒性测试和荧光素酶质粒转染实验得出,含二硫键的交联型阳离子聚合物在测试范围内显示了非常低的细胞毒性,最佳转染效率是PEI25k的4倍,PEG化后其细胞毒性得到进一步改善,转染效率却明显降低.

Comparative study of paclitaxel physically encapsulated in and chemically conjugated with PEG-PLA

Paclitaxel-loaded poly(ethylene glycol)-b-poly(L-lactide (LA)) (PEG-PLA) micelles were prepared by two methods. One is physical encapsulation of paclitaxel in micelles composed of a PEG-PLA block copolymer and the other is based on a PEG-PLA-paclitaxel conjugate, abbreviated as "conjugate micelles" Their physicochemical characteristics, e.g. critical micelle concentration (CMC), morphology, and micelle size distribution were then evaluated by means of fluorescence spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). The results show that the CMC of PEG-PLA-paclitaxel and PEG-PLA are 6.31 x 10(4) and 1.78 x 10(-3) g L-1, respectively. Both micelles assume a spherical shape with comparable diameters and have unimodal size distribution. Moreover, in vitro drug delivery behavior was studied by high performance liquid chromatography (HPLC). The antitumor activity of the paclitaxel-loaded micelles against human liver cancer H7402 cells was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.

Preparation, Surface Properties and Biological Activity of the Composite of Poly (lactide-co-glycolide) and Bioglass Nanoparticles Surface Grafted with Poly(L-lactide)

SiO2-CaO-P2O5 gel bioglass (BG) nanoparticles with the diameter of 40 nm were synthesized by sol-gel approach. The surface of BG nanoparticles was grafted through the ring-open polymerization of the L-lactide to yield poly (L-lactide) (PLLA) grafted gel particle (PLLA-g-BG). The PLLA-g-BG was further blended with poly(lactide-co-glycolide) (PLGA) to prepare the nanocomposites of PLLA-g-BG/PLGA with the various blend ratios of two phases. PLLA-g-BG accounted 10%, 20% and 40% in the composite, respectively. TGA, ESEM and EDX were used to analyze the graft ratio of PLLA-g-BG, the dispersion of nano-particles and the surface elements of the composites respectively. The rabbit osteoblasts were seeded and cultured on the thin films of composites in vitro. The cell adhesion, spreading and growth of osteoblasts were analyzed with FITC staining, NIH Image J software and MTT assay. The change of cell cycle was monitored by flow cytometry (FCM). The results demonstrated that the Surface modification of BG with PLLA could significantly improve the dispersing of the particles in the matrix of PLGA. The nanocomposite with 20% PLLA-g-BG exhibited superior surface properties, including roughness and plenty of silicon, calcium and phosper, to enhance the adhesion, spreading and proliferation of osteoblasts.

Synthesis, self-assembly in water, and cytotoxicity of MPEG-block-PLLA/DX conjugates

Docetaxel (DX) is one of the most effective antineoplastic drugs. Its current clinical administration is limited because of its hydrophobicity and Serious side effects. A polymer/DX conjugate is designed and successfully prepared to solve these problems. It is monomethoxy-poly(ethylene glycol)-block-poly(L-lactide)/DX (MPEG-PLLA/DX) It was synthesized by reacting DX with carboxyl-terminated copolymer MPEG-PLLA, which was prepared by reacting succinic anhydride with hydroxyl-terminated copolymer monomethoxy-poly(ethylene glycol)-block-poly (L-lactide) (MPEG-PLLA). Its structure and molecular weight was confirmed by H-1 NMR and GPC. The MPEG-PLLA/DX micelles in aqueous solution were prepared Using a SO]vent displacement method and characterized by dynamic light scattering for size and size distribution, and by transmission electron microscopy for surface morphology. Its antitumor activity against HeLa cancer cells evaluated by MTT assay showed that it had a similar antitumor activity to Pure D at the same drug content.

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate

A paclitaxel/MPEG-PLA block copolymer conjugate was prepared in three steps: (1) hydroxyl-terminated diblock copolymer of monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA) was synthesized by ring-opening polymerization of L-lactide using MPEG as a maroinitiator, (2) it was converted to carboxyl-terminated MPEG-PLA by reacting with mono-i-butyl ester of diglycolic acid and subsequent deprotecting the t-butyl group with TFA; (3) the latter was reacted with paclitaxel in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Structures of the polymers synthesized were confirmed by H-1 NMR, and their molecular weights were determined by gel permeation chromatography. The antitumor activity of the conjugate against human liver cancer H7402 cells was evaluated by MTT method. The results showed that paclitaxel can be released from the conjugate without losing cytotoxicity.

T-Muurolol Sesquiterpenes from the Marine Streptomyces sp M491 and Revision of the Configuration of Previously Reported Amorphanes

Two new sesquiterpenes, 15-hydroxy-T-muurolol (3d) and 11,15-dihydroxy-T-muurolol (3e), along with the plant cadinenes T-muurolol (3f) and 3 alpha-hydroxy-T-muurolol (3g), were isolated from the marine-derived Streptomyces sp. M491. Their absolute configuration was established via NMR spectroscopy and X-ray crystallography of 3-oxo-T-muurolol (3a), which was reisolated from this strain. In addition, the absolute configuration of further sesquiterpenes previously reported from this strain was revised. These products were tested for their cytotoxicity against 37 human tumor cell lines using the MTT method. Only 3d was cytotoxic against a range of human tumor cell lines with a mean IC50 of 6.7 mu g/mL.