灰树花深层发菌丝体多糖的酶法提取及其抗肿瘤作用

对灰树花深层发酵菌丝体多糖的酶法提取工艺及其抗肿瘤作用进行了研究。结果表明：利用复合酶法处理并结合热水浸提,缩短了提取时间,且能显著提高多糖的提取率,多糖提取率达７．４％,效果优于其他浸提方法。动物体内实验证明,腹腔注射菌丝体多糖对小鼠移植性肿瘤Ｓ１８０有明显的抑制作用,抑制率达５７．６％￣６６．７％。     

苦石莲蛋白分离纯化及抗肿瘤作用探讨

运用硫酸铵分级沉淀、阳离子交换和凝胶层析等方法,分离、纯化得到了一种苦石莲蛋白,其分子量约为29.64kD.采用MTT法考察了其抗肿瘤作用.结果表明,苦石莲蛋白对小鼠黑色素瘤细胞(B16)的体外增殖有明显抑制作用,呈现出明显的量-效关系和时-效关系.作用48 h,该蛋白对B16细胞的半抑制浓度(IC50)约为136μg/mL.     

竹叶多糖对小鼠移植瘤的抑制作用

研究了竹叶多糖对小鼠移植瘤的抑制作用。实验结果表明,竹叶多糖对动物移植性Ｓ１８０肿瘤有抑制作用,抑制率可达５０％。５０％￣７０％醇沉组分抑瘤活性最大,且能显著提高小鼠腹腔巨噬细胞的吞噬能力。     

纤维堆囊菌产物生物学活性的研究

通过分析纤维堆囊菌（Sorangium cellulosum）子实体的性质，子实体提取物的体外抑菌活性，体外肿瘤细胞抑制活性和动物体内毒性及抑瘤活性，探索纤维堆囊菌子实体作为药用菌材料的可行性和药用前景。实验证明，纤维堆囊菌子实体代谢物组成稳定，子实体的提取物对于真菌有显著而广泛的抑制活性，并且对革兰氏阳性细菌也有一定的抑制作用，对于肿瘤细胞如肝癌Bel7402株系具有很强的体外杀伤活性，子实体提取物灌胃对小鼠无毒性反应，并能够明显抑制移植瘤S180的生长。实验结果说明纤维堆囊菌子实体具有作为药用资源的潜力。     

食药兼用真菌姬松茸胞外多糖的结构分析及其生物活性

通过高效液相色谱分析、红外光谱分析、13 C和1 H核磁共振分析对真菌姬松茸液态深层发酵产生的胞外多糖AbEXP1a的单糖组分和分子结构进行了研究,并进行了5、10和20mg/(kg.d)3种剂量的多糖对昆明种小鼠脾指数、胸腺指数和S-180实体瘤抑制作用的动物实验检测。结果表明,姬松茸胞外多糖AbEXP1a分子中葡萄糖和甘露糖的比例为3∶1,主链由3个连续的吡喃型葡萄糖分子通过β-(1→6)糖苷键连在一起,再通过α-(1→6)或β-(1→6)糖苷键与吡喃型甘露糖连接而构成。胞外多糖AbEXP1a对小鼠免疫器官重量有影响,3种剂量的多糖均对小鼠脾指数有显著性影响,但10和20mg/(kg.d)剂量组对小鼠胸腺指数有显著性影响。与对照组相比,3种剂量的胞外多糖均对小鼠S-180移植肿瘤有显著抑瘤效果,其中10mg/(kg.d)剂量组的抑瘤率达到64.04%。     

融合蛋白EGFP-PPA原核表达及标记肿瘤细胞研究

肿瘤细胞糖基化改变是非常普遍的现象,与恶性肿瘤的发生发展有着密切的关系。掌叶半夏凝集素(Pinellia pedatisecta agglutinin,PPA)是一种甘露糖特异性结合凝集素,与增强型绿色荧光蛋白(enhanced green fluorescent protein EGFP),通过基因工程技术形成融合基因,并在大肠杆菌中表达纯化了EGFP-PPA融合蛋白。实验结果表明,此融合蛋白可以识别实体肿瘤细胞表面的一部分标志,如Hep3B、Hu7、PLC、BEL-7404以及A549,而不能识别肝正常细胞L02,提示实体瘤细胞表面显露的甘露糖残基被此融合蛋白所识别,说明PPA识别具有甘露糖表型的实体瘤细胞表面的一部分,这些实验结果为进一步阐明甘露糖显露细胞在肿瘤发生发展中的作用提供了新的研究手段。     

Bax蛋白在小鼠睾丸发育过程中的表达

以17组出生后不同发育阶段的昆明种正常小鼠睾丸组织为材料,在其石蜡组织切片上采用免疫组化技术探讨Bax蛋白在小鼠睾丸生后发育全过程中的表达规律.结果发现：Bax蛋白在生后1d直至57d的小鼠睾丸组织中均有广泛性的表达,其表达涉及睾丸曲细精管中各类型生精细胞.上述结果表明：小鼠睾丸生后发育过程中各级生精细胞内均有Bax蛋白的存在,但生精细胞中Bax蛋白的表达可能并不意味着这些细胞最终走向凋亡.     

桑源缓衰液的研制与延缓衰老实验研究

桑源缓衰液以桑椹子、桑叶、桑蚕蛹蛋白水解液为主成分.制作以桑椹桑叶等浸提液与桑蚕蛹蛋白水解液相合,按均质、杀菌、冷却、装瓶等工艺路线进行.以桑源口服液予老龄BABL/C小鼠灌胃45～60天后测定与衰老有关的生化指标,实验结果表明:桑源液能使小鼠脑内B型单胺氧化酶(MAO—B)比活性显著降低(P<0.01);不影响肝内MAO—B的活性;使肝超氧化物歧化酶(SOD)比活性显著增强(P<0.05或0.01);全血谷胱苷肽过氧化物酶(GSH—Px)活性显著提高(P<0.01);心肌脂褐素含量显著升高(P<0.01).可见桑源液具有较明显的抗氧化延缓衰老的作用,亦即具有滋补肝肾,延年益寿之功效.     

增殖型溶瘤腺病毒SG511-pHSP70-IL24体外杀伤肝癌细胞的研究

研究溶瘤腺病毒SG511携带由热激蛋白70(hsp70)启动子调控IL-24构成的增殖型溶瘤腺病毒SG511-pHSP70-IL24对人肝癌细胞株的体外杀伤效果.实验采用ELISA法检测不同MOI值感染肝癌细胞SMMC-7721和Hep3B细胞3 d后IL-24的表达;MTT法检测SG511、Ad11-IL24以及SG511-pHSP70-IL24对肝癌细胞株SMMC-7721、Hep3B以及人正常成纤维细胞株MRC-5及BJ增殖的抑制作用;并通过结晶紫实验检测进一步证明联合用药对肿瘤细胞的杀伤情况.ELISA检测到SG511-pHSP70-IL24感染SMMC-7721和Hep3B细胞后,IL-24有明显表达;MTT实验结果表明SG511-pHSP70-IL24对肿瘤细胞的杀伤能力比SG511和Ad-IL24更强.同时结晶紫实验也证明了SG511-pHSP70-IL24对肿瘤细胞的杀伤能力比SG511和Ad-IL24更强.增殖型溶瘤腺病毒SG511-pHSP70-IL24相比腺病毒Ad11-IL24以及SG511,对肝癌细胞有较明显的生长抑制和凋亡诱导作用.     

T C-1-HPV16 L1细胞模型与动物模型的建立与评价

为了解决人乳头瘤病毒16型L1蛋白(HPV16L1)为主要靶点的疫苗缺乏肿瘤模型细胞来验证疫苗的免疫效果的问题,构建了一个细胞模型并可以利用此细胞在实验动物体内形成肿瘤,用于以HPV16L1为主要靶点疫苗的验证实验。利用这个模型细胞或肿瘤模型可以在体内外检测疫苗的免疫学性质和保护功效。首先,利用聚合酶链式反应(polymerase chain reaction,PCR)的方法获得目标基因HPV16L1的基因序列并连接到载体质粒中,将质粒转染到细胞TC-1后在杀稻瘟菌素抗性压力筛选下获得稳定表达HPV16L1的细胞株。对TC-1和TC-1-HPV16L1两种细胞的生长增殖和成瘤特性进行比较发现,外源基因的加入对细胞特性没有影响。通过体外杀伤实验证实细胞TC-1-HPV16L1能够被HPV16L1靶点疫苗免疫过的小鼠脾淋巴细胞杀伤。实验动物被疫苗免疫后,进行攻瘤实验发现疫苗只对TC-1-HPV16L1组具有保护作用。综上,在本研究中成功地构建了可以检测HPV16L1疫苗的抑瘤效果的模型细胞TC-1-HPV16L1,为HPV疫苗的研究提供了一个模型细胞。     

A New Type of Nanogel Carrier based on Mixed Pluronic Loaded with Low-Dose Antitumor Drugs

To design a new type of antitumor nanodrug carrier with good biocompatibility, a drug delivery system with a 2.19% drug-loading rate, measured by high-performance liquid chromatography(HPLC), was prepared by membrane hydration using a mixed polymer: Pluronic~? F-127, which binds folic acid(FA), Pluronic~? F-68 and triptolide(TPL)(FA-F-127/F-68-TPL). As a control, another drug delivery system based on a single polymer(FA-F-127-TPL) with a 1.90% drug-loading rate was prepared by substituting F-68 with F-127. The average particle sizes of FA-F-127/F-68-TPL and FA-F-127-TPL measured by a particle size analyzer were 30.7 nm and 31.6 nm, respectively. Their morphology was observed by atomic force microscopy(AFM). The results showed that FA-F-127-TPL self-assembled into nanomicelles, whereas FA-F-127/F-68-TPL self-assembled into nanogels. An MTT assay showed that a very low concentration of FA-F-127/F-68-TPL or FA-F-127-TPL could significantly inhibit the proliferation of multidrug-resistant(MDR) breast cancer cells(MCF-7/ADR cells) and induce cell death. The effects were significantly different from those of free TPL(P 0.01). Using the fluorescent probe Nile red(Nr) as the drug model, FA-F-127/F-68-Nr nanogels and FAF-127-Nr nanomicelles were prepared and then incubated with human hepatocarcinoma(HepG2) and MCF-7/ADR cells, and the fluorescence intensity in the cells was measured by a multifunctional microplate reader. The results indicated that both FA-F-127/F-68-Nr and FA-F-127-Nr had sustained release in the cells, but HepG2 and MCF-7/ADR cells exhibited significantly higher endocytosis of FA-F-127/F-68-Nr than that of FA-F-127-Nr(P 0.01). A nude mice transplanted tumor model was prepared to monitor FA-F-127/F-68-Nr in the tumor tissue and organs by whole-body fluorescent imaging. The results showed that FA-F-127/F-68-Nr targeted tumor tissues. The prepared nanogels had small particle size, were easy to swallow, exhibited slow release property,targeted tumor cells, and could improve the antitumor effects of TPL; hence, they are ideal carriers for low-dose antineoplastic drugs.     

Effects of Antihepatocarcinoma with Apatite Nanoparticles in vivo

The inhibition effect of hydroxyapatite （ HAP ） nanoparticles on hepatocarcinoma was investigated in vivo. The human hepatocarcinoma cell line Bel- 7402 was transplanted subcutaneously into nude mice. Hydroxyapatite nanoparticles suspension at a dose of 0. 2 mL was injected into the transplanted tumors every day for 2 weeks, and saline was used us control. The efficacy of hydroxyapatite nanoparticles on this carcinoma was surveyed and morphological changes of tissue and cells were observed by light microscopy and transmission electron microscopy （TEM）. Experimental results show that hydroxyapatite nanoparticles have a visible destructive effect on the structures of hepatocarcinoma cells and tissue. The inhibition rates of tumor growth were 77.21% and 51. 32% after intra-tumor injection of hydroxyapatite nanoparticles for 1 week and 2 weeks, respectively. Compared with the control group, hydroxyapatite nanoparticles can also prolong the survival time of the nude mice bearing this cancer significantly. This indicates that hydroxyapatite nanoparticles have the therapeutic potential on hepatoma in vivo.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.     

Magnetic biliary stent targeting to treat hepatoma combining with magnetic nanoparticles

To evaluate the effect of targeting to hepatoma treated by magnetic biliary stent combining with magnetic nanoparticle containing 5-fluorouracil （5-FU）, thirty-two nude mice modes with transplanted hepatoma were divided equally into four groups randomly. Experimental group received magnetic biliary stent and magnetic nanoparticles containing 5-FU. The tumor volume and pathomorphology of all groups was measured. The tumor control rate of the experimental group provided magnetic biliary stent wires and magnetic nanoparticles containing 5-FU is remarkably higher than three other control groups, showing significant curative effect. More apoptosis of tumor cells could be detected easily in experimental group. There are more apoptotic bodies and phagotrophic magnetic particle in apoptosis cells of experimental group under electron microscope. Magnetic biliary stent combining with magnetic nanoparticle containing 5-FU could inhibit the growth of hepatoma, and its curative effect is more remarkable than the traditional methods based on external magnetic fields.     

胡椒碱对人胃癌SGC-7901细胞抗肿瘤活性的体外实验研究

胡椒碱（Piperine）是一种从胡椒属植物中提取的生物碱,具有镇静、抗炎、抗肿瘤等多种药理活性.探讨胡椒碱对人胃癌SGC-7901细胞的增殖抑制和诱导凋亡的作用.采用MTT比色法测定其对SGC-7901细胞增殖抑制率;通过免疫荧光法、流式细胞术、Western blot法对其进行抗癌机制研究.MTT结果显示Piperine处理细胞72h的IC50值为37.41 μmol·L-1,且呈现时间剂量依赖性; Hoechst33258染色荧光显微镜观察发现Piperine能诱导SGC-7901细胞核形态学改变,部分细胞呈现典型的凋亡形态学特征;Annexin V-FITC/PI荧光双染结果亦证实Piperine可以诱导SGC-7901细胞发生凋亡;DAPI和DCFH-DA染色流式细胞术分析显示Piperine诱导SGC-7901细胞G2/M期阻滞、细胞活性氧产生增加; Western blot分析发现Bcl-2蛋白表达减少,Bax蛋白表达逐渐增加,呈现剂量依赖性,且Bax/Bcl-2比例增加.因此,Piperine具有抑制SGC-7901细胞增殖和诱导凋亡的抗肿瘤活性.     

凋亡蛋白对Bcl-2家族几个基因表达影响

凋亡蛋白（Apoptin）只诱导肿瘤细胞或转化的细胞凋亡,但其作用机制还不十分清楚．从鸡贫血病毒总DNA中克隆出凋亡蛋白基因,构建pcDNA3．1／CT—Apoptin—GFP—TOPO融合表达的载体．用该载体转染HHCC单层传代细胞24h后,观察到绿色荧光蛋白表达,同时观察到细胞形态呈凋亡特征变化．提取细胞总RNA,按SuperArray芯片操作方法检测到实验组Bcl-2家族中的BimL,Bcl-2,Bax和Bak基因上调表达．Bcl-2家族在细胞凋亡的过程中作用重要．凋亡蛋白诱导HHCC细胞凋亡过程中,促凋亡的BimL,Bax和Bak基因上调,抗凋亡的Bcl-2基因也上调．可能BimL与Bcl-2竞争性结合导致Bax或Bak释放,最终诱导细胞凋亡．揭示Bcl-2家族的基因参与凋亡蛋白诱导的HHCC细胞凋亡过程．     

纳秒脉冲电场诱导B16细胞凋亡的机制研究

纳秒脉冲电场具有诱导癌细胞凋亡、抑制肿瘤生长和诱导抗肿瘤免疫的作用。由于纳秒脉冲诱导治疗涉及的通路过多和常规检测特异性生物标志物技术的局限性,其凋亡机制尚未达成共识且暂时没有高效的研究方法。为了深入研究凋亡机制,该文利用蛋白芯片技术和富集分析技术研究了纳秒脉冲电场诱导小鼠黑色素瘤B16细胞的凋亡机制,并推导了纳秒脉冲诱导治疗的凋亡机制。该研究方法对于有此类靶点数目众多的机制研究具有独特的优势,可为设计未来的纳秒脉冲诱导治疗方案提供参考,优化最佳的脉冲参数或增强/抑制特定的分子以获得最佳治疗效果。     

凋亡因子（TRAIL）对癌细胞凋亡的机制研究

凋亡因子TRAIL（Tumor necrosis factor related apoptosis inducing ligand）能诱导肿瘤和转化细胞凋亡而不伤害正常细胞.TRAIL与细胞膜上死亡受体（death receptors,DR）结合,引发受体蛋白构象变化,经胞膜内受体死亡域（death domain,DD）募集接头蛋白FADD（Fas associateddeath domain）,再经过FADD的死亡效应域（death effector domain,DED）与凋亡蛋白解酶原-8/10（procaspase-8/10）的DED相互结合,形成DISC（death-inducing signaling complex）,引起凋亡蛋白解酶（caspase）级联反应,促使癌细胞凋亡.目前,可溶性重组人性化TRAIL（rhTRAIL）和抗DR4/5单克隆抗体已进入临床Ⅰ/Ⅱ期实验阶段.但绝大部分癌细胞对TRAIL有耐受或引起耐受,耐受机制主要是由于IAP（Inhibitor of Apoptosis Protein）,Bcl-2（B-cell lymphoma-2）及cFLIP（cellular FLICE/caspase 8-like inhibitory protein,胞质凋亡抑制蛋白）家族蛋白的过表达而产生.因此,为了克服TRAIL的耐受,已有多种小分子IAP和Bcl-2拮抗剂在研发并进行临床实验.至今尚无cFLIP拮抗剂的报导.     

IRES连接双基因在溶瘤腺病毒中的抗肝癌作用

单基因治疗癌症可能杀伤力还嫌不够，故使用两个基因在癌症治疗中具有重要价值。利用IRES将双基因连接起来在溶瘤腺病毒中表达，构成ZD55-IL-24-IRES-TRAIL。研究发现，该病毒在多种肝癌细胞中能有效地介导外源双基因表达，并在很大程度上杀死肿瘤细胞，而对正常细胞WI38无明显杀伤力。并且，该病毒可以有效地抑制体内肿瘤生长，有的肿瘤甚至完全消失。