摘要
小球藻是一种人工培养的营养保健品,含有多种营养成分。近年来的研究表明,小球藻热水抽提物和干粉具有抗肿瘤及其它多种生物功能,其中起主要作用的是糖蛋白提取物。但目前的研究主要集中在糖蛋白粗提物及其在动物的体内试验,对小球藻糖蛋白的分离纯化和预防肿瘤作用报道甚少。同时传统的肿瘤药物筛选方法是针对疾病或者针对化合物,只适合于随机筛选。本论文对小球藻糖蛋白进行了分离纯化,并根据肿瘤预防的机理建立一个快速、灵敏、高通量而又经济的体外细胞筛选模型,对小球藻糖蛋白预防肿瘤的作用进行了筛选,不仅对小球藻预防肿瘤药物的开发利用具有一定的理论意义和应用价值,同时也为筛选利用其它天然药物提供了借鉴作用。
首先对小球藻糖蛋白进行了分离纯化。运用响应面分析确定了小球藻糖蛋白水提法的最佳提取条什:料水比1:21.9,在温度86.0℃下提取5.0 h;运用正交试验确定了Sevag法的最佳脱蛋白条件:样液与试剂比2:1,氯仿与正丁醇比4:1,脱蛋白次数3次,脱蛋白时间15 min;经Sephadex-75层析进一步纯化、透析、浓缩、冷冻干燥后,得到两种白色棉花状糖蛋白物质(CGPⅠ和CGPⅡ)。SephadexG-200凝胶柱层析证明CGPⅠ为均一组分,CGPⅡ组分不均一,高压凝胶过滤色谱证实CGPⅠ纯度为98.35%,CGPⅡ的纯度为78.84%。
对小球藻糖蛋白CGPⅠ的化学组成进行了研究。通过SDS-PAGE,显示CGPⅠ呈现一条带,相对分子质量为63,700:氨基酸自动分析仪测定CGPⅠ含有全部17种氨基酸,并富含酸性氨基酸。气相色谱显示CGPⅠ主要单糖组成为葡萄糖、半乳糖、鼠李糖。紫外可见光谱分析显示CGPⅠ有蛋白质和糖吸收峰,没有核酸吸收峰;红外光谱分析表明CGPⅠ的糖苷类型为吡喃型。β-消去反应说明CGPⅠ糖组分和蛋白质组分是通过0-糖肽键连接的。
测定了小球藻糖蛋白CGP I的体外抗氧化活性。通过清除羟自由基、超氧阴离子自由基、DPPH自由基、烷基自由基、还原力等试验,测定小球藻糖蛋白的抗氧化活性。结果显示,不同浓度CGPⅠ有一定的还原能力,且具有清除羟自由基、超氧阴离子自由基、DPPH自由基、烷基自由基的能力,其还原能力和清除自由基的能力与剂量有一定的效应关系。CGPⅠ消除羟自由基的能力高于Vc,其还原能力、清除超氧阴离子自由基、DPPH自由基、烷基自由基的能力均低于Vc。
建立了体外筛选化学预防剂的转基因细胞模型筛选小球藻糖蛋白的肿瘤预防作用。应用重组PCR技术,从pRL-TK上扩增重组胸腺嘧啶核苷激酶(TK)启动子,并在其基本启动子上游创建特异性酶切位点SacⅠ。第一轮PCR获得500 bp和150 bp两个片段,第二轮PCR获得635bp的目的片段,克隆入pMD18-T载体中。而后扩增TK基本启动子克隆入pEGFP中构建载体pTK-GFP,并在其上游插入抗氧化反应元件(ARE)片段,构建pARE-TK-GFP。最后从以上两载体中扩增TK和ARE-TK目的片段克隆入载体pEGFP-N_1中,构建最终表达载体pTK-GFP/neo和pARE-TK-GFF/neo。两表达载体分别用脂质体法转染HepG2细胞后,用800μg/ml G418筛选出细胞克隆HepG2-TK-GFP和HepG2-ARE-TK-GFP,在荧光显微镜下可见明显的绿色荧光。将阳性克隆细胞扩大培养,加入糖蛋白溶液,使其终浓度分别为0.32、1.6、8、40、200(μg/ml),同时用PDTC和香菇多糖作阳性对照。结果显示,糖蛋白在200μg/ml时诱导效果最好,且糖蛋白浓度与GFP相对荧光强度存在剂量效应关系,而在对照克隆细胞中未发现与受试物有剂量效应关系。
本研究从小球藻中分离得到一种糖蛋白,该糖蛋白具有抗氧化活性;根据肿瘤预防的抗氧化机理构建了一个带有抗氧化反应元件(ARE)和绿色荧光蛋白基因(GFP)载体的转基因细胞模型;小球藻糖蛋白能诱导ARE的顺式表达作用,促进GFP的荧光强度,并有剂量的依赖关系,说明小球藻糖蛋白具有预防肿瘤的作用。
Cklorella pyrenoidosa is man-cultured health supplement contained kinds of compounds.The hot water extracts(CVE) and power of Chlorella pyrenoidosa have been reported to possess various pharmacological effects including antitumor activities and potent biological response modifiers that modulate immune responses against bacteria and viruses,and it mainly is the glycoprotein extracts that play an important role.The study on Chlorella pyrenoidosa focused on the extracts and its functions in vivo,but the report on the and tumor-prevention is rare.Traditional methods of screening the drug for antienoplastic in the light of dieases and compounds suited only to random screening.In order to use this resource,the isolation,purifition of glycoprotein from Chlorella pyrenoidosa were carried and a rapid,sensitive,high throughout and cheap screening method in vitro was constructed to study the tumor-prevention of glycoprotein from Chlorella pyrenoidosa.It will be of scientific significance in researching and developing the new synthesized and natural tumor-prevention.
The glycoprotein from Chlorella pyrenoidosa were isolated and purifed firstly. The technology and condition of glycoprotein extraction from Chlorella pyrenoidosa were studied.Water extraction was used to extract glycoprotein,the optimum extraction conditions of glycoprotein were chosen by considering the effect of each factor and subsequently doing response surface experiment.The better condition of glycoprotein extraction is:the ratio of raw material to water is 1:21.9,extracting temperature is 86.0℃and extracting time is 5.0 h;Sevag method was used to remove protein.The optimum conditions was chosen by considering the effect of each factor and subsequently doing orthogonal experiment,and the better condition of removing protein is:the ratio of sample liquid to Sevag liquid is 2,the ratio of chloroform to 1-butanol is 4,removing protein times is 3,the time of removing protein is 15 min.
The crude glycoprotein was further purified by Sephadex G75 gel,dialysis, concentration.After vacuum freeze-drying,two white flocculent purified glycolproteins were obtained.Elution patterns on sephadexG-200 column chromatography proven CGP I was homogeneous and CGPⅡwas not homogeneous.The results of HPLC indicated the purity of CGPⅠand CGPⅡwere 98.35%and 78.84% respectively.
Chemical composition of CGPⅠwas studied.The results of SDS-PAGE of CGPⅠshowed a single spot and the molelular weight is 63,700;CGPⅠcontains 17 amino acids and two acidic amino acids(Asp and Glu) is higher using amino acid analyzer;Lack of absorption at 260.00nm by UV scaning indicated that CGPⅠcontained no nucleic acid.It was proved that the main monosacchrides were glucose, galactose and xylose by gas chromatography.The type of the glucosidic bonds was pyranose by IR and O-glycosidic linkage was found byβ-elimination reaction.
The antioxidant activities in vitro of glycoprotein from Chlorella pyrenoidosa were researched through determinations of reducing power and scavenging hydroxyl free radical,superoxide free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical,the results showed that the different concentrations CGPⅠhad stronger reducing power and scavenging effect on superoxide free radical,hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical in a concentration of dose-dependant manner.The scavenging capacity of CGPⅠin the hydroxyl free radical was stronger than Vc,but the scavenging capacity to superoxide free radical, hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical was lower than Vc.
A transgenic cell model was established to screen preliminarily in vitro the tumor prevention of glycoprotein from Chlorella pyrenoidosa.The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the SacⅠenzyme site was added.Then the 500 bp and 150 bp segments obtained from the first round of the recombinant PCR and the 635 bp segment from the second round PCR were both identified then cloned into pMD18-T vector.The TK promoter was amplified from it and cloned into pEGFP to construct pTK-GFP,the ARE enhancer was inserted into the upstream of TK to construct the vector pARE-TK-GFP.At last the TK and ARE-TK segments were amplified and cloned into pEGFP-N1 so the eukaryotic expression vectors pTK-GFP/Neo and pARE-TK-GFP/Neo were constructed.The two vectors were transfected into HepG2 cells and clones resistant to 800μg/ml G418 were isolated and named as HepG2-TK-GFP and HepG2-ARE-TK- GFP of which the green fluorescence was obvious under the fluorescent microscope.The different concentration(0.32,1.6,8,40,200μg/ml)of glycoprotein was added into the transgenic cells and the PDTC and lentinan were as controls.The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dosedependency with the different concentrations of glycoprotein in a certain range while the controls have not.
In the study,a glycoprotein from Chlorella pyrenoidosa was obtained,and it anti-oxidation activities was proved;According to the the antioxidant mechanism of tumor-prevention,A green fluorescent protein(GFP) transgenic cell model under the transcriptional control of TK promoter and ARE was constructed to evaluate the tumor-preventive activity of glycoprotein from Chlorella pyrenoidosa.The glycoprotein could induce the cis-expression of ARE and promote the fluorescence intensity of GFP,and the induced level of GFP have dose-dependency with the concentrations of glycoprotein.So,the tumor-prevention of the glycoprotein from Chlorella pyrenoidosa was made sure preliminarily.
引文
1 Beeley J G.Glycoprotein and proteoglycan techniques,as laboratory techniques in biochemistry and molecular biology,Vol 16,Elsevier.Amsterdam:New York:Osford,1985
2 吴东儒.糖类的生物化学[M].北京:高等教育出版社,1987,687-755
3 仲娜,郝林华,于小如.糖蛋白药物的研究进展[J].中国新药杂志,2005,14(12):1400-1403
4 黄琳娟,田庚元,齐春会,等.枸杞子糖缀合物LbGp4糖链的结构及其免疫活性的研究[J].高等学校化学学报,2001,22(3):407-411
5 Kusano S,Abe H,Tamura H.Isolation of antidiabetic components from white-skinned sweet potato(ipomoea batatas L.)[J].Biosci Biotechnol Biochem,2001,65(1):109-114
6 高荫榆,霍光华,何小立,等.乌桕叶水溶性多糖蛋白复合体及其组成的研究[J].食品科学,2003,24(9):23-26
7 张以芳,刘旭川.螺旋藻及其多糖、多糖蛋白提取物抗疲劳作用试验研究[J].海洋通报.1999,18(3):90-92
8 杨真威,姜瑞芝,陈英红.耙齿茵糖蛋白的提取分离、理化性质及抗炎活性[J].天然产物研究与开发,2005,17(3):280-282
9 杜良成,王世林,李英,等.珠子参抗真菌糖蛋白的研究[J].云南植物研究,1992,14(4):430-436
10 阮继红,郭宝江,苏亮衡.螺旋藻(Spirulina Platensis)多糖蛋白对辐射诱发小鼠PCE微核率及白细胞数的影响[J].辐射研究与辐射工艺学报,1990,8(4):210-213
11 王平.糖蛋白工程及艾滋病疫苗研究新进展[J].现代实用医学杂志,2000,13(5):844-845
12 牛纪晓,张天一,林琳,等.96KD糖蛋白诱导肿瘤免疫的试验研究[J].实用肿瘤学杂志,2003,18(2):112-114
13 胡开辉,汪世华.小球藻的研究开发进展[J].武汉工业学院学报,2005,24(3):27-30
14 汪炬,蒲含林,洪岸,等.蛋白核小球藻提取物的抑瘤作用及对免疫功能的影响[J].营养学报,2004,26(2):136-143
15 王业勤,李勤生.小球藻应用研究动态[J].微生物学通报,1985,12(6):275-277
16 陈颖,李文彬,孙勇如.小球藻生物技术研究应用现状及展望[J].生物工程进展,1998,18(6):12-16
17 宋汉晓.抗癌药的研究 化学预防时方向[J].中国医药指南,2006,3:54-57
18 郭婧,李强.肝细胞癌的病因及化学预防[J].世界华人消化杂志,2005,13:2021-2025
19 孙哲,吕秋军,温利清,等.基于报告基因的雌激素受体α亚型配体筛选模型的建立和应用[J].药学学报,2000,35(10):747-751
20 Kawagushi K,Microalgae production system in Asia,in "Algae Biomass Production and Use",1980
21 李师翁、李虎乾.植物单细胞蛋白资源——小球藻开发利用研究的现状[J].生物技术,1997,7(3):45-48
22 Yamaguchi K.Recent advance in microalgal bioscience in Japan[J].Journal of Applied Phycology,1997(8):487-490
23 陈颖,李文彬,孙勇如.小球藻生物技术研究应用现状及展望[J].生物工程进展,1998,18(6):12-16
24 Hasegawa T,ItoK,Ueno S,et al.Oral administration of hot water Chlorella vulgaris reduces IgE production against milk casein in mice Int[J].Immunopharmacology,1999,21(5):311-323
25 Hasegaw T,Kimura Y,Hiromatsu K,et al.Effect of hot water extract of Chlorella vulgaris on cytokine expression patterns in mice with murine acquired immuno- deficiency syndrome after infection with Listeria monocytogenes[J].Immunopharmacology,1997,35(3):273-282
26 刘海琴,韩士群.小球藻提取物的生物活性研究[J].海洋科学,2005,29(9):23-26
27 Dantas D C,Queiroz ML.Effects of Chlorella vulgaris on bone marrow progenitor cells of mice infected with Listeria monocytogenes[J].Immunopharmacology,1999,21(8):499-508
28 Dantas D C,Kaneno R,Queiroz,et al.The effects of Chlorella vulgaris in the protection of mice infected with Listeria monocytogenes[J].Immunopharmacology lmmunotoxicology,1999,21(3):609-619
29 Negishi T,Rai H,Hayatsu H,et al.Antigenotoxic activity of natural chlorophylls[J]. Mutation Reseach,1997,376:97-100
30 Konishi F,Tanaka K,Himeno K,et al.Antitumor effect induced by a hot water extract of Chlorella vulgaris(CE):Resistance to Meth-A tumor growth mediated by CE-induced polymorphonuclear liukocytes[J].Cancer Immunol Immunother.1985,19(2):73-78
31 Tanaka K,Tomita Y,Tsuruta M et al.Oral administration of Chlorella vulgaris augments concomitant antitumor immunity[J].Immunopharmacology Immunotoxicology,1990,129(2):277-291
32 Tanaka K,Yamada A,Noda K,et al.novel glycoprotein obtained from Cbolrella vulgaris strain CK22 shows antimetastatic immunopotentialtion[J].Cancer Immunol Immunother,1998(45):313-320
33 Sano T,Tanaka Y.Effect of dried powdered Chlorella vulgaris on experimental atherosclerosis and alimentary hypercholesterolemia in cholesterolfed rabbits[J].Artery,1987,14(2):76-81
34 Merchant R E,Andre C A A.review of recent clinical trials of the nutuitional supplement Chlorella vulgaris in the treatment of fibromyalgia,hypertension and ulcerative colitis[J].Altern Ther Health Med,2001,7(3):79-91
35 Honjoh K,Yoshimoto M,Job T,et al.Isolation and characterization of hardening-induced proteins in Chlorella vulgaris C-27:identification of late embryogenesis abundant proteins[J].Plant Cell Physiol,1995,36(8):1421-1430
36 Opekarova M,Caspari T,Tanner W.The HUP1guen product of Chlorella Kessleri:H+/glucose symport studied in vitro[J].Biochemica et Biophysica Acta,1994,1194(1):149-154
37 Tanaka K,Koga T,konishi F,Nakamura M,et al.Augmentation of host defense by a unicellular green alga,Chlorella vulgaris,to.Escherichia coli infection[J].Infection and Immunity,1986,53(2):267-271
38 中国人民解放军总医院营养科.小球藻E-25辅助肿瘤放射治疗临床试验总结报告[J].引进国外医技术与设备,1999,5(3):62-64
39 Singh S,Tiku A B,Kesavan P C.Post-exposure radioprotection by Chlorella vulgaris (E-25)in mice[J].Indian J Exp Boil,1995,33(8):612
40 Tanaka K,Yamada A,Noda K,t al.Oral administration of a unicellular green algae,Chlorella vulgaris,prevents stress-induced ulcer[J].Planta Medica,1997,63(5):465-466
41 Hu Q H,Pan B S,Xu J,et al.Effects of supercritical carbon dioxide extraction conditions on yields and antioxidant activity of Chlorella pyrenoidose extracts[J].Journal of Food Engineering.2007,80:997-1001
42 Vijayavel C,Anbuselvaw,M P.Balasubramanian.Antioxidant effect of the marine algae Chlorella vulgaris against naphthalene-induced oxidative stress in the albino rats[J].Molecular and Cellular Biology.2007,303:39-44
43 Morita K,Matsueda T,Lida T,et al.Chlorella accelerates dioxin excretion in rats[J].Nutrition,1999,129(9):1731-1736
44 Hasegawa T,Noda K,Kumanmoto S,et al.Chlorella vulgaris culture supernatant(CVS)reduces phychological stress-induced apoptosis in thymocytes of mice[J].Immunopharmacology,2000,22(11):877-885
45 Merchant R E,Carmack C A,Wise C M.Nutritional supplementation with Chlorella pyrenoidosa for with fibromyalgia syndrome:a pilot study[J].Phytotherapy Research,2000,14(3):167-173
46 林芃,刘艳,杨海波.对小球藻蛋白质的提取及分离的研究.大连大学学报,2002,23(4):70-73
47 陈晓清,郑怡,林雄平.二种微藻多糖与蛋白质提取物的抗菌活性.福建师范大学学报(自然科学版),2005,21(2):76-79
48 郑怡,余萍,刘艳如.蛋白核小球藻凝集素的分离纯化及部分性质研究.水生生物学报,2003,27(1):36-40
49 Sheng J C,Yu F,Xin Z H,et al.Preparation,identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa[J].Food Chemistry,2007,105:533-539
50 Suarez E R,Jaroslav A.Kralovec,Miguel D.Noseda,et al.Isolation,characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa[J].Carbohydrate Research,2005,340:1489-1498
51 江红霞,郑怡,林雄平.蛋白核小球藻脂溶性化合物的抑菌活性及成分分析[J].植物资源与环境学报,2003,12(1):1-5
52 桂林,李琳,胡松青,等.蛋白核小球藻中叶黄素提取工艺的研究[J].食品研究与开发,2005,26(5):71-74
53 Hua-Bin Li,Feng Chen,Tian-You Zhang,et al.Preparative isolation and purification of lutein from the microalga Chlorella vulgaris by high-speed counter-current chromatography[J].Journal of Chromatography A,2001:151-155
54 李亚娜,林永成,佘志刚,等.小球藻糖蛋白的分离、纯化和结构分析[J].天然产物研究与开发,2004,16(6):503-506
55 Suarez E R,Kralovec J A,Noseda M D,Isolation,characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa[J].Carbohydrate Research,2005,340:1489-1498
56 Hasegawa T,Matsuguchi T,Noda K.Toll-like receptor at least partly involved in the antitumor activity of glycoprotein from Chlorella vulgaris[J].International Immunopharmacology,2002,2:579-589
57 Tanaka K,Yamada A,Noda K,et al.A novel glycoprotein obtained from Cholrella vulgaris strain CK22 shows antimetastatic immunopotentialtion[J].Cancer Immunol Immunother,1998,45:313-320
58 Tetsuya M,Kiyoshi N,Kuniaki T,et al.Toll-like recepter 2 is at least partly involved in the antitumor activity of glycoprotein from Chlorella vulgaris[J].International Immunopharmacology,2002,2:579-589
59 Konishi F,Mitsuyama M,Okuda M,et al.Protective effect of an acidic glycoprotein obtained from culture of Chlorells vulgaris against myelosuppression by 5-fluorouracil[J].Cancer Immunol Immunother,1996,42(5):268-274
60 梁世乐.抗癌药物的筛选及其作用机制的初步研究[D]:[硕士学位论文].天津:天津大学,2001
61 景永奎.抗肿瘤药物筛选方法的进展[J].国外医学药学分册,1991,18(1):1-4
62 谢天培,顾心彬,朱洪莉,等.抗肿瘤药物高通量体内筛选模型——中空纤维测定法[J].中国药学杂志,2003,40(6):469-470
63 姚新生.海洋抗肿瘤药物研究展望[J].第二军医大学学报,2002,23(3):233-235
64 侯毅鞠,袁忠海,李艳,等.冻存复苏条件对髓性白血病细胞株生物学特性的影响[J].中国组织工程研究与临床康复,2007,11(24):4714-4717
65 李雯.抗肿瘤药物敏感试验的研究进展[J].中国医院用药评价与分析,2004,4(1):61-62
66 张擎,邓宁,贺国安,等.耳壳藻内酯分子合成及其抗肿瘤活性的研究[J].热带海洋学报,2004,23(3):1-5
67 Mosmann T.Rapid colorimetric assay for celluar growth and surival:application to proliferation and cytotoxicty assays.Journal of Immunological Methods,1983,65:55
68 Carmicbaiel J,Degraff W G,Gandar A F,et al.Evaluation of a tetrazolium based semiautomated colori metric assay:assessmen of chemosemitivity testing.Cancer Reseach,1987,47(4):936-942
69 Sargent J M,Taylor C G.Appraisal of the MTT assay as a rapid test of Chemosensitivity in acute myeloid leukaemia.Br J Cancer,1989,60(2):206-210
70 鲁迎年,王秋波,吴虹,等.MTT比色法在测定化疗药物敏感试验中的条件选择[J].青岛医学院学报,1997,33(3):224-226
71 肖大江,朱国臣,王晓岚,等.田基黄、冬凌草甲素对人鼻咽癌细胞系CNE细胞毒作用的研究[J].齐齐哈尔医学院学报,2005,26(12):1396-1398
72 Lei S,Chien P Y,Sheikh S,et al.Enhanced therapeutic efficacy of a novel liposome-based formulation of SN-38 against human tumor models in SCID mice Ⅲ.Anticancer Drugs,2004,15(8):773-778
73 金静君,魏一生,何萍,等.SRB法急性白血病体外药敏试验适用性研究[J].福建医药杂志,1999,21(4):84-85
74 王兴旺,胥彬.抗癌药物筛选模型与筛选方法的研究[J].中国医药工业杂志,1997,28(1):39-43
75 Hawser S P,Jessup C,Vitullo J.et al.Utility of 2,3-bis(2-methoxy-4-nilro-5-sulfophenyl-5-[(phenyl-amino)carbonyl]-2H-tetrazolium Hydroxide(XTT) and minimum effective concentration assays in the determination of antifungal susceptibility of aspergillus fumigatus to the lipopeptide class of compounds[J].J Clinical Microbio,2001,39(7):2738-2746
76 吴楠.XTT比色法在细胞活性检测中的应用[J].国外医学临床生物化学与检验学分册,1998,19(5):204-206
77 王中平,鲍朗,张会东,等.问号钩端螺旋体毒力基因mviN的表达及细胞毒性作用研究[J].中国人畜共患病学报,2007,23(1):23-26
78 Gillies R G,Didier N,Fenton M.Determination of cell number in monolayer culter[J].Analytical Biochemistry,1989,159:109-113
79 陈军,战洪生.结晶紫染色测定法在抗肿瘤药物筛选中的作用[J].癌症,1997,16(3):231-232
80 Weisanthel L M,Mersden J A,Dm D A,et al.Comparison of dye exclusion assays with a clonogenic in the determination of drum induced cytotoxicity.Canccr Research,1983, 43(1):258-264
81 Rolf L and Nygren P.Chemotherapeutic drug sensitivity testing of human leukemia cells in vitra using a semiautomated fluorometri assay[J].Leukemia,1990,4(8):567-571
82 陈军,战洪生.快速荧光素测定法在抗肿瘤药物筛选中的应用[J].中国肿瘤临床,1996,23(7):495-497
83 刘文虎,贺殿,胥桂贤.抗癌新靶点及其靶向药物的研究现状[J].药学进展,2007,31(12):535-542
84 Kim N W,Piatyszek M A,Prowse K R,et al.Specific association of human telomerase activity with immortal cells in cancer[J].Science,1994,266:2011
85 Hiyamak H.Telomerase activity in small cell and non small celllung cancer[J].J Natl Cancer Inst,1995,87(12):895
86 李耀武.作用于微管的抗肿瘤药物研究进展[J].国外医学药学分册,2005,32(1):1-5
87 李越中,胡玮,周璐.具有促微管聚合活性的抗肿瘤天然化合物[J].药学学报,2001,36(2):155-160
88 Kwak M Y,Kensler T W,Casero R A.Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2:effect of thepolyamine metabolite acrolein[J].Biochemical and Biophysical Research Communications,2003,305:662-670
89 Kwak M Y,Patricia A E,Patrick M D,et al.Role of phase 2 enzyme induction in chemoprotection by dithiolethiones[]].Mutation Reseach,2001,481:305-315
90 谢玲,黄海潇,张浩,等.基于抗氧化反应元件的药物筛选模型的建立[J].中国药理学通报,2006,22(12):1525-1528
91 刘琼,吕秋军,温利清,等.基于报告基因的雌激素受体α亚型配体筛选模型的建立和应用[J].药学学报,2000,35(10):747
92 刘学铭,梁世中.小球藻的保健和药理作用[J].中草药,1999,5:383-386
93 Ansley D.Cancer Institte turns to cel line streening[J].The Scientist,2001,4:3-9
94 陈晓光.抗肿瘤药物研究策略与方向[J].首都医药,2003,22:11-15
95 李宜明,沈业寿,王清,等.抗肿瘤药物的筛选方法[J].安徽大学学报(自然科学版),2005,29(1):90-96
1 王利军,吕杰.南瓜中糖蛋白提取工艺的研究[J].粮油食品科技,2006,14(1):46-47
2 Nisson M,Saulnier L,Andersson R,et al.Water unextractable polysaccharides from three milling fractions of rye grain[J].Carbohydrate polymers,1996,30:229-237
3 Maes C,Delcour J A.Alkaline hydrogen peroxide extraction of wheat bran non-starch polysaccharides[J].J cereal science,2001,34:29-35
4 雷晓凌,范秀萍,赵树进,等.章鱼干糖蛋白的提取与分级分离的初步研究[J].陕西科技大学学报:自然科学版,2006,24(4):45-49
5 赵梅,丁霄霖.甘薯糖蛋白提取工艺研究[J].食品研究与开发,2005,26(1):77-79
6 郭辉,李善玲,张红旭,等.红毛五加粗多糖脱蛋白工艺比较[J].现代中药研究与实践,2004,18(6):53-55
7 马丽,覃小林,刘雄民,等.不同的脱蛋白方法用于螺旋藻多糖提取工艺的研究[J].食品科学,2004,25(6):116-119
8 陈倩超.鲍鱼多糖的提取及抗肿瘤试验[J].中国现代应用药学,1998,15(1):8-11
9 李布青,张慧铃,等.中低档绿茶中茶多糖的提取及降血糖作用[J].茶叶科学,1996,16(1):67-72
10 Sunil S M,Kiran K M,Gadag J R.Purification and properties of hyaluronidase from Palamneus gravimanus(Indian black scorpion) venom[J].Toxicon,2006,47(2):188-195
11 韩冰,朱蓓薇.阴离子交换柱(DEAE-52)层析纯化海参自溶酶[J].大连轻工业学院学报.2004,23(2):118-121
12 张玉香,尹卓容.甘露糖蛋白的提纯及分子量测定[J].酿酒科技,2005,4:72-74
13 Mohapatra B R,Harris N,Nordin R,et al.Purification and characterization of a novel caffeine oxidase from Alcaligenes species[J].Journal of Biotechnology,2006,125(3):319-327
14 任金山,吴梧桐,李颖,等.花粉多糖的分离纯化及其组成单糖分析[J].中国药科大学学报,1990,21(3):173-175
15 陈怡.多糖的研究概况[J].世界科学技术-中药现代化,2000,2(6):52-55
16 方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报,1984,19(10):46-49
17 王明兹,施巧琴,陈必链,等.紫球藻生长周期可见光吸收光谱与生化变化[J].食品与发酵工业,29(4):23-26
18 吴叶玲.测定叶绿素a方法的改进[J].福建分析测试,2006,15(2):38-39
19 郭晓娜,姚惠源,陈正行.苦荞粉蛋白质的分级制备及理化性质[J].食品与生物技术学报,2006,25(3):88-92
20 张惟杰主编.复合多糖生化研究技术[M].上海:上海科学技术出版社,1987:6-7
21 李建武,余瑞元,袁明秀,等.生物化学试验原理和方法[M].北京:北京大学出版社,1997:174-176
22 张惟杰主编.复合多糖生化研究技术[M].上海:上海科学技术出版社,1987:6-7
23 杨鹭生,李国平,陈林水.蛋白核小球藻粉的蛋白质、氨基酸含量及营养价值评价[J].亚热带植物科学,2003,32(1):36-38
24 韦进钟.小球藻的营养价值及其开发利用简述[J].畜牧兽医科技信息,2004,8:12-13
25 中国预防医学科学院营养与食品卫生研究所.食物成分表(全国代表值)[M].北京:人民卫生出版社,1991.42
26 Pellet P L,Young V R.Nutritional Evaluation of Protein Foods[J].The United National University,Printed in Japan,1980.26-29
27 Padhye V W,Salunkhe D K.Extraction and characterization of rice proteins.Cereal Chem,1973,56(5):389-395
28 刘学铭,梁世中.小球藻在食品工业中的应用及小球藻食品的研制[J].武汉食品工业学院学报,1999,1:47-52
29 刘秀河,吴艳,艾连中.山药水溶性多糖提取工艺的研究[J].食品与机械,2006,22(2):21-23
30 周浓.荔枝粗多糖提取工艺的研究[J].现代食品科技,2006,22(3):121-123
31 蒋秋燕,乔旭光,张振华.影响大蒜多糖提取的因素及其条件优化(Ⅰ)[J].中国食品学报,2005,5(4):101-105
32 姜帆,高慧颖,陈秀妹等.枇杷叶水溶性多糖提取工艺的研究[J].福建果树,2005,135:1-3
1 武金霞,赵晓瑜.糖蛋白的结构、功能及分析方法[J].生物技术通报,2004,1:31-34
2 Hasegawa T,Matsuguchi T,Noda K.Toll-like receptor at least partly involved in the antitumor activity of glycoprotein from Chlorella vulgaris[J].International Immunopharmacology,2002,2:579-589
3 李亚娜,林永成,佘志刚,等.小球藻糖蛋白的分离、纯化和结构分析[J].天然产物研究与开发,2004,16(6):503-506
4 周志刚,刘志礼,刘雪娴.极大螺旋藻多糖的分离、纯化及其抗氧化特性的研究[J].植物学报,1997,39(1):77-81
5 张龙翔,张庭芳,李令媛.生化试验方法和技术[M].北京:高等教育出版社,1981:112-118
6 张惟杰主编.复合多糖生化研究技术[M].上海:上海科学技术出版社,1987:6-7
7 李建武,余瑞元,袁明秀,等.生物化学试验原理和方法[M].北京:北京大学出版社,1997:174-176
8 殷钢,刘铮,李琛等.螺旋藻蛋白的分离纯化及其性质研究[J].高等学校化学学报,1999,20(4):565-568
9 Sofya N.Senchenkova,Alexander S.Sshashkov,Mihaly L.Kecskes,et al.Structure of the O-specific polysaccharides of the lipopolysaccharides of Xanthomonas campestris vignicola GSPB 2795 and GSPB 2796[J].Carbohydrate Research,2000,329:831-838
10 郭玫,余晓晖.多糖的研究概况[J].甘肃中医学院学报,1994,11(2):55-56
11 余晓斌,尤蓉.云芝菌丝体多糖的分离纯化研究[J].天然产物研究与开发,2003,15(1):29-31
12 孙玉军,陈彦.茶多糖的分离纯化及其结构分析概述[J].中国茶叶加工,2004,4:38-40
13 Taylor,Carol M.Glycopeptides and glycoproteins:Focus on the glycosidee linkage[J].Tetrahedron,1998,54:11317-11362
14 Chapin M F,Kennedy J F.Carbohydrate analysis[M].Oxford,Washington DC:IRL Press,1986:151
1 方允中,郑宋梁主编.自由基生物学的理论与应用[M].北京:科学出版社,2002:4
2 Vendemiale G,Grattagliano I,Altomare E.An update on the role of free radicals and antioxidation defense in human disease[J].International Journal of Clinical & Laboratory Research,1999,29:49-55
3 Bu Q H,Pan B S,Xu J,et al.Effects of supercritical carbon dioxide extraction conditions on yields and antioxidant activity of Chlorella pyrenoidosa extracts[J].Journal of Food Engineering.2007,80:997-1001
4 Vijayavel C,Anbuselvam,M P.Balasubramanian.Antioxidant effect of the marine algae Chlorella vulgaris against naphthalene-induced oxidative stress in the albino rats[J].Molecular and Cellular Biology.2007,303:39-44
5 Gouveia L G,Raymundo A R,Batista A P,et al.Chlorella vulgaris and Haematococcus pluvialis biomass as colouring and antioxidant in food emulsions[J].European Food Research Technology,2006,222:362-367
6 Hinneburg I,Damien Dorman H J,Hiltunen R.Antioxidant activities of extracts from selected culinary herbs and spices[J].Food chemistry,2006,97:122-129
7 周站平,刘鲁宁,陈秀兰,等.光照、变性剂和pH对钝顶螺旋藻别藻蓝蛋白抗氧化活性的影响[J].海洋与湖沼,2005,36(2):179-185
8 程超,李伟.几种植物水溶性多糖的体外抗氧化作用[J].食品工业科技,2006,27(9):63-65
9 Mau J L,Tsai S Y,Tseng Y H,et al.Antioxidant properties of methanolic extracts from Ganoderma tsugae[J].Food chemistry,2005,93:641-649
10 周志刚,刘志礼,刘血娴.极大螺旋藻多糖的分离、纯化及其抗氧化特性的研究[J].植物学报,1997,39(1):77-81
11 Oyaizu M.Studies on product of browing reaction prepared from glucose amine[J].Japanese journal of Nutrition,1986,44:307-315
12 Yen C C,Chen H Y.Antioxidant activity of various tea extracts in relation to their antimutagenicity[J].Journal of Agricultural and Food Chemistry,1995,43(1):27-32
13 Siddhuraju P,Mohan P S,Becker K.Studies on the antioxidant activity of Indian Laburnum(Cassia fistula L.):A prediminary assessment of crude extracts from stem bark,leaves,flowers and fruit pulp[J].Food chemistry,2002,79(1):61-67
14 Wang Q J,Ding F,Zhu N N,et al.Determination of hydroxyl radical by capillary zone electrophoresis with amperometric detection[J].Journal of Chromotography A,2003,10(16):123-128.
15 欧荣,邹国林.维生素C或H_2O_2系统中HO对DNA的损伤作用[J].武汉大学学报(自然科学版),1997,43(2):238-242.
16 袁倬斌,邹洪.电化学方法测定羟自由基反应速度常数[J].中国科学技术大学学报,2002,32(3):287-292
17 黄沛力,曾昭晖.银杏叶和山楂叶的抗氧化作用[J].中国药学杂志,1996,31(5):274-276
18 Yang T,Mieko.S,Shinjiro K,et al.A facilitated electron transfer of copper-zinc superoxide dismutase(SOD) based on a cysteine-bridged SOD electrode[J].Biochemica et Biophysica Acta,2002,1569(1):151-158
19 许申鸿,杭瑚.溶剂及pH值对1,1-二苯基-2-苦肼基自由基分析法的影响[J].分析测试学报,2000,19(3):50-51
20 彭长连,陈少薇,林植芳,等.用清除有机自由基DPPH法评价植物抗氧化能力[J].生物化学与生物物理进展,2000,27(6):658-661.
21 丁红秀,高荫榆,晁红娟,等.毛竹叶柄多糖体外抗氧化作用研究[J].食品科学,2007,28(11):122-125
22 范晓,严小军,房国明,等.高分子褐藻多糖抗氧化性质研究[J].水生生物学报,1999,23(5):494-499
23 阎琪,于起福,任桂凤,等.硫酸化高山红景天多糖对感染病毒小鼠血液SOD和LPO的影响[J].中国中医基础医学杂志,1997,3(6):28-30
24 Zhou M,Chen Y,Ouyang Q,et al.Reduction of the oxidative injury to the rabbits with established atberosclerosis by protein bound polysaccharide from coriolus vesicolor[J].Am J Chin Med.2000,28(2):239-249
25 Volpi N,Tarugi P.Influence of chondroitin sulfate charge density,sulfate group position and molecular mass on Ca~(2+)-mediated oxidation of human lowdensity lipoproteins:effect of normal human plasma-derived chondroitin sulfate[J].Journal of Biochemistry,1999,125(2):297-304
26 钦传光,周军,赵文,等.泥鳅多糖清除活性氧和保护DNA链的作用[J].生物化学与生物物理学报,2001,33(2):215-218
27 Lee J M,Kwon H,Jeong H,et al.Inhibition of lipid peroxidation and oxidative DNA damage by Ganoderma incidum[J].Phytotherapy Research,2001,15(3):245-249
28 肖湘,俞丽君,邱玉莹,等.油柑多糖的提取与清除氧自由基作用研究[J].中国药学杂志,1998,33(5):279-281
29 张静丽,王宏勋,张雯,等.灵芝、枸杞多糖复合抗氧化作用[J].食品与机械,2004,20(6):11-12
30 何书英,詹彤,王淑如.山药水溶性多糖的化学及体外抗氧化活性[J].中国药科大学学报,1994,25(6):369-372
31 严奉伟,罗祖友,吴季勤,等.菜籽多糖的抗氧化作用与机理研究[J].中国农业科学 2005,38(1): 157-162
32 刘少平,董卫国,吴东方,等.当归多糖对免疫性结肠炎大鼠结肠损伤的保护作用研究[J].中国药理学通报,2003,19(6):693-696
33 Ye Y N,Liu E S L,Li Y,et al.Protective effect of polysaccharides-enriched fraction from Angelica Sinensis on hepatic injury[J].Life Sciences,2001,69:637-646
34 陈炅然,胡庭俊,程富胜,等.鬼臼多糖对免疫功能低下小鼠自由基产生酶活性的影响[J].动物医学进展,2005,26(8):41-44
35 黄克和.自由基与肿瘤的关系[J].中国兽医杂志,1995,21(2):42-45
36 郑美莲,毛志达.Cu~(2+)Zn~(2+)-SOD在鼻咽癌中的研究[J].微量元素与健康研究,1998,15(4):34-36
37 Yen G C,Chen H Y.Antioxidant activity of various tea extracts in relation to their antimutagenics[J].Journal of Agricultural and Food Chemistry,1995,43(1):27-32
38 滕霞,丛建波,田晓华,等.海藻硫酸多糖抗氧化与抗肿瘤作用的试验研究[J].营养学报,1998,20(1):48-52
1 Chua Y L,Zhang D W,Boelsterli U,et al.Oltipraz induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA[J].Biochemical and Biophysical Research Communications,2005,337:375-381
2 Cao Z X,Li Y B.The chemical inducibility of mouse cardiac antioxidants and phase 2 enzymes in vivo[J].Biochemical and Biophysical Research Communications,2004,317:1080-1088
3 Li Y B,Cao Z X,Zhu H.Upregulation of endogenous antioxidants and phase 2 enzymes by the red wine polyphenol,resveratrol in cultured aortic smooth muscle cells leads to cytoprotection against oxidative and electrophilic stress[J].Pharmacological Research,2006,53:6-15
4 Moriarty R M,Naithani R,Kosmeder J,et al.Cancer chemopreventive activity of sulforamate derivatives[J].European Journal of Medicinal Chemistry,2006,41:121-124
5 Keum Y S,Owuor E D,Kim B R,et al.Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element(ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC)[J].Pharmaceutical Research,2003,20(9):1351-1356
6 Kwak M Y,Kensler T W,Casero R A.Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2:effect of thepolyamine metabolite acrolein[J].Biochemical and Biophysical Research Communications,2003,305:662-670
7 Kwak M Y,Patricia A E,Patrick M D,et al.Role of phase 2 enzyme induction in chemoprotection by dithiolethiones[J].Mutation Reseach,2001,481:305-315
8 Kwak M Y,Kensler T W,Casero R A.Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2:effect of thepolyamine metabolite acrolein[J].Biochemical and Biophysical Research Communications,2003,305:662-670
9 Kwak M Y,Egner P A,Dolan P M,et al.Role of phase 2 enzyme induction in chemoprotection bydithiolethiones[J].Mutation Research,2001,481:305-315
10 Hayes J D,Ellis E M,Neal G E,et al.Cellular response to cancer chemopreventive agents:contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress[J].Biochemical Society Symposium,1999,64:141-168
11 金春元,张学,王筠,等.PCR产物真核TA克隆法构建基因表达重组体[J].中华医学遗传学杂志,1997,14(2):115-116
12 于永利,麻彤,杨贵贞.TA克隆及双链DNA测序:介绍一种快速克隆及分析PCR产物的方法[J].中国免疫学杂志,1994,10(1):5-7
13 Cubitt A B,Heim R,Adams S R,et al.Understanding,improving and using green fluorescent proteins[J].Trends in Biochemical Sciences,1995,20(11):448-455
14 Shimomura O,Hohnson F H,Saiga Y,et al.Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea[J].J Cell Comp Physiol,1962,59:223-233
15 逄越,李庆伟.鸡卵清蛋白基因启动子调控GFP基因在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞的表达[J].生物工程学报,2005,21(1):154-158
16 Hechler D,Nitsch R,Hendrix S.Green fluorescent protein expressing mice as models for the study of axonal growth and regeneration in vitro[J].Brain Research Reviews,2006,52(1):160-169
17 Marx J C,Allay J A,Persons D A,et al.High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells[J].Human Gene Therapy,1999,10(7):1163-1173
18 Alberts D,Colvin O M,Conney A H,et al.Prevention of cancer in the next millennium:report of the chemoprevention working group to the American Association for Cancer Research.Cancer Research,1999,59:4743-4758
19 江永浩,叶钢.TK基因治疗肿瘤的研究现状[J].四川医学,2006,27(4):358-360
20 陈红兵,许杨.应用体外重组PCR技术构建鸡单链抗体基因的研究[J].中国预防兽医学报,2003,25(3):175-178
1 Steele V E,Kelloff G J.Development of cancer chemopreventive drugs based on mechanistic Approaches[J].Mutation Research,2005,(591):16-23
2 王兴旺,胥彬.抗癌药物筛选模型与筛选方法的研究[J].中国医药工业杂志,1997,28(1):39-43
3 Tanaka K,Yamada A,Noda K,et al.A novel glycoprotein obtained from Cholrella vulgaris strain CK22 shows antimetastatic immunopotentialtion[J].Cancer Immunol Immunother,1998,45:313-320
4 Tetsuya M,Kiyoshi N,Kuniaki T,et al.Toll-like recepter 2 is at least partly involved in the antitumor activity of glycoprotein from Chlorella vulgaris[J].International Immunopharmacology,2002,2:579-589
5 Konishi F,Mitsuyama M,Okuda M,et 21.Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil[J].Cancer Immunol Immunother,1996,42(5):268-274
6 Holtzclaw W D,Dinkova-Kostova A T,Talalay P.Protection against electrophile and oxidative stress by induction of phase 2 genes:the quest for the elusive sensor that responds to inducers[J].Advances in Enzyme Regulation,2004,(44):335-367
7 Kong A T,Yu R,Hebbar V,et al.Signal transduction events elicited by cancer prevention compounds[J].Mutation Research,2001,481:231-241
8 卜丽佳,张素梅,王雪,等.质粒的制备[J].安徽医科大学学报,2003,38(1):76-78
9 贺竹梅,刘秋云.一种提取质粒DNA的改良方法[J].生物技术,1996,6(1):37-38
10 倪志华,赵晓瑜,刘建荣,等.碱裂解提取质粒DNA的改进方法[J].河北大学学报(自然科学版),2005,25(2):222-224
11 邹湘辉,庄东红,钟名其,等.大肠杆菌质粒DNA不同提取方法的比较[J].汕头大学学报(自然科学版).2003,18(2):20-24
12 王丽.基因工程原理与技术[M].长春:东北师范大学出版社:155-159
13 邓宁.重组质粒DNA的提取和纯化方法研究[J].黄淮学刊,1996,12(4):61-63
14 J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译.分子克隆试验指南[M].北京:科学出版社,2002,1181-1185
15 卜丽佳,张素梅,王雪,等.质粒的制备[J].安徽医科大学学报,2003,38(1):76-78
16 刘云海,钟英丽,任凯,等.群细胞转染条件的优化[J].湖南师范大学自然科学学报,2004,27(2):84-88
17 刘新宇,梁东春,张镜宇.三种非病毒载体转染方法的比较[J].天津医科大学学报,2003,9(4):456-458
18 Thompson J S,Asmis R,Glass J,et al.P53 status influences regulation of HSPs and ribosomal proteins by PDTC and radiation[J].Biochemical and Biophysical Research Communications,2006,343:435-442
19 Zhu M,Fahl W E.Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents[J]. Analytical Biochemistry. 2000, 287: 210-217
20 Rose W C, Reed F C, Simanroff P, et al. Immunotherapy of Madison 109 Lung carcinoma and murine tumours using Lentinan[J].Cancer Research, 1984, 44: 1368-1373
21 Zhang L N, Li X L, Xu X J. Correlation between antitumor activity, molecular weight, and conformation of lentinan[J]. Carbohydrate Research, 2005, 340: 1515-1521
22 Maeda Y, Takahama S, Kohara Y. Polygenic Control of the expression of biological Activities of an antitumor polysaccharide, lentinan[J]. International Journal of mmunopharmacology, 1997, 19(9/ 10): 469-472
23 Cao Z X, Li Y B. The chemical inducibility of mouse cardiac antioxidants and phase 2 enzymes in vivo[J]. Biochemical and Biophysical Research Communications, 2004, 317: 1080-1088
24 Jaiswal A K. Regulation of genes encoding NAD (P) H:Quinone oxidoreductases. Free Radical Biology and Medicine, 2000, 29: 254-262.
25 Nguyen T, Sherratt P J, Pickett C B. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element[J]. Annu Rev Pharmacol Toxicol, 2003, 43: 233-260
26 Kwak M K, Wakabayashi N, Itoh K, et al. Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keapl-Nrf2 pathway: Identification of novel gene clusters for cell survival [J]. Journal of Biological Chemistry, 2003, 278 (10): 8135-8145
27 Dinkova-Kostova A T, Hohzcla W D, Colew R N, et al. Direct evidence that sulfhydryl groups of Keapl are the sensors regularing induction of phase 2 enzymes that protect against carcinogens and oxidants[J]. Proc Natl Acad Sci USA, 2002, 99(18): 11908-11913
28 Wakabayashi N, Dinkova Kostova A T, HohzclawWD, et al. Protection against electrophile and oxidant stress by induction ofthe phase 2 response: fate of cysteines of the Keapl sensor modified by inducers[J]. Proc Natl Acad Sci U S A, 2004, 101(7): 2040-2045
29 Surh Y J. Cancer chemoprevention with dietary phytochemicals[J]. Nature Rev Cancer, 2003, 3(10): 768-780