【作者】 张景芝；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2007， 硕士

【摘要】 为深入研究旋毛虫抗肿瘤的机理,本试验首先利用ELISA方法初步确定了旋毛虫与SP2/0骨髓瘤细胞间存在相关抗原,并利用Western-bloting方法测得了相关抗原的分子量。运用离心超滤装置及透析等技术对相关抗原进行了分离,免疫组化SABC法对其进行了定位分析。在此基础上,还进行了相关抗原的体内和体外抗肿瘤实验,并利用双向电泳分离技术及串联质谱获得了相关抗原的具体氨基酸序列。实验结果显示:旋毛虫与SP2/0骨髓瘤细胞间存在较强的免疫交叉反应;旋毛虫抗原与SP2/0骨髓瘤高免血清反应的分子量分别是:66.2、39、31、27kDa,其中33KDa抗原产生的免疫反应最强;免疫组化定位分析相关抗原为表面抗原;应用相关抗原免疫小鼠进行的体内抗肿瘤试验表明,实验组小鼠产生了特异的体液和细胞免疫应答,CD4+和CD8+等T淋巴细胞以及CD19+等B淋巴细胞均进行了有效增值。统计学分析表明,相关抗原抑瘤效果与佐剂对照组比较差异显著(P<0.01),与旋毛虫活虫组比较差异不显著(P>0.05)。氨基酸测序结果显示,旋毛虫与SP2/0骨髓瘤间存在的相关抗原是一种混合物,90%以上的成分是原肌球蛋白(TM),它是一个由284个氨基酸组成的绳状、酸性的保守蛋白。分子量为33.211kDa、等电点为4.64。本试验首次以相关抗原为切入点研究旋毛虫抗肿瘤的机理,为生物抗肿瘤制剂的研究与开发提供了理论依据。更多还原

【Abstract】 It’s well known that tumor has become the primary threat to human life.In 2000,the statistical data from World Health Organization indicated that there were 20 000 000 people infected with cancers globally and 7 000 000 people died.It has been estimated that the incidence of cancer will increase by 50 percent in 2020 and there will be 15 million cancer patients in 2010. In China,there will be 2 million emerging cancer patients every year and the number is increasing at the rate of 3 percent per year.It takes on the trend that younger people are involved in. Now,it’s the crucial time to prevent,control and cure cancer.With the development of biotechnology and thorough study in molecular mechanism of tumor genesis, biotherapy has become the fourth comprehensive mode to cure cancer and is being paid more attention to.It’s very important to improve the therapeutic effect and life quality by combining biotherapy and other therapeutic techniques,which is bene -ficial to biotherapy in the combined therapy to tumor.International studies have demonstrated that there were anti-tumor active components in many marine animals and plants.Lots of viruses and parasites also have anti-tumor effects. Until recently, some studies have shown that a lot of parasites have anti-tumor effect including Trichinella spiralis,acantho-amoeba trophozoite,plasmodium et al but the mech -anism involved is unclear.Trichinosis a globalized zoontic parasitic disease.Trichinella spiralis that is the most frequent and food-transmitted helminth zoonosis can infect the human and 150 kinds of animals,what is more,cause people’s death.In the previous study ,some scholars discovered that when the hosts are infested with Trichinella spiralis their immunity can be enhanced,hower,when hosts are infected with other nematodes,the host’s immunity is not remarkble improved. It is suggested that Trichinella spiralis can activate the host’s immune system,moreover,It can contain some antitumor components.In order to study antitumor mechanism of Trichinella spirali ,the experiments was designed to investigate the anti-tumor mechanism of Trichinella spiralis and SP2/0 myeloma cell related antigens,furthermore,pave the way for inventing some new antitumor biological agent.This reserch includes five following sections:The primary part, The reactions between SP2/0 myeloma cell antigen (S.mcAg)and the serum of mouse immunized with Trichinella spiralis antigen (T.sAg ),also T.sAg and the sera of mouse immunized with S.mcAg were analyzed by ELISA. The second part,The related protein between SP2/0 myeloma cell and Trichinella spiralis were analyzed by SDS-PAGE.The molecular weight of related antigens were determined by Western-bolting.The third part,The localization SP2/0 myeloma cell was examined by SABC and using antibodys specific for related antigens.The fourthion part,Antitumor action of Trichinella spiralis and extraction of solid tumor model was evaluated in vivo. Antiproliferation effect of related antigens was observed by MTT and SP2/0 myeloma cell’s morphology. change was observed by optic microscope.The fifth part,The relate antigens has been separated by two-dinensional gel electrophoresis and identified by matrix-massited laser desorption ionization tandem time-of-flight mass spctrometry(MALDI-TOF/TOF MS).Main methods include four sections as follow:①ELISA,ELISA was prepared to according to the method described before with a slight odification. Briefly,ELISA was performed by coating 96-well-plates with antigens of T.spiralis and yeloma cell separately overnight at 4℃.After blocking with 5% bovine serum albumin (BSA) diluted in PBS for 1 hour,the plates were incubated with positive serum of myeloma cell, positive serum of T.spiralis and standard negative serum diluted in PBS and kept for 1.5 hours at 37℃. Once the plates were washed, 100μL well of goat anti-mouse IgG-HPR,at the appropriated dilution,was added and incubated for 1.5 hours at 37℃. Substrate OPD was added at 4% with 3% hydrogen peroxide (H2O2) as eveloper. The reaction was stopped with 2M sulphuric -acid five minutes later and the plates were read at 492nm.②SDS-PAGE and Western- bloting. SDS-PAGE and Western blotting was prepared according to the menthd described before with a slight modification.SDS-PAGE was performed by the method of moleculer cloning using 12% Bis-Tris Gel.The gels were then stained with Simple Blue Safestain or were transferred to PVDF Immobilon transfer membranes with a Trans-Blot electrophoreic cell system.The membrane was blocked by incubation in casein blocking buffer overnight at 4℃,then incubated with control serum and serum containin Each strip contain SP2/0 myeloma cells antigens and T.spiralis antigens tracks. The reagents in culture dishes were incubated at room temperature for 2 hours on horizontal wagging platform, added goat anti-mouse IgG-HRP diluted with milk power blocking solution. After reacting at room temperature for 1 hour, DAB colour reagent kit was used then the reaction was stopped by TBS 5 minutes later. The molecular weight of reactive protein zone was obtained.③SABC, SP2/0 myeloma cells at exponential phase of growth were collected and spreaded uniformly on POLY-L-LYSINE treated glass slide. They were divided into positive, experimental and negative group based on the different added antibodies. The procedure was carried out according to the instruction of SABC kit.④2-D and MS/MS, The relate antigens has been sepatated by two-dinensional gel electro -phoresis and identified by matrix-massited laser desorption ionization tandem time-of-flight mass spctrometry(MALDI-TOF/TOF MS). The amino acid sequences of related antigens to d obtained.Mian results includes three fllowing sections:Firstly, To ascertain the related antigens between SP2/0 myeloma cell and Trichinella spiralis. and related antigen’s molecular weight that is 33,12 and pI that is 4.64.Secondly, the experiment find that the related antigen is superfic and have remarkable antitumor funtion in vivo.Thirdly,.The related antigens is Tropomyosin,includes 284 amino acid sequences of related antigens. Trichinella spiralis and the extract and antitumor fuction and the extract could probably inhibition tumor cell by E-cadherin,VEGF and endostatin.更多还原