【作者】 许杜娟；

【导师】 陈敏珠； 李卫平； 杨雁；

【作者基本信息】 安徽医科大学 ， 药理学， 2003， 博士

【摘要】 黄芪是肿瘤临床的常用中药，现代医学表明，黄芪及其某些有效组分有免疫增强及抗肿瘤作用，但其制剂仍属粗放。黄芪总提物（Total Astragalus Extract, TAE）是从黄芪中提取的有效部位群，主要由黄芪总苷（AST）与黄芪多糖（APS）组成。鉴于目前肿瘤的治疗尚不尽人意，寻找新型抗肿瘤药仍是当务之急。据此，我们采用体内、外相结合的方法，分别从整体、细胞、分子水平上，研究了TAE的抗肿瘤作用及其作用机理。主要内容概括如下： 1．对动物移植性肿瘤的抑制作用 采用小鼠肝癌（HepA）和肉瘤（S180）的实体型以及小鼠Ehrlich腹水癌。每一种瘤株的实验均重复三批。三批实验结果表明，TAE低、中、高3个剂量(15，50和150mg·kg^-1，ig，d1-d11)治疗小鼠HepA实体型的平均抑瘤率分别为40.8％，45.8％和43.0％。治疗小鼠S180实体型的平均抑瘤率分别为57.1％，54.5％和45.9％。对Ehrlich腹水癌小鼠的生存期，TAE中、高剂量可使存活20天以上的小鼠数明显增加，20天存活率分别为54.5％和69.7％。提示TAE不仅对小鼠HepA与S180的实体型有明显的抑制作用，而且对Ehrlich腹水癌小鼠的生存期亦有一定的延长作用。对TAE两个主要部位——AST和APS的抗肿瘤作用的研究表明，AST低、中、高3个剂量(3，10和30mg·kg^-1，ig，d1-d11)治疗小鼠2003年安徽医科大学博士学位论文HePA实体型的平均抑瘤率分别为35 .2%，40.8%和43.6%;治疗小鼠5180实体型的平均抑瘤率分别为44.6%，502%和54.1%。APS低、中、高3个剂量（12、40和120mg’k梦，ig，dl一dll）治疗小鼠HePA实体型的平均抑瘤率分别为41.3%，53.6%和45.3%。说明AST和APS均为TAE抗肿瘤作用的活性部位。2.免疫调节作用 用环磷酞胺（CyeloPhosphamide，CTX）或5一氟尿喀陡（5一Fluorouraeil，5一Fu）制备正常及荷瘤小鼠的免疫抑制模型，通过体内给药和体外检测的方法，观察TAE的免疫调节作用，实验结果表明，TAE中、高剂量均可明显升高CIX降低正常或荷5180小鼠的胸腺指数。TAE与5一Fu合用，可使5一Fu降低的HePA小鼠胸腺指数和血清IL-2含量上升。本文结果还表明，TAE3个剂量（15，50，150mg’kg一1，19，d1一dll）与cTx合并治疗荷5180小鼠不仅可减轻cTx对荷瘤小鼠脾细胞COnA增殖反应及IL一2诱生的抑制作用，还可对CTX降低腹腔巨噬细胞产生TNF一。有明显的提升作用。体外实验结果表明，TAE（25mg·L一’）可明显促进亚适量ConA诱导正常小鼠脾细胞增殖反应以及IL一2的产生，还可促进亚适量LPS诱导正常小鼠腹腔巨噬细胞产生r1NF一Q。提示TAE不仅能上调正常和荷瘤小鼠低下的免疫功能，而且对二类免疫细胞的增殖和/或分泌均有增强作用。3.对化疗药抗肿瘤的增效作用 TAE3个剂量（10，20和40mg’kg一，，19，dl一dll）分别与cTX3个剂量（5，10和20mg·kg，ip，dl，4，7，10）联合治疗小鼠5180实体型，或分别与5一Fu3个剂量（5，20和Zomg·kg一，，ip，dl，4，7，10）联合治疗小鼠HepA实体型，实验结果经药物相互作用动力学软件分析，Q值均在一1一1之间，提示TAE与CTX或5一Fu合用均有相加作用。2003年安徽医科大学博士学位论文4.对肿瘤化疗药降低外周血白细胞的减毒作用 实验结果表明，TAE3个剂量（15，50和1 somg·kg”，19，dl一dll）与cTx（80mg’kg一l，iP，d6与d7）合用于正常小鼠，可明显提升cTx降低的外周血白细胞。此外，TAE3个剂量（15，50和1 somg·kg”，19，dl一dll）与eTx（somg·kg一，，ip，d一，4，7，10）合并治疗荷5150小鼠，在保持eTx疗效的同时，对其降低外周血白细胞数，亦有明显的提升作用。提示TAE对CTX降低正常与荷瘤小鼠外周血白细胞有明显的减毒作用。5.抗肿瘤作用的机理 TAE不仅有抗肿瘤作用和免疫调节作用，而且有增效与减毒作用，符合生物反应调节剂（BRM）的要求。实验也提示AST与APS均是TAE的活性部位。那么，TAE及其活性部位是如何发挥抗肿瘤作用的呢?采用MTT比色法或护H]TdR渗入法分别检测了TAE、AST和APS体外对人肝癌Bel一7404细胞株、人宫颈癌HeLa细胞株和人白血病K562细胞株生长的影响。结果显示，TAE（10，20，40，50，160或32omg·L一，）对Bel一7404细胞、HeLa细胞和K562细胞均有明显的抑制作用。AsT（10，20，40，50，160或32omg·L”）对Bel一7404细胞、HeLa细胞和K562细胞的生长亦有剂量依赖性的抑制作用，但APS（10、20、40、80、160或320mg’L一’）对Bel一7404细胞、HeLa细胞和K562细胞的生长均没有直接抑制作用。APS（160，320，640mg·L一’）与小鼠脾细胞和腹腔巨噬细胞分别共培养的两种上清，对Bel一7404细胞的生长均有显著的抑制作用。用小鼠IFN一Y ELISA试剂盒和TNF一Q放免药盒检测培养上清，实验结果表明，随着APS浓度的增加，APS分别增加腹腔巨噬细胞培养上清中TNF一a的含量和脾细胞培养上清中IFN一Y的含量。提示APS通过促进IFN一Y和TNF一Q的分泌等免疫反应而实现其抗肿瘤作用。 TAE和AST是如何直接抑制肿瘤细胞的生长?本文采用流式细胞术检测和分析了TAE和AST对Bel一7404细胞和HeLa细胞周期的影响，结果显示，TAE与2003年安徽医科大学博士?更多还原

【Abstract】 Total Astragalus Extract (TAE),composed of astragalosides(AST) and astragalus polysaccharides(APS), are active compounds extracted from the root of Astragalus membranceus. As the tumor therapy at present is still unsatisfactory,finding new antitumor drugs is a urgent matter. The present study was therefore designed to investigate the antitumor effect of TAE both in vivo and in vitro and to explore its mechanisms of action at gross , celluar and molecular levels.The main points are concluded as follows:l.Inhibitory effect of TAE on implanted tumors in miceThe models of hepatoma (HepA),sarcoma(S180) and Ehrlich ascilic carcinoma(EAC) in mice were established.Experiments of studing the effect of TAE on the above three animal models were repeated by three times.The results showed that TAE(15,50,and 150mg kg-1, ig ,d1 to d11) could inhibit the growth of HepA tumor cells in mice at the inhibitory rate of 40.8%,45.8% and 43.0% ,respectively.It could also inhibit the growthof S180 tumor cells in mice at the inhibitory rate of 57.1%,54.5% and 45.9%,respectively.Also,TAE (50and 150mg kg-1,ig)could increase the number of the survival EAC mice whose life span exceeded20 days at the survival rate of 54.5% and 69.7%. These suggested that treatment with TAE could not only inhibit the growth of HepA and S180 in mice, but also could prolong the life span of EAC mice.In addition ,we also investigated the antitumor effects of two active parts TAE-AST and APS. The results showed that AST(3,10and30mg kg-1 ,ig ,d1 to d11) inhibited the growth of HepA in mice at the inhibitory rate of 35.2%, 40.8% and 43.6% respectively. It could also inhibit the growth of S180 in mice at inhibitory rate of 44.6% , 54.1% and 50.1%,respectively. Moreover, APS(12,40and 120mg kg-1 ,ig, d1 to d11) could inhibit the growth of HepA in mice at the inhibitory rate of 41.3% ,53.6% and 45.3%, respectively.These suggested that AST and APS are both active parts of TAE in tumor-therapy.2.1mmunomodulatory effect of TAEThe immunosuppressine model induced by Cyclophosphamide (CTX) or 5-Fluorouracil (5-Fu) in normal and tumor bearing mice were established to investigate the immunomodulatory effect of TAE. The results showed that TAE(50 and 150mg kg-1 ,ig ,d1 to d11) could enhance the thymus index in normal or S180 tumor bearing mice suppressed by CTX. TAE(50,150mg kg-1, ig ,d1 to d11l) could also enhance the low thymus index and the IL-2 production of splenocytes from HepA bearing mice treated by 5-Fu .Furthermore ,TAE could not only enhance ConA induced splenocytes proliferation and IL-2 production ,but also enhance the low TNF- a production of peritoneal macrophage of S180 tumor bearing mice treated by CTX. The experiments in vitro showed that TAE (25mg L-1) could enhance the proliferation response and IL-2 production of splenocytes induced by suboptional dose of ConA, it can also enhanceTNF- a production of peritoneal macrophages induced by suboptional dose of LPS from normal mice.These suggested that TAE could not only improve low immune function of normal and tumor-bearing mice,but also could enhance proliferation and/or secretion of two kinds of immunocytes(splenocytes , M s).3.Synergetic effect of TAE on antitumor effect of chemotherapeutic agentsIn combination with TAE(10 , 20 , and 40 mg kg-1 , ig ,d1 to d11) and CTX( 5, 10 ,and 20 mg kg-1,ip ,d1,4,7,10) or 5-Fu (5 ,10 and 20mg kg-1, ip ,d1,4,7,10) treated S180 or HepA tumor in mice, respectively. Data were analyzed with a software of the dynamics of drug interaction . Q values were all from -1 to 1 .This suggested that TAE had synergetic effect in combination with CTX or 5-Fu.4.Effect of TAE on the total leucocyte count in peripheral blood suppressed by CTXThe results showed that TAE(15,50 and 150mg kg-1 ,ig ,dl to dl 1) could amelionate the decreased blood leucocyte count induced by CTX(80mg kg-1 ,ip ,d6and d7) in normal mice. Furthermore, TAE(15 , 50 and 150mg kg-1 ,ig ,dl to dl 1) could amelionate the decreased blood leucocyte count induced by CTX(50mg kg-1 ,ip,d1,4,7,10) in S18更多还原