灵芝孢子油抗肿瘤药效学研究及相关机理探讨

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1、灵芝孢子油抗肿瘤药效学研究及相关机理探讨项目名称：灵芝孢子油抗肿瘤药效学研究及相关机理探讨Researches on the Antineoplastic Pharmacodynamics and Relative Mechanisms of Ganoderma Lucidum Spore Oil主管单位： 流动站名：基础医学二级学科：药理学合作导师： 目 录中文摘要3Abstract5一. 前 言81 背 景82 计划及目标16二. 博士后期间需要解决的问题及思路17三. 计划进度及完成情况18四. 研究内容19（一） 祛邪作用（直接抑制肿瘤细胞生长的作用）201. 灵芝孢子油注射乳剂的急性

2、毒性试验202. 体内抗肿瘤作用小鼠移植瘤试验232.1 灵芝孢子油注射乳剂iv对S180小鼠肉瘤的抑制作用232.2 灵芝孢子油注射乳剂iv对H22小鼠肝癌的抑制作用273. 体外抗肿瘤试验灵芝孢子油注射乳剂对体外培养人癌细胞的生长抑制作用294. 体内抗肿瘤作用人癌裸小鼠移植瘤试验34灵芝孢子油注射乳剂iv对人胃癌SGC-7901裸小鼠移植瘤的抑制作用灵芝孢子油注射乳剂iv对人卵巢癌HO-8910裸小鼠移植瘤的抑制作用（二） 扶正作用（调节机体免疫功能）灵芝孢子油注射乳剂的扶正作用研究451. 对动物免疫功能的影响451.1 对H22荷瘤小鼠体液免疫功能的影响451.2 对正常小鼠体液免疫

3、功能的影响461.3 对H22荷瘤小鼠网状内皮系统(RES)吞噬功能的影响471.4 对正常小鼠网状内皮系统(RES)吞噬功能的影响491.5 对H22荷瘤小鼠特异性细胞免疫功能的影响 (DTH试验法)501.6 对正常小鼠延迟性超敏反应的影响 (DTH试验法) 512. 对荷瘤动物生物调节因子的影响灵芝孢子油注射乳剂对H22荷瘤小鼠生物调节因子的影响523. 对荷瘤动物的抗癌作用（见（一）祛邪作用）53（三） 减 毒 作 用灵芝孢子油注射乳剂对放、化疗毒副反应的减毒作用541 灵芝孢子油注射乳剂 iv对H22荷瘤小鼠环磷酰胺(CTX)化疗的减毒作用542 灵芝孢子油注射乳剂 iv对正常小鼠环

4、磷酰胺(CTX)化疗的减毒作用543 灵芝孢子油注射乳剂 iv对H22荷瘤小鼠60Co放疗的减毒作用554 灵芝孢子油注射乳剂 iv对正常小鼠60Co放疗的减毒作用56（四） 作用机理的研究591. 灵芝孢子油注射乳剂对肿瘤细胞增殖的影响592. 灵芝孢子油注射乳剂对肿瘤细胞调亡的研究64（五） 其它实验研究（口服灵芝孢子油等系列研究）731. 口服灵芝孢子油的体内抗肿瘤作用（小鼠和裸小鼠模型）732. 口服灵芝孢子油对动物免疫功能的影响813. 口服灵芝孢子油对荷瘤动物生物调节因子的影响854. 口服灵芝孢子油对放、化疗毒副反应的减毒作用855. 康莱特注射液的体内抗肿瘤作用（小鼠及人癌裸小

5、鼠移植瘤实验）896. 口服灵芝孢子油对正常小鼠的免疫调节作用90五、研究工作总结104六、讨 论106七、参考文献109八、博士后期间主要工作内容汇总113九、缩 略 语114十、博士后期间发表论著汇总117十一、致 谢118灵芝孢子油抗肿瘤药效学研究及相关机理探讨中文摘要灵芝是担子菌纲多孔菌科（Polyproraceae）灵芝属（Ganoderma）真菌的总称，中国药典（2005版一部）收载了赤芝Ganoderma lucidum (Ley ss. Ex Fr.)Karst和紫芝Ganoderma sinense Zhao, Xu et Zhang。自古以来，灵芝是祖国中医药宝库中的珍品，

6、素有仙草之誉。近年来，对灵芝属真菌的化学成分及临床作用的研究颇多，对灵芝的生殖细胞-灵芝孢子药用价值的研究也日益加强,特别是由灵芝孢子提取出来的灵芝孢子油已经成为现代研究的焦点。本课题以灵芝孢子油注射乳剂为主要研究对象，对灵芝孢子油的抗肿瘤药效学及相关机理进行了系统而全面的研究。不仅从离体和整体层面阐述了灵芝孢子油的抗肿瘤药效作用，而且从分子水平深入探讨了其抗肿瘤的作用机理。(一) 祛邪作用（直接抑制肿瘤细胞生长的作用）在ICR小鼠移植瘤模型上的实验结果表明，灵芝孢子油注射乳剂（0.25，0.5，1.0 g/kg，i.v）对小鼠移植瘤H22（肝癌）和S180（肉瘤）具有确切的抑制作用，肿瘤生长

7、抑制率在40 %以上（P0.05）。在BALB/cA 裸小鼠人癌移植瘤模型上的实验结果表明，灵芝孢子油注射乳剂（1 g/kg，i.v）均对人胃癌SGC-7901和人卵巢癌HO-8910有确切的抑制作用，相对肿瘤增殖率T/C（%）均在60 % 以下（P 0.05）。灵芝孢子油注射乳剂对体外培养的SGC-7901，HO-8910 ，Hu-7等15种人癌细胞具有不同程度的生长抑制作用。(二) 扶正作用（增强机体免疫功能的作用）1. 灵芝孢子油注射乳剂对动物免疫功能的影响灵芝孢子油注射乳剂（0.25，0.5，1.0 g/kg）i.v给药可显著提高H22荷瘤和正常小鼠的廓清指数K 、吞噬系数和脾脏系数；

8、灵芝孢子油注射乳剂高、中剂量iv给药可显著提高H22荷瘤和正常小鼠由2,4-二硝基氟苯（DNFB）引起的迟发性过敏反应；灵芝孢子油注射乳剂iv给药对H22荷瘤和正常小鼠血清半数溶血值无显著影响。2. 灵芝孢子有注射乳剂对H22荷瘤小鼠生物调节因子的影响灵芝孢子油注射乳剂高剂量(1.0 g/kg)iv给药明显提高H22荷瘤小鼠血清IFN-水平（P 0.05）。扶正作用实验结果表明，灵芝孢子油注射乳剂可显著增强机体的免疫功能。(三) 减毒作用（对放、化疗毒副反应的减毒作用）灵芝孢子油注射乳剂高剂量（1.0 g/kg）iv给药可显著对抗由环磷酰胺（CTX）和60Co造成的H22荷瘤和正常小鼠外周血白

9、细胞、骨髓有核细胞数和脾脏系数的下降。减毒实验结果表明，灵芝孢子油注射乳剂对放化疗引起的毒性反应有一定的减毒作用。(四) 作用机理的研究用MTT法对灵芝孢子油注射乳剂的作用进行了检测，结果表明灵芝孢子油注射乳剂对各种组织来源的肿瘤细胞的生长具有不同程度的抑制作用。用流式细胞仪和DNA Ladder的方法检测了灵芝孢子油注射乳剂作用后DNA片段形成，一种细胞凋亡的典型表现。结果灵芝孢子油注射乳剂对能明显抑制细胞增殖的肿瘤细胞发现DNA片段形成，而灵芝孢子油注射乳剂对不能抑制细胞增殖的肿瘤细胞则不出现DNA片段形成。结果表明灵芝孢子油注射乳剂可通过诱导细胞凋亡杀伤肿瘤细胞，抑制肿瘤细胞增殖。灵芝孢

10、子油注射乳剂可以直接抑制肿瘤细胞的增殖，诱导肿瘤细胞调亡，这是灵芝孢子油注射乳剂抗肿瘤作用的机理之一。(五) 其它实验研究（口服灵芝孢子油等系列研究，限于篇幅，在此略）关键词：灵芝孢子油，注射乳剂，抗肿瘤，免疫功能，祛邪作用，扶正作用，减毒作用，细胞凋亡.Researches on the Antineoplastic Pharmacodynamics and Relative Mechanisms of Ganoderma Lucidum Spore OilAbstractGanoderma (Ling Zhi or Renzhi in Chinese; Saruno Koshikake o

11、r Mannendake in Japanese), a medicinal mushroom, has been used extensively as “the mushroom of immortality” in China and other Asian countries. This medicinal mushroom is highly ranked in traditional Chinese medicine and has been used as a panacea for many types of chronic diseases, as described 200

12、0 years ago in Shen Nungs Herbal. These diseases include cancer, neurasthenia, hypertension, type II diabetes, and hepatopathy. Ganoderma is the generic name of fungous, which belongs to Ganoderma genus, Polyproraceae, Bas diomycetes. It is received in Chinese Pharmacopoeia (2005) that Ganoderma luc

13、idum (Ley ss. Ex Fr.) Karstand Ganoderma sinense Zhao, Xu et Zhang. Recently, a lot of researches on the chemical constituents, the pharmacologically active new compounds and clinical uses of Ganoderma Lucidum have been made. Studies on the germocyte of Ganoderma Lucidum (spore) are also increased d

14、ramatically, especially on sporoderm-broken Ganoderma Lucidum Spore Oil. Focused on the Ganoderma Lucidum Spore Oil injection emulsion (here named HFL, the same below), we have carried out systematic and full-scale experiments on the antineoplastic pharmacodynamics and mechanisms of Ganoderma Lucidu

15、m Spore Oil. Our goals are to elucidate its antineoplastic pharmacodynamics from ex vivo and in vivo, furthermore, to make deep insights into the molecular mechanism.1. Effect on eliminating pathogenic factors (Directly inhibit tumor cells growth)In vivo animal models, tumor transplantation in ICR m

16、ouse，HFL (0.25，0.5，1.0 g/kg，i.v) showed significant inhibition effects in mice bearing transplanted hepatoma H22 and sarcoma 180, the inhibition rate of tumor growth all above 40 % (P0.05).On models of human cancer (human gastric carcinoma SGC-7901 and ovarian cancer HO-8910, respectively) transplan

17、ted into BALB/cA nude mice, HFL (1 g/kg, i.v) both showed remarkably inhibition activity, their relative tumor growth rates all below 60 % (P0.05).By using MTT method, in ex vivo experiments of different human cancer cells, such as SGC-7901，HO-8910，Hu-7,etc 15 human cancer strains, HFL has been foun

18、d different growth inhibition effects on different cancer cells. 2. Effect on reinforcing the healthy qi (Enhance the body immune function)(1) Effects on the host immune function of normal and bearing cancer mouseHFL（0.25，0.5，1.0 g/kg, i.v）could notably raise clearance index K, phagotrophic coeffici

19、ent and spleen coefficient in normal and bearing H22 cancer mice, respectively. The high and middle groups of HFL significantly boosted the delayed hypersensitivity reaction induced by 2,4-dinitro-1-fluorobenzene (DNFB) in bearing cancer and normal mice. HFL showed no significant influence on the se

20、rum HC50 of bearing cancer and normal mice.(2) Effects of HFL on biological regulatory factors in bearing H22 cancer mouseThe high dose group of HFL(1.0 g/kg, i.v) increased the IFN- level in the serum of H22 bearing cancer mouse(P0.05).All data in experiments of strengthening the body resistance sh

21、owed that HFL could enhance the host immune function significantly.3. Effect on reducing the toxicity (Reduce the toxicity of radiotherapy and chemotherapy)The high dose group of HFL (1.0 g/kg, i.v) could counteract the decrease of peripheral white blood cells (PWBC), marrow nucleated cells and sple

22、en index of bearing cancer and normal mice induced by Cytoxan (CTX) or 60Co.Results here showed that HFL could counteract the toxicity of radiotherapy and chemotherapy in some way.4. Researches on the mechanismsIn MTT reduction assay experiments, HFL showed different growth inhibition effects on dif

23、ferent human cancer cells.By using the methods of flow cytometry and DNA Ladder, the formation of DNA fragment after using HFL was detected. Its a typical appearance of apoptosis. The formations of DNA fragments have been detected in cancer cells, which are inhibited by HFL significantly. On the con

24、trary, no fragments could be found. These results showed that HFL could kill the cancer cell by inducing apoptosis and inhibit cell proliferation.HFL could inhibit cancer cells proliferation directly and induce cancer cells apoptosis. These might be one of the antineoplastic mechanisms. 5. Other exp

25、eriments (Series studies of oral Ganoderma Lucidum Spore Oil, etc, details not been showed here.)Key Words: Ganoderma Lucidium Spore Oil, Injection Emulsion, Antineoplastic, Immune Function, Eliminate Pathogenic Factors，Reinforce the Healthy Qi， Reduce Toxicity, Apoptosis.十一、致 谢本项目是在合作导师雷林生教授（南方医科大学）、刘菊妍（广州医药集团总工程师、教授级高工）、葛发欢（广州汉方现代中药研究开发有限公司 副总经理教授级高工）的共同悉心指导下完成的。他们高尚的人格、渊博的学识、严谨的治学态度使我获益匪浅，衷心感谢他们对我的谆谆教诲和无微不至的关怀。 感谢广州医药集团人事科陈颂明科长、技质部吴义忠科长等领导，南方医科大学博士后管理处钟军、许兆峰等老师在工作和生活上的诸多指导、关心和帮助。工作中得到广州汉方现代中药研究开发有限公司各位同事的热情帮助和极大支持，在此深表谢忱。最后，我还要向我的妻子王缨、女儿蒋天美表示衷心的感谢，是她们在生活和工作中给予了我无私的奉献和支持。9