摘要
研究背景
恶性肿瘤是当今威胁人类健康的主要疾病。中国每年死于癌症的人数为140万到150万。药物治疗、外科治疗和放射治疗是当今肿瘤的三大治疗手段。肿瘤药物治疗涉及化学治疗、生物治疗、基因治疗、靶向治疗、传统中药及天然产物中有效抗癌成分治疗等。近半个世纪来从中药中提取有效成分应用于临床抗癌治疗已取得令人瞩目的突破性进展,应用中药中的有效抗癌成分治疗肿瘤,已越来越被广大学者和患者接受,并逐渐受到重视,是目前恶性肿瘤综合治疗的有效手段之一。因此,从中药中研究、开发抗肿瘤新药早已成为广大医药研究工作者的共识。
大量临床和试验研究证明,中医药在肿瘤防治和康复中具有显著作用。中医药不仅对化疗、放疗具有显著的增效减毒作用、对肿瘤患者良好的免疫调节作用;对肿瘤切除后的抗复发、抗转移作用;对肿瘤症状的良好治疗等作用之外,而且具有一定的抑制肿瘤细胞作用。中医药和中西医结合疗法在新世纪前夕已成为抗癌治疗的一支不可缺少的力量。从天然物质中寻找毒副作用小、安全有效的抗肿瘤药物已成为世界范围内的研究热点。近年来,中药及其有效成分抗肿瘤作用的分子机制研究取得较大进展,认为可通过诱导细胞凋亡、细胞毒性作用、调节细胞信号转导、诱导细胞分化、逆转多药耐药、抑制端粒酶活性等多种机制发挥抗肿瘤作用。
雷公藤是卫矛科植物雷公藤Triptreygium Wilferdii Hook.f的根,主要成分有生物碱类、二萜类、三萜类和倍半萜类。至今已从中分离出100多种有效成分,其中雷公藤红素(tripterine)、雷公藤内脂酮(triptonide)、雷公藤甲索(triptolide)、雷公藤乙素(tripdiolide)活性最高。雷公藤甲素是从雷公藤中分离出的二萜类内酯,是雷公藤中的最具代表性成分之一,经药理和临床试验表明其具有免疫抑制、抗炎、抗肿瘤等生物活性,我国临床上应用的各种雷公藤制剂多数以它作为主要活性成分,生药中也以雷公藤甲素的含量多少宋评价药材质量的好坏,并被广泛用于治疗类风湿性关节炎、肾炎、肝炎、各种皮肤病和肿瘤等常见病和疑难病。
MC002(2-二甲胺基乙酸宙公藤甲素酯柠檬酸盐)是雷公藤甲素(又称雷公藤内酯醇)的半合成衍生物,进入机体后在酯酶的催化作用下生成雷公藤甲素而发挥细胞毒作用。由于其具有水溶性,在制剂和临床给药方面具有优势。本论文研究内容属于北京市新药创制重点课题任务,根据2007年国家食品药品监督管理局(SFDA)颁布的《细胞毒类抗肿瘤新药研究技术指导原则》的要求,体外试验采用MTT法检测了MC002对12种人肿瘤细胞增殖的抑制作用;体内试验采用4种人肿瘤细胞裸鼠异种移植瘤模型探讨了MC002的体内抗癌作用,在此基础上我们采用TUNEL细胞凋亡检测和免疫组化方法、EMSA法探讨了MC002对人肺腺癌细胞系A-549的抗肿瘤作用机理。本论文将就体内外试验的3种人肿瘤细胞:人肺腺癌细胞系A-549、人纤维肉瘤细胞系HT-1080、人结肠癌细胞系HCT-8的试验结果及初步探讨的作用机理进行报道。
研究内容
研究一血清孵化MC002对肿瘤细胞的体外抑制作用研究
将一定浓度的MC002与人血清孵育2小时,以顺铂为阳性对照药,应用四甲基偶氮唑蓝(MTT)细胞活性检测方法观察MC002对人肺腺癌细胞系A-549、人纤维肉瘤细胞系HT-1080、人结肠癌细胞系HCT-8的细胞毒性作用,并计算其IC_(50)。采用6个10倍稀释浓度(1×10~(-5)M、1×10~(-6)M、1×10~(-7)M、1×10~(-8)M、1×10~(-9)M、1×10~(-10)M)的MC002处理3种肿瘤细胞24h后,酶标仪检测OD值。结果显示:与细胞对照组比较,经人血清孵化后MC002对所试3种人类肿瘤细胞的增殖均有显著抑制作用(P＜0.05),且有良好的量效相关性,IC_(50)分别为1.45×10~(-7)M、6.81×10~(-7)M、3.07×10~(-7)M,重复试验的IC_(50)分别为9.64×10~(-8)M、2.88×10~(-7)M、1.16×10~(-7)M。
研究二MC002对人异种移植裸鼠模型肿瘤生长抑制作用的研究
采用BALB/C裸小鼠接种人纤维肉瘤HT-1080瘤株、人结肠癌HCT-8瘤株、人肺腺癌A-549瘤株建立裸鼠异种移植肿瘤模型,设立MC002大、中、小剂量组(1.5mg·kg~(-1)、1.0 mg·kg~(-1)、0.75 mg·kg~(-1)),以环磷酰胺为阳性对照,5%葡萄糖溶液为溶剂对照,腹腔注射,隔天一次,分别给药七次、十一次、九次。末次给药后24小时处死各组动物,剥出的肿瘤并测量瘤重,计算各组平均瘤重及抑瘤率。观察MC002对荷瘤小鼠肿瘤生长的抑制作用,以相对肿瘤增殖率(T/C)、瘤重、抑瘤率作为评价指标。试验重复一次。结果显示:对于三种不同瘤株,MC002大、中、小三个剂量组瘤体积明显小于模型对照组,瘤重均显著降低(P＜0.05),MC002对人纤维肉瘤HT-1080的抑瘤率分别为41.74%、24.83%、22.27%,对人结肠癌HCT-8的抑瘤率分别为75.27%、57%、53.67%,对人肺腺癌A-549的抑瘤率分别为58.35%、54.52%、43.92%,具有良好的量效相关性。重复试验亦获得相似结果。
研究三MC002对裸鼠肿瘤组织诱导凋亡作用及对核因子κB活化的影响
文献报道,雷公藤甲素能够诱导具有野生型p53的胃癌细胞系AGS细胞凋亡并且抑制其核转录因子κB(NF-κB)和激活蛋白-1(AP-1)的反式激活,而对具有突变型p53的MKN-28和SGC-7901细胞却不能诱导其凋亡:抑制p53的表达或过度表达无活性的p53能抑制雷公藤甲素对NF-κB激活的抑制作用;提示雷公藤甲素诱导凋亡可能与促进p53的表达及抑制NF-κB激活有关。
为了进一步明确雷公藤甲素的半合成衍生物MC002的抗肿瘤作用机理,本课题采用TUNEL法检测MC002对A549细胞异种移植裸鼠肿瘤组织细胞凋亡的影响、采用免疫组化技术检测MC002在产生抑瘤作用的同时对A-549肿瘤组织中核转录因子kappaB表达的影响、采用凝胶迁移试验探讨MC002对A549细胞异种移植裸鼠肿瘤组织细胞NF-κB与DNA结合活性的影响,结果提示:与模型对照组相比,给药组肿瘤细胞发生凋亡,MC002大、中、小剂量组的凋亡指数分别为46.65%、27.58%、13.32%:NF-κB的阳性表达率分别为2.75±1.51%、3.52±1.79%、5.64±1.60%、7.28±2.53%,均呈现出良好的量效关系。试验结果表明MC002可能是通过抑制NF-κB活化来诱导肿瘤细胞的凋亡。
Research Backgroud
Malignant tumor is one of major diseases that threat to human health in nowadays.In China,1,400,000 to 1,500,000 people dying of cancer each year.Drug treatment,surgical treatment and radiation therapy are the major treatments of tumors.Tumor treatment involving chemotherapy, biological therapy,gene therapy,targeted therapy,traditional Chinese medicine and the effective natural product of anti-cancer therapy.Nearly half a century active ingredients come from Chinese medicines has been made remarkable breakthroughs in using in clinical anti-cancer treatment. Application of effective Chinese medicine in anti- tumors treatments,has been more and more accepted by scholars and the majority of patients.It is an effective means of Comprehensive treatment in malignant tumors. Therefore,there has been the consensus that we shoule do some research and develop the anti-tumor drugs from Chinese herbals by a vast number of medical workers.
A large number of clinical and experimental studies have shown that Chinese medicine in cancer prevention,treatment and rehabilitation has played an important role.Chinese medicine in addition to have a significantly attenuated toxic effect on chemotherapy and radiation therapy,have a effect on anti-recurring and anti-transferring after operation on tumor,with good effect on tumor symptoms treatment,there is a certain role in the inhibition of tumor cells.Chinese medicine and traditional Chinese and western medicine in the new century on the eve has become indispensable force in anti-cancertreatment.Finding anticancer drugs from natural substances with small toxic side effects and small,safe and effective effects has become the research hot-spot worldwide.The anti-tumor molecular mechanism of Chinese medicines and its active ingredients have made a larger progress.That variety of mechanisms such as anti-tumor effects may be through inducing apoptosis, cytotoxicity,regulation of cell signal transduction,induction of cell differentiation,reversal of multidrug resistance,inhibition of telomerase activity.
Triptolide is the root of Celastraceae plant Tripterygium Triptreygium Wilferdii Hook.f.The major components are alkaloids, diterpenoid,triterpenoid and sesquiterpenoid.It has been isolated from more than 100 kinds of active ingredients,which tripterine,triptolide in Schisanlignone,triptolide(triptolide),tripterygium B are the highest activity.Triptolide isolated diterpenoid lactone from Tripterygium,which is one of the most representative components of tripterygium.Pharmacological and clinical trials show that it has some biological activity on immuno-suppressive,anti-inflammatory, anti-tumor.It used as the main active ingredient,which in clinical application of various tripterygium.The content of triptolide also used to assess the number of good quality ingredients in natural medicine and are widely used to treat rheumatoid arthritis,nephritis,hepatitis,skin diseases and tumors,such as a variety of common and difficult disease.
Triptolide MC002,a derivative of triptolide,is catalyzed into triptolide by esterase in the body.lt has advantages in the preparation and clinical administration because of its water-soluble.This paper studies is the tasks of creation of new drugs in Beijing.According to State Food and Drug Administration requirements issued in 2007 <>,in vitro MTT assay using the MC002 for 12 kinds of human tumor cells;in vivo test using four kinds of human tumor ceils in nude mice xenograft tumor model of anti-cancer effect of MC002.On this basis, we used the detection of apoptosis TUNEL and immunohistochemical method, EMSA method of MC002 on human lung adenocarcinoma cell line A-549 to detect anti-tumor mechanism.In this paper,in vitro and in vivo tests on three kinds of human tumor cells:human lung adenocarcinoma cell line A-549, human fibrosarcoma cell line HT-1080,human colon cancer cell line HCT-8 and use preliminary test to explore its anti-tumor mechanism of action.
Research Content
Research 1 MC002 serum incubation of tumor cells in vitro study
A certain concentration of MC002 has incubated for 2 hours with human serum in 37℃.Cisplatin as a positive control.Application of methyl thiazolyl tetrazolium(MTT) method to observe the cell activity of MC002 on human lung adenocarcinoma cell line A-549,human fibrosarcoma cell line HT-1080,human colon cancer cell line HCT-8 cytotoxicity,and calculate the IC_(50).We used six 10-fold dilution of the concentration of MC002(1×10~(-5)M、1×10~(-6)M、1×10~(-7)M、1×10~(-8)M、1×10~(-9)M、1×10~(10)M) to deal with three kinds of tumor cells 24h,after that OD values were detected by microplate reader.The results showed that:compared with the cells in the control group,MC002 after incubated by human serum,three kinds of human tumor cell proliferation inhibitory effects were significantly(P＜0.05),and has a good dose-effect relationship,IC_(50) were 1.45×10~(-7)M,6.81×10~(-7)M, 3.07×10~(-7)M,repeat the test IC_(50) of 9.64×10~(-8)M,2.88×10~(-7)M,1.16×10~(-7)M.
Research 2 MC002 in human tumor xenograft model in nude mice inhibited the growth of research
Using BALB / C nude mice inoculated human lung adenocarcinoma A-549 tumor line,the HT-1080 fibrosarcoma tumor line,HCT-8 human colon tumor line to establish tumors in nude mice xenograft model,set up MC002 large, medium and small dose group(1.5mg·kg~(-1)、1.0mg·kg~(-1)、0.75mg·kg~(-1)),,and cyclophosphamide as a positive control,5%glucose solution as a solvent control,by intraperitoneal injection,once the next day and administered seven times,eleven times and nine times.Last 24 hours after administration of each group,animals were put to death,stripped of the tumor and taken tumor weight measurements,and then calculate the average tumor weight and inhibition rate.To observe the tumor growth inhibition of MC002,relative tumor proliferation rate,tumor weight,inhibition rate used as the evaluation index.The results showed that:For the three different tumor strains,the tumor volume of MC002 large,medium and small three-dose group tumor size is smaller than the model control group,tumor weight were significantly lower,and has a good dose-effect relationship.
Research 3 MC002 tumor tissues of nude mice induced apoptosis and nuclear factor-κB activation
Reported in the literature,anti-tumor effects of Tripterygium wilfordii has a multi-target effect.Triptolide can induce apoptosis with wild-type p53 in gastric cancer cell line AGS cell and inhibite its nuclear transcription factorκB(NF-κB) and activator protein -1(AP-1) of the trans-activation,and with mutant p53 of the MKN-28 and SGC-7901 cells can not induce apoptosis;Inhibit the expression of p53 or over-expression of p53 can inhibit the activity of TP activation of NF-κB inhibition; Prompted triptolide induced apoptosis may be related to the promotion of p53 expression and inhibit activation of NF-κB.
In order to further clarify the triptolide MC002 of semi-synthetic derivatives of the anti-tumor mechanism of action,the subject of the use of TUNEL method,immunohistochemistry,gel shift experiment tried to detect the induction of apoptosis in human lung adenocarcinoma may be the mechanism.Treatment group results suggest that compared with model control group,treatment group of MC002 large,medium and small dose induced tumor cells apoptosis,the apoptotic index are 46.65%、27.58%、13.32%;Expression of NF-κB positive rates were 2.75±1.51%、3.52±1.79%、5.64±1.60%、7.28±2.53%,and showed good dose-effect relationship;At the same time,NF-κB activation was inhibited,indicating that MC002 might induce apoptosis on tumor cells through inhibiting NF-κB activation.
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