【作者】 玛依努尔·艾力；

【导师】 哈木拉提·吾甫尔；

【作者基本信息】 新疆医科大学 ， 内科学， 2008， 博士

【摘要】 肿瘤是严重威胁人类健康的一类疾病。寻找有效的抗肿瘤药物与方法,彻底攻克肿瘤是世界医学界的重要研究课题。肿瘤治疗强调综合治疗,因中医中药的应用可使患者症状改善显著,全身状态保持较好,病情发展较慢,肿瘤亦可缩小或带瘤长期生存,故其在中晚期肿瘤的综合治疗中占有重要地位[1]。肿瘤化疗过程中,出现多药耐药(Multidrug resistance MDR)是化疗失败的主要原因之一。当今抗肿瘤药物发展战略的重点之一是从天然药物中寻找活性成分,而我国有丰富的中药材资源以及数千年的中医药理论与实践经验,对中药抗肿瘤作用的研究与开发前景良好。目前已筛选出一些具有抗肿瘤作用的天然中药及单体,如苦参碱、三羟异黄酮、白藜芦醇、紫杉醇等,其中一些已广泛地应用于临床,显示出良好的效果。同样,天然药物中寻找逆转肿瘤耐药性、增加化疗药物作用、有效低毒的逆转剂,长期以来一直是医学界关注的的热点。维吾尔医学认为,异常黑胆质作为胆液质、血液质、粘液质、黑胆质被“燃烧”,继而“沉淀”的最终病理产物或表现形式,经常导致肿瘤等难治性疾病。临床大部分肿瘤患者也表现为异常黑胆质性[2]。我区名老维医巴黑.玉素甫和吐尔逊·托乎提阿吉等多年来根据维医理论和临床治疗原则将维医异常黑胆质成熟剂应用于癌症患者的治疗,取得良好的疗效,被患者誉为“神医”。肿瘤的维医异常黑胆质成熟剂治疗有良好效果,其促使我们,采用现代科学技术对异常黑胆质成熟剂的抗肿瘤作用进行深入研究,揭示其作用机理。十几年来,哈木拉提教授为首的课题组采用现代科学技术对异常黑胆质成熟剂作用机理进行了较深入的研究。动物实验研究表明,该复方能够促进免疫细胞的生长和增殖,提高机体的免疫功能,增强免疫器官中抗氧化酶的活性,增强机体的抗癌能力[3-8]。研究发现,异常黑胆质成熟剂具有明显的抗氧化应激对淋巴细胞DNA的损伤作用及减轻氧化造成的淋巴细胞毒性,能上调淋巴细胞Bcl-2蛋白的表达水平,能显著抑制氧化应激诱导的淋巴细胞的凋亡,能明显降低氧化诱导对淋巴细胞NF-κB的激活作用,同时异常黑胆质成熟剂本身也能激活淋巴细胞的NF-κB作用。异常黑胆质成熟剂中可能存在作用于细胞增殖凋亡信号传导通路和影响相关基因表达调节的活性成份,这些仅在已发生癌变的细胞中发挥着诱导细胞凋亡、上调抑癌基因表达和下调凋亡抑制基因的作用,而且在正常细胞中发挥着抗氧化应激对细胞DNA的损伤作用及减轻氧化造成的细胞毒性,通过上调淋巴细胞Bcl-2蛋白的表达水平,抑制氧化应激诱导的淋巴细胞的凋亡、降低氧化诱导对淋巴细胞NF-κB的激活作用,从而预防细胞发生癌变[9-13]。通过动物实验[14]观察受照射小鼠体重、脾脏指数、骨髓功能和SOD等指标的变化,发现辐射后各组小鼠体质量均下降;异常黑胆质成熟剂干预各组脾脏指数明显低于对照,各组异常黑胆质成熟剂干预后受照射小鼠的SOD值、白细胞数、红细胞数和30d生存率均有明显的升高。认为在活体组织中异常黑胆质成熟剂对辐射有确切的保护作用。同时,以往工作中,发现异常黑胆质成熟剂对HepG2(人肝癌细胞)[15]、Hela(人宫颈癌细胞)[16]、T淋巴瘤细胞[17]、乳腺癌细胞[18]体外具有抗癌作用,已初步确定异常黑胆质成熟剂总黄酮是异常黑胆质成熟剂重要的抗癌活性成分。我们应用运用体外细胞培养技术,研究异常黑胆质成熟剂总黄酮对结肠癌细胞增殖、凋亡、分化的影响;运用动物实验来研究总黄酮对结肠癌癌前期病变的预防和抑制作用;以进一步明确抗癌谱的同时探讨异常黑胆质成熟剂总黄酮逆转肿瘤细胞耐药性的作用机制,主要内容如下:1.异常黑胆质成熟剂总黄酮对人结肠癌细胞株(Caco-2)生长、凋亡及DNA链断裂的影响本研究中以人结肠癌细胞株(Caco-2)等作为研究抗癌活性的体外细胞模型,采用体外细胞培养技术、琼脂糖凝胶电泳技术、流式细胞术研究异常黑胆质成熟剂总黄酮对癌细胞体外生长和凋亡调控的影响,从而探讨异常黑胆质成熟剂总黄酮诱导细胞凋亡的作用机理。探索维吾尔医异常黑胆质成熟剂总黄酮的抗肿瘤作用机理。MTT实验结果表明,异常黑胆质成熟剂总黄酮不同作用时间对Caco-2细胞抑制作用的IC50分别为5.99mg/ml(48h)及3.02mg/ml(72h)。异常黑胆质成熟剂总黄酮与Caco-2细胞共同培育48h后,对Caco-2细胞DNA链断裂的作用有所不同。当样品浓度在0.5～5.0mg/ml时,总黄酮对DNA没有产生明显的影响,即凝胶图谱上没有出现明显的“梯子”样DNA条带。但在7.5mg/ml浓度时,对Caco-2细胞DNA链产生明显的作用,出现了较明显的“梯子”样DNA条带。流式细胞术观察表明,当Caco-2细胞与异常黑胆质成熟剂总黄酮共同培养48小时后,与空白对照组比较,表现出浓度依赖性得阻滞细胞周期,使细胞滞留于sub-G1期,癌细胞凋亡率逐渐提高(P<0.05),尤其是当浓度为7.5mg/ml时,明显提高Caco-2细胞在sub-G1期的百分比。2.异常黑胆质成熟剂总黄酮对1,2-二甲肼(DMH)诱导的大鼠结肠异常隐窝灶的影响1,2-二甲肼(1,2-dimethylhydrazine,DMH)诱导的大鼠结肠癌前期病变是一个长期、多因素共同参与的过程,其引起的细胞动力学、组织病理学变化和肿瘤形成的分子机制的改变等方面均与人类的结肠癌前期的形成过程比较近似,是研究结肠癌形成的一个良好模型。实验结果表明,不同剂量的异常黑胆质成熟剂总黄酮预防组和抑制组可以明显抑制DMH诱发的大鼠体重减轻的趋势,明显抑制DMH诱发的大鼠ACF、AC总数及其多样性,明显抑制由DMH攻击引起的大鼠免疫器官重量减轻,提高DMH诱发大鼠免疫器官(胸腺及脾脏)指数。本研究发现异常黑胆质成熟剂总黄酮抑制DMH诱发的大鼠结肠癌癌前期病变的作用,这与其抑制ACF形成有关,而且证实了异常黑胆质成熟剂总黄酮具有较强的体内预防及抑制肿瘤形成作用。3.异常黑胆质成熟剂总黄酮对人肝癌耐药细胞株Bel/Fu逆转肿瘤多药耐药机制的研究我们应用MTT实验方法测定Bel/Fu的IC50、耐药细胞集落实验观察异常黑胆质成熟剂总黄酮对耐药细胞株Bel/Fu(肝癌耐药细胞)逆转作用,同时观察异常黑胆质成熟剂总黄酮和氟尿嘧啶协同效果。FCM(流式细胞技术)观察细胞周期改变和凋亡。免疫组织化学法(S-P)法检测细胞P糖蛋白的表达。逆转录PCR法检测细胞内MDR1、GSTπ、TopoⅡ、凋亡基因mRNA的表达。研究异常黑胆质成熟剂总黄酮对人肝癌细胞株Bel/Fu逆转多药耐药作用机制。异常黑胆质成熟剂总黄酮对肝癌耐药细胞Bel/Fu的IC50是2000.68μg/ml,用药安全范围很大。异常黑胆质成熟剂总黄酮在390μg/ml浓度时对细胞无明显的杀伤作用,但与抗癌药物5-Fu合用时,合并用药组的细胞毒性作用与单独用5-Fu抗癌药物的相比有显著性差异(P<0.01),表明异常黑胆质成熟剂总黄酮与抗癌药物有协同作用,逆转倍数为7.8。随异常黑胆质成熟剂总黄酮浓度的加大集落逐渐变小、集落形成率减少,390μg/ml时表现为极小的集落,集落形成率仅为3.5%、细胞形状不规则有中毒颗粒,460μg/ml基本无集落形成。免疫组织化学法(S-P)实验证实,异常黑胆质成熟剂总黄酮抑制P-gp的表达。因此我们认为异常黑胆质成熟剂总黄酮很可能是通过抑制P-170糖蛋白的作用而升高耐药细胞内药物浓度,达到增敏目的。实验还发现异常黑胆质成熟剂总黄酮对MDR1/mRNA和P-gp的表达有明显的抑制作用。对MRP、GSTπ和TopoⅡ基因表达进行了同步观察,发现异常黑胆质成熟剂总黄酮同样可以升高的TopoⅡαmRNA水平,提示异常黑胆质成熟剂总黄酮也是TopoⅡα耐药逆转剂。流式细胞技术观察结果,异常黑胆质成熟剂总黄酮以不同的浓度作用于Bel/Fu细胞48h后,对细胞周期产生不同的影响。在2.5、5.0和7.5mg/ml时,凋亡细胞所特有的sub-G1期的百分比分别为0.9%、4.0%、7.8%,在2.5及7.5mg/ml时分别与空白对照组(0.4%)比较具有显著性差异(P<0.05),同时我们发现异常黑胆质成熟剂总黄酮对Bel/Fu细胞凋亡(sub-G1)的诱导作用具有明显的浓度依赖性。异常黑胆质成熟剂总黄酮逆转作用提高抑癌基因p53及促凋亡基因p21、Bax mRNA表达、抑制抗凋亡基因Bcl-2 mRNA表达等有关。4.结论4.1由维吾尔医复方衍生的异常黑胆质成熟剂总黄酮不仅具有体内外抗肿瘤作用,而且具有体外逆转肿瘤细胞多药耐药性之功效。4.2异常黑胆质成熟剂总黄酮对结肠癌细胞(Caco-2)体外抗癌作用可能主要通过诱导癌细胞凋亡等途径来实现。即引起癌细胞DNA链断裂,诱导细胞凋亡,癌细胞滞留于sub-G1期,使癌细胞凋亡率逐渐增高。4.3异常黑胆质成熟剂总黄酮抑制DMH诱发的大鼠结肠ACF形成,具有预防和抑制肿瘤发生作用。其部分机理在于提高使用DMH诱发的异常隐窝大鼠的胸腺指数达正常水平,抑制DMH诱发的异常隐窝大鼠体重的减轻,提高DMH诱发的异常隐窝大鼠免疫器官指数。4.4异常黑胆质成熟剂总黄酮对肝癌耐药细胞(Bel/Fu)具有逆转多药耐药MDR的作用,其机制可能与抑制Bel/Fu细胞的MDR1/mRNA,降低Pgp的表达和功能,升高TopoⅡα的mRNA水平有关。4.5异常黑胆质成熟剂总黄酮抑制肿瘤耐药细胞耐药集落形成、减少集落形成率、增加细胞中毒现象,从而抑制耐药细胞的生长。4.6异常黑胆质成熟剂总黄酮可能通过肿瘤耐药细胞滞留在sub-G1期,提高抑癌基因p53及促凋亡基因p21、Bax mRNA表达、抑制抗凋亡基因Bcl-2 mRNA表达等作用来诱导肿瘤耐药细胞凋亡。更多还原

【Abstract】 A large numer of anticancer agents, including alkylating agents, antitumor antibioltics, antimetabolites, and various other agents such as platinum derivatives, procarbazine, and emetine, have been tried, alone and in a variety of combinations and by different routes of administration, to treat carcinoma, but the predictable response rate has always been less than 20%. Additionally, drug toxicity is frequent. Current trials are investigateing the use of plant derivatives , much attention has been paid to Chinese herd, because it has little toxicity, not susceptible to get drug resistance, has multimeans in antitumor.Multidrug resistance (MDR) is the protection of tumor cell population against numerous drugs differing in structure and mechanisms of influence on the cells. MDR is also one of the major causes of failure of chemotherapy of human maligancies. It is related to many active mechanisms. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The active mechanisms read as follows: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of glutathione detoxification system; alteration of the genes and the proteins involved into the control of apoptosis (especially P53 and Bcl-2); changes of activity and quantity of topoisomerases. The tumor cell MDR and looking for little toxic and more effective chemotherapy drug has long been an interesting subject.Uighur medicine considers that Abnormal Savda is a pathological production resulted from the combustion of different body fluids known as Savda, Sapra, Belghem and Kan. It is main leading cause of cancers, and about 90% of caners attribute to Abnormal Savda in clinical reports. Bahki Yusup and Tursun Tohti Haji, two of the famous Uighur Doctor,according to the traditional therapy, have applied the ASMq-TF for treatment of cancers, which demonstrated remarkable clinical results, there by gaining the honor of“highly skilled doctor”by the local people.To date, the anticancer effects of ASMq-TF have only been little studied and its potential mechanism of action is still unclear, so a possible anticancer effect of Munziq of Abnoraml Savda clearly warrants further investigation.In the present study, we used human clone cells and Caco-2 to examine the anticancer effects of ASMq-TF, and also examine in vivo by used 1,2-dimethylhydrazine (DMH) induced rat colon carcinogenesis.We used Reversal of resistance to 5-Fu in human hepatocarcinoma cell line BEL /Fu toexamine reversal effects of ASMq-TF.Part1 The effect of Abnormal Savda Munziq Total Flavonoids on the proliferation, apop- tosis in Caco-2Colon cancer cells line of Caco-2 was cultured by cell culture technique. The effect of ASMq-TF, on the proliferation of Caco-2 cells after exposure to different concentration and different time was evaluated by MTT. Apoptosis rate was determined by using flow cytometry, DNA fragmentation assay. The study the effects of Abnormal Savda Munziq on the proliferation, apoptosis and correlative gene expression in cancer cells line of Caco-2.The results of MTT assay demonstrated that water soluble of ASMq-TF ethanol extract showed different effects on Caco-2 cell grows at 48h and72h incubation. The IC50, they were IC50 5.99mg/ml(48h)and 3.02mg/ml(72h). DNA fragmentation observed by gel electrophoresis showed that there were no significant DNA ladders in Caco-2 after treatment with different water soluble of ASMq-TF 0.5~5.0mg/ml , and seems to be no detectable difference between control cultures. But ethyl acetate extract induced the typical pattern of DNA ladders in Caco-2 cells at 7.5mg/ml. To study the acting mechanism of the fractions of ASMq-TF ethanol extract, flow cytometry was used to detect the cell cycle distribution of Caco-2 cells after 48h treatment with each fraction, respectively. The percentage of HepG2 cells in phase sub-G1 was most high concentration of 7.5mg/ml.Part2 Effects of Abnormal Savda Munziq Total Flavonoids on DMH induced colon carcinogenesisIn vivo experiment was designed to assess the potential preventive and curative effect of ASMq-TF on 1,2-dimethylhydrazine (DMH) induced rat colon carcinogenesis. Obserb number of aberrant crypt foci (ACF) and aberrant crypt (AC) and crypt multiplicity. The study on the preventive and treatment effects of Abnormal Savda Munziq on colon carcinogenesis.The results showed that all doses (400, 800 and 1600 mg/kg b. w.) of ASMq-TF ethanol extract in the pre-treatment group as well as in the treatment group significantly reduced the number of aberrant crypt foci (ACF), aberrant crypt (AC) and crypt multiplicity (P<0.05), the organ relative weights in pre-treatment and treatment groups were regained by ASMq-TF ethanol extract administration when compared to negative control group (P<0.05). These findings suggested that ASMq-TF ethanol extract had a chemoprotective effects on DMH-induced colon carcinogenesis, by suppressing the development of preneoplastic lesions, and probably exert protection against the initiation and promotion steps of colon carcinogenesis.Part3 Reversal mechanism of resistance to 5-Fu in human hepatocar- cinoma cell line BEL/Fu by Abnormal Savda Munziq Total FlavonoidsA drug-resistant hepatocarcinoma cell line BEL/Fu was used in this study. The aim of this study was to observe the reversal effect of 5-Fu resistance and its potential mechanism in hepatocarcinoma cell line BEL/Fu by Abnormal Savda Munziq.Sentivity to 5-Fu of BEL-/Fu cells was assessed by MTT assay. The reversal action of ASMq-TF, IC50 value and synergic action of vincristine and ASMq-TF were determined by MTT assay. The reversal action of ASMq-TF was also observed by clone forming text. MDR1 expression of Pgp was examined by immunochemical assay and determination of gene expression of MDR1 ,MRP, glutathione S-transferase(GSTpi) and topoisomeraseⅡαof Bel-7402 cell line were investigated using reverse transcription polymerase chain reaction (RT-PCR). Expression of bcl-2 protein and percentage of cell apoptosis was detected by flow cytometry. MDR1 expression of Pgp was examined by immunochemical assay.The reversal effect of ASMq-TF was evaluated using Bel/Fu cell line with innate resistance in this study. The IC50 value of ASMq-TF crude extract was 2000.68μg/ml. ASMq-TF at 390μg/ml concentration was no toxic but had a toxic effect on Bel/Fucell line with 5-FU. The cytotoxicity of 390μg/ml ASMq-TF and 5-FU to Bel/Fu cellline was more remarkable than only with 5-FU (P<0.01). The fold reversal of ASMq-TF combining with 5-FU was 7.8.While ASMq-TF concentrations lower than 390μg/ml had less toxic, it had no synergetic effect with 5-FU. ASMq-TF concentrations higher than 400μg/ml were not suitable due to cytotoxicitiy to tumor cells .The reversal effect of ASMq-TF was confirmed by clone forming text. The inhibited effect of ASMq-TF on Bel/Fu cell line correlated well with ASMq-TF concentration. However, Bel/Fu cell line treated with 5-FU and ASMq-TF showed significantly inhibited effect as comparing with ASMq-TF treatment. Bel/Fu cells treated with 390μg/ml ASMq-TF,The clone forming size is much smaller and the clone forming rate was only 3.5%. At same time, the irregular shaped cells, intracellular toxic granules, very loosely arranged cell and bizarre cells were observed. There was not clone forming and a few of dispersed cells in cells treated with 460μg/ml ASMq-TF .Human Pgp is coded by gene MDR1 and related to MDR of tumor cells. ASMq-TF has been found to downed-regulat MDR1/Pgp gene and proteinexpression. And simultaneously it could up-regulate TopoⅡαmRNA.Taken together, the present study suggested that the apoptosis triggered by ASMq-TF total flavonoids was mediated through DNA fragmentation, cell cycle arrest at sub-G1, up-regulation of p53, p21 and Bax gene expression, down-regulation of Bcl-2 gene expression in concentration- dependent manner. We concluded that the anticancer properties of ASMq-TF was possibly mediated through multiple pathways, suggesting multiple ingredients, rather than single component The acting ingredients in ASMq-TF that exerted the anticancer effect may include total flavonoids which are abundant in ASMq-TF.更多还原