【作者】 彭程；

【导师】 牛军； 徐克森；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 研究背景：结肠癌是临床常见的消化道恶性肿瘤,随着我国经济水平的提高和人民群众膳食结构的变化,其发病率呈现逐年升高的趋势。传统的外科手术虽然是治疗结肠癌的最有效手段,但由于结肠癌发病的隐匿性和症状的不典型性,很多患者就诊时已达中晚期,错过了手术治疗的最佳时机。因此,化疗成为了中晚期结肠癌首选辅助治疗方案。遗憾地是化疗会产生严重的毒副反应,患者不易耐受。如何在确保治疗效果的前提下减少不良反应,是当前和今后一段时间内治疗中晚期结肠癌的研究热点。在此背景下,传统中医中药所具有的抗癌作用强、不良反应小、患者易耐受的特点逐渐受到了人们的重视。去甲斑蝥素(Norcantharidin, NCTD)是中药斑蝥的提取物。国内多家医院临床观察证实,将NCTD和常规化疗方案联合应用治疗晚期结肠癌与单独化疗相比,不但近期疗效好,不良反应轻,而且中位生存时间延长,生活质量明显提高。NCTD已经成为晚期结肠癌一线化疗的最优辅助用药。NCTD如何发挥抗癌作用,目前虽有各种解释,但尚无决定性结论。整合素是一类细胞表面黏附因子,由α和β两种亚基通过非共价键连接而成,构成了细胞外基质与细胞内骨架蛋白之间结构与功能的桥梁。αvβ6是一种特殊的整合素亚型,在健康人上皮组织中不表达或极少量表达,但在胚胎形成、损伤修复和一系列上皮来源的恶性肿瘤中呈现高表达状态。目前,关于整合素αvβ6在恶性肿瘤发生发展过程中的生物学作用研究取得了丰硕的成果,尤其是在结肠癌研究方面更加突出。已有文献报道,整合素αvβ6可以促进结肠癌细胞增殖、侵袭,诱导其抵御凋亡,增强癌细胞成瘤能力,对结肠癌的恶性进展具有明显的调控作用。我们前期实验证实,NCTD具有抑制结肠癌细胞表达整合素αvβ6的药理作用。本研究(第一部分)是在前期实验的基础上深入探讨NCTD抑制结肠癌细胞生长的具体机制,并为根据此药理机制开发新型靶向药物提供理论依据。一直以来,关于肿瘤的起源问题,医学界始终存在两种理论：随机理论认为所有肿瘤细胞的形状、大小及体积都是相同的,任何一个瘤细胞都可分化形成新的肿瘤,但其发生过程是一些低概率的随机事件。等级理论则认为,在所有肿瘤细胞中只有极其少量的细胞可以分裂形成新的肿瘤。这些少量的具有可分裂增殖能力的细胞被称为肿瘤干细胞。它具有自我更新和分化能力,是肿瘤不断生长、侵袭迁移的根源。目前肿瘤干细胞理论得到了越来越多的实验结果支持。结肠癌干细胞是消化系统恶性肿瘤中第一个被发现的肿瘤干细胞。按照肿瘤干细胞理论,肿瘤组织中干细胞的持续存在与肿瘤的恶性程度及预后密切相关；肿瘤干细胞大多处于G0／G1静止期,对化疗药物的耐受性远强于快速增殖期的其它肿瘤细胞；肿瘤干细胞所特有的成瘤能力是恶性肿瘤复发转移的重要因素。结合我们前期在组织学、细胞学、动物实验中的研究发现,表达整合素αvβ6的结肠癌细胞表现出的抵御凋亡、增殖侵袭和成瘤能力与肿瘤干细胞的强耐药性、无限增殖、高致瘤性等特性相一致。本研究(第二部分)初步探讨整合素αvβ6在结肠癌干细胞上的表达情况,明确其作为结肠癌干细胞新型表面标记的可能性,为分选、鉴定并深入研究该类细胞的特性奠定坚实的基础。第一部分去甲斑蝥素通过αvβ6-ERK信号通路诱导HT-29结肠癌细胞凋亡的实验研究目的研究去甲斑蝥素(Norcantharidin, NCTD)诱导结肠癌细胞凋亡的药理学机制。方法利用MTT法检测NCTD对结肠癌HT-29细胞生长的抑制效应；明胶酶谱分析NCTD对结肠癌HT-29细胞分泌基质金属蛋白酶(MMPs)量的影响；Biotrak MMPs活性检测NCTD处理后结肠癌细胞分泌的MMPs活性变化；Hoechst 33258荧光染色检测NCTD处理后结肠癌细胞凋亡程度；流式细胞检测NCTD对结肠癌细胞表面多种整合素表达情况的影响；MTT检测多种功能性阻抗对NCTD处理后HT-29细胞生长的影响；Western blotting分析NCTD对HT-29细胞内各种丝裂原活化的蛋白激酶(MAPK)表达量及磷酸化程度的影响；MTT检测多种MAPK抑制剂对NCTD处理后HT-29细胞生长的影响；免疫共沉淀实验检测NCTD对αvβ6-ERK直接连接的影响。结果NCTD能明显抑制人结肠癌HT-29细胞生长,且这种抑制效应与药物浓度和作用时间正相关；NCTD能够抑制HT-29细胞MMP-9分泌及活性,但对MMP-3无明显抑制效应；NCTD能够诱导HT-29细胞凋亡,60μmol/L NCTD处理12小时,细胞凋亡率即达35.6%；流式细胞分析表明NCTD能抑制结肠癌细胞表面整合素αvβ6的表达,但对αvβ3、αvβ5的表达无明显影响,进一步Western blotting分析显示,仅有p6亚基蛋白含量明显降低,αv、β3、β5亚基蛋白含量未见明显变化；MTT实验证实αvβ6功能性阻抗10D5能够增强NCTD抑制结肠癌HT-29细胞生长的效应,整合素αvβ3、αvβ5的单克隆抗体LM609和F1P6无明显抑制细胞生长的能力；Western blotting分析证实,HT-29细胞经NCTD处理后仅有磷酸化-ERK含量随NCTD浓度增加或作用时间延长而逐渐降低,细胞内ERK、JNK、p-JNK(磷酸化.-JNK)、P38和p-P38(磷酸化-ERK)水平无明显变化；ERK抑制剂PD98059能明显抑制结肠癌HT-29细胞的生长,而JNK抑制剂(SP600125)和P38抑制剂(SB203580)则无此抑制效应；免疫共沉淀证实NCTD干扰αvβ6-ERK直接连接的形成。结论NCTD通过降低HT-29细胞表达整合素αvβ6,阻碍了ERK的磷酸化过程,从而干扰αvβ6-ERK直接连接形成,阻断了αvβ6介导的恶性生物学信号向胞内的传导,最终达到诱导HT-29细胞凋亡,抑制肿瘤细胞生长的作用。意义揭示了NCTD诱导结肠癌细胞凋亡、抑制肿瘤细胞生长的具体机制,进一步证实了整合素αvβ6及avp6-ERK直接连接在调控结肠癌细胞恶性生物学行为中的作用,为今后针对αvβ6或avp6-ERK直接连接开展靶向治疗提供了确切的理论依据。第二部分整合素αvβ6在结肠癌干细胞向高侵袭性结肠癌细胞分化中的作用研究目的阐明整合素αvβ6的表达与结肠癌干细胞的相关性,并探索其在调控结肠癌干细胞向高侵袭性结肠癌细胞分化过程中的作用。方法免疫组化分析整合素αvβ6和结肠癌干细胞表面标记分子CD133在结肠癌肿瘤组织中的表达相关性；流式细胞分析HT-29结肠癌细胞中表达CD133阳性表达的细胞数,并利用CD133分子的单克隆抗体ANC9C5分选CD133+ HT-29细胞。将分选后的CD133+结肠癌细胞置于无血清培养基中孵育,并观察结肠癌干细胞形态学特征。利用siRNA技术干扰CD133+结肠癌细胞中β6基因的表达后,进行MTT实验观察αvβ6的表达对结肠癌细胞生长增殖的影响,明胶酶谱分析检测αvβ6的表达对结肠癌细胞分泌MMP-9的影响。结果免疫组化染色证实,结肠癌组织中整合素αvβ6和CD133的表达具有明显的一致性。其阳性表达率大致相等,并均在肿瘤边缘高表达,中心低表达。对细胞行流式分析证实,HT-29结肠癌细胞中CD133阳性表达率为1.6%,利用抗CD133分子的单克隆抗体ANC9C5分选HT-29细胞后,富集的CD133+HT-29结肠癌细胞达92.6%。将分选后的CD133+HT-29结肠癌细胞置于无血清培养基中孵育,细胞在72小时后能形成明显的肿瘤细胞球,呈现出典型的干细胞特征。在无血清培养基中加入胎牛血清后,肿瘤细胞球逐渐消失,瘤细胞再次贴壁生长,符合已分化肿瘤细胞特征。构建表达anti-p6 shRNA的质粒,并将其导入CD133+HT-29结肠癌细胞后,行RT-PCR和Western Blotting分析证实,(αvβ6 mRNA和蛋白表达水平明显减低。MTT检测证实(αvβ6+ CD133+ HT-29结肠癌细胞在无血清培养基中能正常分裂增殖,而(αvβ6- CD133+ HT-29细胞则几乎无增殖。明胶酶谱分析也证实(αvβ6+ CD133+ HT-29结肠癌细胞分泌MMP-9水平明显高于(αvβ6- CD133+ HT-29细胞。结论整合素αvβ6和CD133分子在结肠癌肿瘤组织中共表达、共定位。整合素αvβ6能够促进CD133+的HT-29结肠癌细胞增殖生长,并诱导其分泌MMP-9,在调控结肠癌干细胞向高侵袭结肠癌分化中发挥了关键作用。意义揭示了整合素αvβ6调控结肠癌干细胞向高侵袭结肠癌分化中的关键作用。由于肿瘤组织边缘富集高侵袭力的结肠癌细胞,因此整合素αvβ6也可以作为一种高侵袭性结肠癌细胞的一种分子标记。更多还原

【Abstract】 Background:Colon cancer is one of the most frequent digestive tract cancers. It’s incidence rate is elevating year by year with the economy raising and the changes of people’s food. The traditional surgical operation is the best method to treat the colon cancer, but most patients are on the middle-advanced stage and have missed the chance of accepting the operation. Hence, chemotherapy has already been the first adjuvant therapy to the advanced stage colon cancer. Unfortunately, chemotherapy will produce severe side reaction, patients cannot be tolerant. It is a research heat-point in colon cancer treatment in current and future that how to decrease the adverse reaction with the premise of good treatment effectiveness. On this background, the traditional Chinese medicine has been through highly because they have many good characteristics, such as stronger anti-tumor effects, lighter adverse reaction and easy to be tolerant.Norcantharidin (NCTD) is the extract of cantharis. Many domestic hospitals have proven that taking the NCTD with the classical chemotherapy in colon cancer treatment is better than only thermotherapy. Comprehensive treatment has many good qualities, for example, better short-term curative effect, lighter adverse reaction, longer meta-live time, better life quality. NCTD has been the best adjuvant drug in first line thermotherapy of colon cancer treatment. However, the mechanism of NCTD anti-tumor is unclear. There are many explanations to its mechanism, but no decisive conclusion.Integrin is one kind of cell adhesion factors. They are composed byα&βsubunits which are linked through non-covalent bond. Integrin is the bridge between extra-cell matrix and intra-cellular skeleton protein.αvβ6 is a special isoform of integrin. They do not or almost not express in epithelial tissue of health people. But they highly express in embryogenesis, trauma repairing and a series of malignant epithelial tumors. Now, the researches about integrinαvβ6 how regulate the malignant tumor biological behavior have gotten plentiful results, especially in the researches on colon cancer. Some paper has been reported that integrinαvβ6 could promote colon cancer cell proliferate, invasive, resisting apoptosis and accelerate tumor cell form cancer. Our previous research has already proven that NCTD could restrain colon cancer express the integrin avP6. This research (part 1) is focuing on investigating the specific mechanism of NCTD inhibits colon cancer growth, and developing new target drugs according to the pharmaco-mechanism.For many years, about the original question of tumor, there are two theories in medical field. Stochastical theory presumes that all of tumor cells are like each other in shape, size and volume. Any one of tumor cells can be differentiated and form a new tumor, but it is some low probability random event. Hierarchy theory deems that only an exceeding small amount cell can be differentiated and form a new tumor. Those cells which can be differentiated and form a new tumor are called cancer stem cell. They have the ability self-renewal ability and differentiation, and are the source of tumor cell keep growing and migrating. Recently, the cancer stem cell theory acquires more and more study results to support. Colon cancer stem cell is the first discovered digestive tract malignant tumor stem cell.Based on the cancer stem cell theory, stem cell continuing existing is tightly related to tumor malignant extent level and prognosis. Most cancer stem cell is on the G0/G1 resting phase, chemotherapy survivability of those stem cells is obviously stronger than quick proliferative phase tumor cells. Special form of new tumor capability is the main reason of malignant tumor recurrence and metastasis. Combining our previous research results in histology, cytology and animal experiments, we believe that the characteristic ofαvβ6 expressing colon cancer cell including resisting apoptosis, proliferation, invasion, and tumor forming, are coincide with those characteristic of cancer stem cell including strong drug survivability, infinity proliferation, and easily tumor forming. This research (part 2) focus on investigating the expression of integrin avP6 on colon cancer stem cells, and identifying the possibility of integrinαvβ6 to become the colon cancer stem cell molecular marker and establish a substantial base for future research, including sorting, identifying, and lucubrating those cancer stem cell.PartⅠNorcantharidin induces HT-29 colon cancer cell apoptosis through theαvβ6-ERK signaling pathwayObjectiveInvestigate the pharmaco-mechanism of Norcantharidin (NCTD) restrain colon cancer cell growing.MethodsMTT assay was used to detect the inhibition effect of NCTD on HT-29 colon cancer cell growth. The matrix metalloproteinase (MMPs) content secreted by HT-29 cell treated by NCTD was detected by the gelatin zymography assay. Biotrak MMPs activity assay system was used to inspect the MMPs activity changing when HT-29 cell treated by NCTD. Hoechst 33258 fluorescent staining was used to sense the degree of HT-29 colon cancer cells apoptosis. Flow cytometry was used to detect some kinds of integrin expression variance produced by NCTD. MTT assay was used again for detecting the growth inhibition effects of several functional blocking antibodies on NCTD treated HT-29 colon cancer cells. Western Blotting assay analyze the expression and phosphorylation degree of Mitogen Activated Protein Kinases (MAPKs) in the HT-29 colon cancer cell treated by NCTD. MTT assay was used to detect the growth inhibition effects of several MAPKs inhibitors on NCTD treated HT-29 colon cancer cells. Co-immunoprecipitation assay was used for detecting the effect onαvβ6-ERK direct linkage produced by NCTD.ResultsNCTD can obviously restrain the HT-29 colon cancer cells growing, and this kind of inhibition effect is positive correlation with NCTD dose and treatment time. NCTD can also restrain the HT-29 colon cancer cells secrete the MMP-9 and its activity, but no inhibition effect on MMP-3. NCTD can induce HT-29 colon cancer cells apoptosis, using 60μmol/L NCTD treat cell for 12 hours, the apoptosis rate is 35.6%. Flow cytometry assay show that NCTD can restrain the HT-29 colon cancer cells expresses the integrinαvβ6, but the expression ofαvβ3 andαvβ5 were not affected. The Western Blotting assay further confirmed that only the content of (36 subunit decreased obviously, the levels ofαv,β3 andβ5 subunits did not change. MTT assay show only 10D5 (a functional blocking antibody toαvβ6) can strengthen growth inhibition effect on HT-29 colon cancer cells produced by NCTD, LM609 (functional blocking antibody toαvβ3) and P1F6 (functional blocking antibody toαvβ5) cannot. Western Blotting analysis showed, only the content of phosphorylate-ERK (p-ERK) decreased accompanying with the NCTD dose richer or treatment time longer. But the levels of ERK, JNK, p-JNK, P-38 and p-P38 did not change. The inhibitor of ERK PD98059 can inhibit HT-29 colon cancer cells grow, but both the SP600125 (JNK inhibitor) and SB 203580 (P38 inhibitor) cannot. Co-immunoprecipitation assay certified that the formation of avP6-ERK direct linkage was disturbed by NCTD.ConclusionNCTD interfere with the phosphorylation of ERK because it decreases the expression of integrinαvβ6. Hence, the direct linkage betweenαvβ6 and ERK was broken, and the signal mediated byαvβ6 was blocked. Thus, NCTD induce the HT-29 colon cancer cells apoptosis, and inhibit the cancer cells growing.Significance This research disclosed the mechanism of NCTD induce colon cancer cell apoptosis and inhibit cancer cells growth; further confirmed the important role of integrin av(36 andαvβ6-ERK direct linkage in regulating the malignant biological behavior of cancer cells; provided the precise theory for future target treatment toαvβ6 andαvβ6-ERK direct linkage in colon cancer treatment.PartⅡThe Role of Integrinαvβ6 in Regulating Colon Cancer Stem Cell differentiates to powerful invasive colon cancer cellObjectiveIdentify the relationship between the integrinαvβ6 expression and colon cancer stem cell, and investigate the role of integrinαvβ6 in regulating the colon cancer stem cell malignant biological behavior.MethodsImmunohistochemisty staining analyzes the expression correlation between the integrinαvβ6 and CD133 in colon cancer tumor tissue. FACScan were carried out to detect the percentage of CD133+ cell in HT-29 colon cancer cell, and sorted those CD133+ cell with its mAb ANC9C5. Cultured those CD133+ cell in serum-free medium, and observed cancer stem cell morphocytology characteristic. Using siRNA technology to interfere with the expression of avP6 followed MTT assay was carried out to inspect if the avP6 expression can affect the colon cancer stem cell growth. Zymogram analysis was used to detect the effect ofαvβ6 expression on colon cancer cell MMP-9 secretion.ResultsImmunohistochemisty staining confirmed that the expression ofαvβ6 and CD 133 are consistency, and both of them are prone to highly express in the leading edge of tumor tissue. The FACScan analysis showed that the percentage of CD133+ cell in HT-29 colon cancer stem cell was 1.6%. After Sorting CD133+ cell by FACScan with mAb ANC9C5, the percentage increased to 92.6%. Cultured those CD133+ cell in serum-free medium, those cell can form several small tumor cell globes in 72 hours, present classical stem cell characteristic. Added fetal calf serum into serum-free medium, tumor cell globes disappear gradually, cancer cell grow adherence again, showed out the differentiated characteristic. Constructed anti-β6shRNA plasmid, and transduced the plasmid into CD133+ cell, followed by RT-PCR assay and Western Blotting analysis. Both of the two experiments conformed that the mRNA and protein levels of av(36 decreased obviously. MTT assay showed that only theαvβ6+ CD133+ HT-29 colon cancer cells can normally divide and proliferate in serum-free medium, but the avP6 CD133+ HT-29 cannot. Gelatinase zymogram analysis also confirmed that MMP-9 secreted amount ofαvβ6+ CD133+ HT-29 cell were obviously more thanαvβ6- CD133+ HT-29 colon cancer cell.ConclusionIntegrin avP6 and CD133 express in same period and same area in colon cancer tumor tissue. Integrinαvβ6 can promote CD133+ HT-29 colon cancer cell proliferation, and induce them secrete MMP-9. Integrinαvβ6 play an important role in regulating colon cancer stem cell differentiates to powerful invasive colon cancer cell.SignificanceDisclosed the important role of integrinαvβ6 in regulating colon cancer stem cell differentiates to powerful invasive colon cancer cell. Because tumor surrounding tissues enrich powerful invasive tumor cells, integrin avP6 also should be deemed as a molecular marker of invasive colon cancer stem cell.更多还原