【作者】 贾友鹏；

【导师】 王忠裕；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 结直肠癌是最常见的消化道肿瘤,据统计,自2002年至2007年,美国结直肠癌的死亡率占每年恶性肿瘤总死亡率的10%左右,连续5年排在第三位。在每年新发癌症患者中,结直肠癌同样以10%左右的发生率牢牢占据前三位。与此同时,结肠癌的发病年龄呈年轻化趋势,术后转移率、复发率逐年升高,已经引起疾病控制部门和临床医生的高度重视。结肠癌的局部浸润和淋巴转移都在早期发生。因临床症状发生较晚、表现不典型及检查手段的局限性,使结肠癌的早期诊断愈加困难,客观上给结肠癌的浸润、转移提供了时间。恶性肿瘤的浸润、转移过程是由多因素参与的复杂过程,涉及细胞游走、定植、增殖、新生血管形成、免疫监视等多个方面,目前还没有确切的手段可以对这个过程进行监测和控制。结肠癌的局部浸润首先是肠壁内扩散,可以在2年左右的时间里环绕肠壁一周,还可以侵袭肠系膜甚至肠周围器官,直接与结肠癌的临床分期相关,对结肠癌的预后有重大的影响。淋巴转移则在结肠癌的转移过程中占主要地位,约占60%,在早期即有发生,不仅与结肠癌的临床分期有关,还可以指导术后进一步的抗肿瘤治疗,也是影响结肠癌患者预后的主要因素之一。因此,针对结肠癌浸润、淋巴转移的研究是相当重要和必要的。目前针对结肠癌公认的治疗方案是以根治性手术切除为基础的肿瘤综合治疗,但是对于肿瘤部位相近、病理类型和分化程度相近、具有相同Dukes分期、同样施行了根治性手术和区域淋巴结清扫的患者,其预后和生存期限却有着显著的不同。这显然无法单纯用患者的年龄、身体状况和环境因素等来解释。因此,多数学者认为肿瘤的复发、转移是直接影响结肠癌患者预后的主要因素。如果可以对肿瘤细胞的浸润范围和能力进行严密的测定和监视,就可以有效地预防肿瘤局部复发,同样,如果能够对肿瘤的淋巴转移情况甚至淋巴微转移的情况进行监测和控制,则可以有效地预防和治疗术中、术后肿瘤转移的发生。近年来恶性肿瘤微转移的研究成为热门课题,微转移主要是指一种肿瘤状态:使用常规的临床及病理诊断方法无法检测出来,但却保持了肿瘤细胞的生物学活性,具有增殖分化潜能,最终可以使肿瘤发生转移或复发,而且这种状态不能单纯用转移灶大小、部位等来定义。微转移涵盖微小的浸润、血行微转移、淋巴微转移和骨髓微转移等多个方面。在外科根治性手术已经进行的前提下,临床可见的肿瘤组织已经基本清除,常规病理学检查检测不到的微转移就成为影响肿瘤患者预后的重要因素。例如对于临床诊断为Dukes A、B期的结肠癌患者,因常规病理检查无淋巴结转移,在治疗规范中没有对这部分患者做出明确的术后辅助放疗、化疗的要求,如果忽略了可能已经发生的微转移的存在,就可能延误治疗,造成患者生存期限的差异。因此,微转移目前被认作是准确临床病理分期的必要补充,是指导采取何种治疗方式的关键。细胞因子失衡被认为是恶性肿瘤生长、浸润、转移过程中必要的条件之一,近年来针对细胞因子与恶性肿瘤关系的研究逐渐增多。比较明确的有肿瘤坏死因子家族、白介素家族、生长因子家族和干扰素家族等。肿瘤坏死因子-α(tumor necrosis factor,TNF-α)是与恶性肿瘤关系密切的一种重要的细胞因子,主要由单核—巨噬细胞系统分泌,近年来的研究表明肿瘤细胞也可以产生,它不仅参与机体的免疫反应,还参与肿瘤的发生和发展过程,对肿瘤的进展和转移有着重要影响。细胞角蛋白(cytokeratins, CKs)是一类多基因编码的非水溶性纤维性多肽,存在于上皮细胞的细胞质内,包括20多种亚型,其表达方式反映了上皮细胞分化的不同途径,其中部分亚型的单克隆抗体能识别特异表型的腺癌。癌细胞大多保留起源细胞的CKs类型,由此便可通过测定CKs来决定癌细胞的起源。如CK20具有严格组织特异性,局限于单层上皮及其起源的肿瘤细胞中,不仅在胃肠道、胰腺和胆管等原发部位的腺癌中表达,而且在转移它处后如肝、脊柱和脑转移性腺癌中仍保持其阳性表达特征,因此常作为上述肿瘤的标志物进行检测。本次试验选取与恶性肿瘤关系密切的TNF-α和CK20两种指标,通过对结肠癌患者癌组织、癌旁组织、正常组织和淋巴结中TNF-αmRNA和CK20 mRNA表达情况的检测,试图揭示二者与结肠癌浸润和淋巴转移之间的关系,判断结肠癌的术后复发、亚临床转移发生的可能。提高对结肠癌患者预后的估计,指导结肠癌的临床治疗,并为研究淋巴微转移机制和利用TNF-α抑制结肠癌浸润和淋巴转移等提供一定的帮助。另外,使用Matrigel法研究参芪扶正注射液对结肠癌细胞系侵袭能力的影响、对TNF-αmRNA表达的影响等,为利用祖国传统医学治疗结肠癌提供了理论依据。第一部分TNF-α、CK20在结肠癌浸润过程中表达的研究目的:研究肿瘤坏死因子α(TNF-α)和细胞角蛋白20(CK20)在结肠癌患者组织中的表达和相关性及与肿瘤生物学行为的关系。方法:选取手术切除并经病理证实为结肠癌患者30例,采用RT-PCR法,检测结肠癌患者癌组织、癌旁组织、切缘组织中TNF-αmRNA和CK20 mRNA的表达情况。结果:三种组织中TNF-αmRNA表达阳性率分别为70.0%、43.3%、20.0%,三者比较差异有统计学意义(P<0.01);CK20 mRNA表达阳性率分别为63.3%、33.3%、16.7%,差异有统计学意义(P<0.01),但在癌旁和切缘组织之间表达差异无统计学意义(P>0.05)。TNF-αmRNA和CK20 mRNA的表达之间具有良好的相关性。TNF-αmRNA和CK20 mRNA与结肠癌分化程度无关(P>0.05),但TNF-αmRNA与结肠癌Dukes分期、侵及肠壁范围密切相关(P<0.05)。结论:TNF-αmRNA可以作为反映结肠癌的浸润能力的客观指标。第二部分TNF-α、CK20在结肠癌淋巴微转移中表达的研究目的:研究肿瘤坏死因子α(TNF-α)mRNA和细胞角蛋白20(CK20)mRNA在结肠癌患者淋巴结中的表达情况,探讨二者与结肠癌淋巴微转移的关系。方法:选取手术切除并经病理证实的30例结肠癌患者,收集淋巴结共计398枚,采用连续切片和RT-PCR法,检测淋巴结中微转移灶的存在情况和TNF-αmRNA和CK20 mRNA的表达情况。结果:连续切片结果显示,在常规病理无转移的淋巴结中有20.2%的淋巴结存在微转移,TNF-αmRNA和CK20 mRNA的表达与连续切片的检查结果基本吻合,三者之间具有良好的相关性。TNF-αmRNA和CK20 mRNA的表达随Dukes分期增加而增高,P<0.05。在转移阳性淋巴结中表达高于转移阴性淋巴结,P<0.01。结论:TNF-αmRNA和CK20 mRNA均可以准确地反映结肠癌淋巴微转移的情况,TNF-α具有和CK20一样的反映结肠癌淋巴微转移情况的能力,可能在结肠癌淋巴微转移的过程中起重要作用。第三部分参芪扶正注射液对结肠癌侵袭能力的影响目的:研究参芪扶正注射液对结肠癌细胞系侵袭能力的作用及对TNF-αmRNA、CK20 mRNA表达的影响。方法:用Matrigel法检测不同浓度的参芪扶正注射液对体外培养结肠癌细胞株Caco-2和LS-174T细胞侵袭能力的影响;用RT-PCR法检测不同参芪扶正注射液的浓度状态下结肠癌细胞株TNF-αmRNA、CK20 mRNA的表达情况。结果:在不同浓度的参芪扶正注射液作用下,CK20 mRNA的表达情况在各组之间无明显差异,对照组TNF-αmRNA的表达明显高于用药组,低浓度组明显高于高浓度组。在浓度为10%时,两组细胞系的穿膜细胞数最低,各组间差异有统计学意义。结论:参芪扶正注射液对结肠癌细胞表达TNF-αmRNA的能力有很大影响,不同浓度的参芪扶正注射液可以对结肠癌细胞的侵袭能力产生强弱不同的抑制作用,随着浓度的加大,TNF-αmRNA的表达逐渐减弱,抑制作用逐渐增强。更多还原

【Abstract】 Issue one Expression of tumor necrosis factor-αand cytokeratin 20 the process of invasion of colon cancerObjective: To investigate the expression and the relevance of tumor necrosis factor-α(TNF-α) mRNA and cytokeratin20(CK20) mRNA in the tissues of patients with colon cancer and the relation between the expression and the oncobiological behavior.Methods: Surgically resected and pathologically confirmed 30 cases of colon cancer patients were internalized, RT-PCR technique was used to detect the expression of TNF-αmRNA and CK20 mRNA in the tumor, para-tumor and incisal margin tissues.Resul: The positive rates of TNF-αmRNA expressions in the three groups were 70.0% and 43.3%, 20.0% respectively with a Statistical significant difference (P<0.01), and the positive rates of CK20 mRNA expression were 63.3%, 33.3% and 16.7% also with a Statistical significant difference (P<0.01), but there was no Statistical significant difference between the para-tumor and incisal margin groups (P>0.05). TNF-αmRNA and the CK20 mRNA expression were deeply correlated. TNF-αmRNA and CK20 mRNA was foreign to the differentiation of colon cancer (P>0.05), but TNF-αmRNA is closely related to colon cancer Dukes stages and the invasion extence in the intestinal wall (P<0.01).Conclusion: TNF-αmRNA can be used to speculate the invasional capacity of colon cancer as an objective indicator. Issue two Expression of TNF-αand CK20 in the lymphatic micro-metastasis in colon cancerObjective: To investigate the expressions of TNF-αmRNA and CK20 mRNA in the lymph nodes of patients with colon cancer and to discuss their relations to lymphatic micro-metastasis of colon cancer.Methods: A total of 398 lymph nodes were collected from surgically resected and pathologically confirmed 30 cases of colon cancer patients to be serially sliced and detected of lymphatic micro-metastasis as well as expression of TNF-αmRNA and CK20 mRNA through RT-PCR technique.Results: 20.2 percent of non-lymphatic node metastases excepted by conventional pathologic examination has been found lymph node metastases by serial sections examination. The result of expression of TNF-αmRNA and CK20 mRNA were consistent on the whole to that of serial sections examination and the three presented a close correlation.the expression of TNF-αmRNA and CK20 mRNA were increased positively to the Dukes stages (P<0.05). The expression TNF-αmRNA and CK20 mRNA in positively transfered lymph nodes was superior to that of negative ones (P<0.01).Conclusion: TNF-αmRNA and CK20 mRNA can accurately reflect lymphatic micro-metastases; uniform to CK20, TNF-αmRNA can also reflect the lymphatic micro-transfered capacity and probably plays an important role in the process of lymphatic micro-metastases of colon cancer.Issue three Impact of Shenqi Fuzheng injection to capacity of invasion of colon cancerObjective: To investigate the impact of Shenqi Fuzheng injection to the colon cancer cellular capacity of invasion and to expression of TNF-αmRNA, CK20 mRNA in colon cancer cell lines.Methods: Colon cancer cell lines Caco-2 and LS-174T were cultured in vitro. After adding different concentrations of Shenqi Fuzheng injection, RT-PCR method was used to detect TNF-αmRNA, CK20 mRNA the expression of control group as well as the three Shenqi Fuzheng injection groups of different concentration. Impact of different concentration of Shenqi Fuzheng injection to the ability of cell migration of was detected by Matrigel.Results: The expression of CK20 mRNA was no significant difference among the groups of different concentration of Shenqi Fuzheng injection. To the expression of TNF-αmRNA, the control group was significantly lower than that of drug groups and higher concentration group significantly lower than inferior concentration one (P<0.01).Conclusion: Shenqi Fuzheng injection has great influence on the ability of colon cancer cells’expression of TNF-αmRNA. Different concentration of Shenqi Fuzheng injection can inhibit the abilities of movement or invasion of colon cancer cells in different extents, the inhibit ability is gradually strengthened and the TNF-αmRNA expression is gradually weaker with the concentration increased.更多还原