【作者】 庄大勇；

【导师】 胡三元；

【作者基本信息】 山东大学 ， 外科学， 2008， 博士

【摘要】 第一部分大肠癌患者外周血微转移检测的临床意义目的大肠癌是最常见的恶性肿瘤之一,血行转移是大肠癌根治性手术失败的主要原因之一,也是患者死亡的主要原因。在接受了根治术的患者中,仍有超过50%的患者最终死于肿瘤的复发和转移,提示手术时可能已有大肠癌微转移的存在。本研究以CK20 mRNA作为外周血中大肠癌细胞存在的特异性标志物,探讨了大肠癌患者外周血微转移检测的临床意义,手术操作和外周血微转移的关系,以及大肠癌组织中微血管密度（microvessel density,MVD）和微淋巴管密度（lymphatic microvessel density,LMVD）与大肠癌外周血微转移的关系。方法以CK20 mRNA作为外周血中大肠癌细胞存在的特异性标志物,采用RT-PCR技术检测大肠癌患者术前和术中外周血微转移;采用免疫组织化学方法检测大肠癌组织中MVD和LMVD;分析大肠癌患者外周血微转移、大肠癌组织中MVD和LMVD与临床病理参数的关系以及大肠癌组织中MVD和LMVD与外周血微转移的关系。结果51例大肠癌患者中18例手术前外周血CK 20mRNA呈阳性表达,总阳性表达率为35.29%（18/51）,在Ⅰ～Ⅳ期患者中的阳性表达率分别为12.50%（1/8）、23.53%（4/17）、35.29%（6/17）和77.78%（7/9）,CK20 mRNA在外周血中的阳性表达与TNM分期相关（P＜0.05）;手术中有21例呈阳性表达（21/51）,3例术前阴性表达者术中转为阳性表达,均为直肠癌手术患者;CK20 mRNA在外周血中的阳性表达与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值、肿瘤分化程度、浸润深度无关（P＞0.05）,但与淋巴结转移、远处转移、TNM分期相关（P＜0.05）;MVD与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值无关（P＞0.05）,但与肿瘤分化程度、浸润深度、淋巴结转移、远处转移、TNM分期相关（P＜0.05）;LMVD与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值、肿瘤分化程度、浸润深度无关（P＞0.05）,但与淋巴结转移、远处转移、TNM分期相关（P＜0.01）;大肠癌患者外周血CK20 mRNA阳性表达组MVD和LMVD的值分别为58.83±18.28和9.72±1.64,而外周血CK20mRNA阴性表达组MVD和LMVD的值分别为30.48±9.31和8.21±1.87,显著低于阳性表达组（P＜0.01）。结论大肠癌细胞外周血微转移是大肠癌的早期事件,外周血微转移发生率随临床病理分期的增加而升高,外周血微转移与淋巴结转移、远处转移、TNM分期相关,是反映大肠癌生物学行为的指标之一;手术操作有可能促进直肠癌患者外周血微转移的发生;MVD与肿瘤分化程度、浸润深度、淋巴结转移、远处转移、TNM分期相关;LMVD与淋巴结转移、远处转移、TNM分期相关;大肠癌组织血管化程度和淋巴管生成与外周血微转移密切相关,通过对二者的检测有助于判断外周血微转移发生的情况。第二部分Tiam1、DcR3在大肠癌中表达的临床意义及与大肠癌外周血微转移的关系目的侵袭和转移是恶性肿瘤的重要特征,也是肿瘤患者死亡的主要原因,因此对肿瘤的侵袭和转移及其相关基因的研究具有重要意义。T淋巴瘤侵袭转移诱导因子1（T lymphoma invasion and metastasis factor 1,Tiam1）与肿瘤侵袭和转移密切相关。DcR3（decoy receptor 3）是肿瘤坏死因子受体超家族成员,在多种人类恶性肿瘤中出现过度表达,且DcR3的表达状态与恶性肿瘤的淋巴结转移和病理分期密切相关。关于Tiam1和DcR3与大肠癌外周血微转移的研究报道不多,我们通过检测Tiam1和DcR 3在大肠癌组织和正常大肠粘膜组织中的表达,探讨了Tiam1和DcR3表达与大肠癌临床病理参数的关系以及二者与大肠癌外周血微转移的关系。方法以CK20 mRNA作为外周血中大肠癌细胞存在的特异性标志物,采用RT-PCR方法检测大肠癌患者外周血微转移;采用实时荧光定量RT-PCR方法检测大肠癌组织和正常组织中Tiam1和DcR3的表达;分析大肠癌组织中Tiam1和DcR3表达与临床病理参数以及患者外周血微转移的关系。结果51例大肠癌组织及正常粘膜组织中均见Tiam1 mRNA表达,表达水平分别为（20.61±4.99）×10^-4和（3.49±2.74）×10^-4,大肠癌组织中Tiam1mRNA表达明显高于正常组织（P＜0.01）;Tiam1 mRNA在伴有外周血微转移的大肠癌组织中的表达水平为（72.11±2.38）×10^-4,在不伴外周血微转移的大肠癌组织中的表达水平为（15.78±5.01）×10^-4,二者差异有统计学意义（P＜0.05）;大肠癌组织中Tiam1 mRNA表达水平与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值无关（P＞0.05）,但与肿瘤分化程度、浸润深度、淋巴结转移、远处转移、TNM分期相关（P＜0.05）;51例大肠癌组织及正常粘膜组织中均见DcR3 mRNA的表达,表达水平分别为（42.40±2.83）×10^-4和（12.59±2.55）×10^-4,大肠癌组织中DcR3 mRNA表达明显高于正常组织（P＜0.01）;DcR3 mRNA在伴有外周血微转移的大肠癌组织中表达水平为（61.53±2.60）×10^-4,在不伴外周血微转移的大肠癌组织中表达水平为（34.60±2.84）×10^-4,但二者差异无统计学意义（P＞0.05）;DcR3 mRNA的表达水平与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值无关（P＞0.05）,但与肿瘤分化程度、浸润深度、淋巴结转移、远处转移、TNM分期相关（P＜0.05）。结论大肠癌组织的Tiam1 mRNA和DcR3 mRNA高表达与大肠癌的发生相关;大肠癌组织的Tiam1 mRNA和DcR3 mRNA表达水平与肿瘤分化程度、浸润深度、淋巴结转移、远处转移、TNM分期相关;Tiam1 mRNA高表达是大肠癌患者发生外周血微转移的高危因素;但DcR3 mRNA高表达不能作为大肠癌患者发生外周血微转移的高危因素。第三部分Kallikrein6在大肠癌中表达的临床意义及与大肠癌外周血微转移的关系目的激肽释放酶（Kallikreins,KLK）是一组分泌型丝氨酸蛋白酶,存在于多种组织和生物体液中。人KLK基因家族参与恶性肿瘤的发生发展,多种KLK与前列腺癌、卵巢癌、乳腺癌和睾丸癌等内分泌相关肿瘤的发生发展密切相关,是此类肿瘤的潜在标志物。KLK6异常表达与多种肿瘤相关。国内目前未见有关KLK6和大肠癌关系的报道。我们通过检测KLK6 mRNA在大肠癌组织和正常大肠粘膜组织中的表达,探讨了KLK6表达与大肠癌临床病理资料的关系及其与外周血微转移的关系。方法以CK20 mRNA作为外周血中大肠癌细胞存在的特异性标志物,采用RT-PCR方法检测大肠癌患者外周血微转移;采用实时荧光定量RT-PCR方法检测大肠癌组织和正常组织中KLK6的表达;分析大肠癌组织中KLK6表达与临床病理参数以及患者外周血微转移的关系。结果51例大肠癌组织及正常粘膜组织中均见KLK6 mRNA的表达。大肠癌组织中和正常粘膜组织中KLK6 mRNA表达水平分别为（33.11±2.34）×10^-4和（12.88±2.51）×10^-4,有高度统计学差异（P＜0.01）;伴有外周血微转移的大肠癌组织中和不伴外周血微转移的大肠癌组织中KLK6 mRNA的表达水平分别为（40.64±2.05）×10^-4和（30.06±2.45）×10^-4,差异无统计学意义（P＞0.05）;大肠癌组织的KLK6 mRNA表达与患者的性别、年龄、肿瘤原发部位、肿瘤大小、外周血CEA值、肿瘤分化程度、浸润深度、远处转移无关（P＞0.05）,但与淋巴结转移、TNM分期相关（P＜0.05）。结论大肠癌组织的KLK6 mRNA高表达与大肠癌的发生相关;大肠癌组织的KLK6 mRNA表达水平与淋巴结转移、TNM分期相关,与外周血微转移不相关,大肠癌组织的KLK6 mRNA的高表达不能作为大肠癌患者发生外周血微转移的高危因素。更多还原

【Abstract】 Part One Clinical significance of peripheral blood micro-metastasis in colorectal cancerObjective Peripheral blood micro-metastasis is one of the important factors for failure of surgical resection in colorectal cancer.Above 50%of the patients underwent surgical resection died of cancerous relapse or metastasis,indicating that micro-metastasis may have exited before the sugeries.CK20 mRNA has thought to be a specific biomarker for early diagnosis of micro-metastasis in colorectal cancer.To explore the role of determination of peripheral blood micro-metastasis in prognosis of colorectal cancer and to explore whether microvessel density（MVD） and lymphatic microvessel density（LMVD） have correlation with peripheral blood micro-metastasis.Methods Using CK20 mRNA as a specific biomarker of tumor cells in peripheral blood,we determined its levels before and after surgical resection with the RT-PCR technique,also we determined the MVD and LMVD levels by immunohistochemistry.Results Eighteen of the 51 patients were found positive CK20 mRNA expressions before surgical resection,of them,1（12.50%） was of the 8 patients with stageⅠ,4 （23.53%） was of the 17 patients with stageⅡ,6（35.29%） was of the 17 patients with stageⅢ,and 7（77.78%）was of the 9 patients with stageⅣ.These findings indicated that CK20 mRNA expression in peripheral blood correlated with the TNM classification in colorectal cancer.After surgical resection,3 more patients with rectum cancer were also shown positive CK20 mRNA expressions.CK20 mRNA expression had no relations to age,gender,primary location,tumor size,peripheral blood carcino-embryonic antigen level,and tumor differentiation and invasion,but had relations to lymphonode metastasis,distant metastasis and tumor TNM stage （P＜0.05）.The MVD levels had no relations to age,gender,primary location,tumor size and the peripheral blood carcino-embryonic antigen level,but had relations to tumor differentiation and invasion,lymphonode metastasis,distant metastasis,tumor TNM stage（P＜0.05）.Also the LMVD levels had no relations to age,gender,primary location,tumor size,the peripheral blood carcino-embryonic antigen level,and tumor differentiation and invasion,but had relations to lymphonode metastasis,distant metastasis,and tumor TNM stage（P＜0.05）.The MVD and LMVD levels were 58.83±18.28 and 9.72±1.64 in the positive CK20 mRNA group,however they were 30.48±9.31 and 8.21±1.87 in the negative CK20 mRNA group,the difference was statistically significant（P＜0.01 ）.Conclusions Peripheral blood micro-metastasis is related to lymphnode metastasis, distant metastatis and TNM stage.Surgical procedures may promote the genesis of periphral blood micro-metastasis.Determination of tissue vascularization and genesis of lymphatic vessel is helpful in judging genesis of peripheral blood micro-metastasis in colorectal cancer.Part Two Tiam1 and DcR3 expressions in colorectal cancer and their correlation with peripheral blood micro-metastasisObjective Invasion and metastasis are key characteristics of malignant tumors.It has been shown that T lymphoma invasion and metastasis factor 1（Tiam1） closely correlates with tumor invasion and metastasis.Decoy receptor 3（DcR3） is one member of the tumor necrosin receptor hyperfamily,and it is overexpressed in many human malignant tumors,also its overexpression closely relates to tumor lymphnode metastasis and pathologic staging.To determine the expressions of Tiam1 mRNA and DcR3 mRNA in cancerous tissues and non-cancerous tissues of colorectal cancer,and to explore their correlation with pathologic data and peripheral blood micro-metastasis.Methods Using the RT-PCR method,the CK20 mRNA expression was determined.Using fluorescence quantitative-RT-PCR（FQ-RT-PCR）,the expressions of Tiam1 mRNA and DcR3 mRNA were determined in 51 cases of colorectal carcinoma.Results The Tiam1 mRNA expression level was（20.61±4.99）×10^-4 in cancerous tissues and was（3.49±2.74）×10^-4 in non-cancerous tissues（P＜0.01）.Also it was （72.11±2.38）×10^-4 in cancerous tissues with micro-metastasis and was（15.78±5.01）×10^-4 in cancerous tissues without micro-metastasis,the difference was statistically significant（P＜0.05）.Tiam1 mRNA expression had no relations to age,gender, primary location,tumor size and the peripheral blood carcino-embryonic antigen level,but had relations to tumor differentiation and invasion,lymphnode metastasis, distant metastasis and tumor TNM stage（P＜0.05）.The DcR3 mRNA expression level was（42.40±2.83）×10^-4 in cancerous tissues and was（12.59±2.55）×10^-4 in non-cancerous tissues（P＜0.01）.Also it was（61.53±2.60）×10^-4 in cancerous tissues with micro-metastasis and was（34.60±2.84）×10^-4 in cancerous tissues without micro-metastasis,but the difference was not statistically significant（P＞0.05）.DcR3 mRNA expression had no relations to age,gender,primary location,tumor size and peripheral blood carcino-embryonic antigen level,but had relations to tumor differentiation and invasion,lymphnode metastasis,distant metastasis and tumor TNM stage（P＜0.05）.Conclusions Overexpressions of Tiam1 mRNA and DcR3 mRNA correlate with genesis of colorectal cancer.Tiam1 mRNA overexpression is a high risk factor for peripheral blood micro-metastasis in colorectal cancer,but DcR3 mRNA can not be used as a high risk factor for peripheral blood micro-metastasis in colorectal cancer. Part Three Human KaUikrein6 expression in colorectal cancer and its correlation with peripheral blood micro-metastasisObjective Kallikreins（KLKs） have a similar genomic organization and show significant homology at both the nucleotide and the protein level.Human KLK gene family has participated in genesis and development of cancers.A growing body of evidence suggests that KLKs are involved in human malignancies and that many KLKs are promising biomarkers of prostate,ovarian,breast and testicular cancers.To determine the expression of KLK6 mRNA in colorectal cancer and to explore its relationship to clinicopathologic features and peripheral blood micro-metastasis in colorectal cancer.Methods Using the RT-PCR method,the CK20 mRNA expression was determined.Using the FQ-RT-PCR method,the KLK6 mRNA expression was determined in 51 cases of colorectal carcinoma.Results The KLK6 mRNA expression level was（33.11±2.34）×10^-4 in cancerous tissues and was（12.88±2.51）×10^-4 in non-cancerous tissues（P＜0.01）.Also it was （40.64±2.05）×10^-4 in cancerous tissues with micro-metastasis and was（30.06±2.45）×10^-4 in cancerous tissues without micro-metastasis,the difference was not statistically significant（P＞0.05）.KLK6 mRNA expression had no relations to age, gender,primary location,tumor size,peripheral blood carcino-embryonic antigen level,tumor differentiation and invasion,and distant metastasis,but had relations to lymphnode metastasis and tumor TNM stage（P＜0.05）.Conclusions Overexpression of KLK6 mRNA correlates with genesis of colorectal cancer,however,it can not be used as a high risk factor of peripheral blood micro-metastasis in colorectal cancer.更多还原