【作者】 李晓莉；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 研究背景关于肿瘤的发生、发展、侵袭、转移和治疗一直是医学研究的热点。传统观点认为,肿瘤由成熟细胞突变而来,是由均一的肿瘤细胞共同增殖构成的,所有肿瘤细胞都有无限增殖能力。因此临床上常以肿瘤体积是否缩小或消失作为衡量肿瘤疗效的一个标准.但临床上许多肿瘤患者,在手术切除肿瘤或经过其他治疗,病情已获稳定一段时间后,却又出现肿瘤的复发或转移,不能获得彻底治愈。新近研究发现:肿瘤组织中存在极少量干细胞样亚群,它具有无限增殖的潜能,在启动肿瘤形成和生长中起着决定性的作用,而其余的大多数细胞,经过短暂的分化,最终死亡。肿瘤生长是肿瘤组织中极少量具有特殊表面标志的肿瘤干细胞增殖的结果。这提示肿瘤治疗应针对在肿瘤发生、发展中起决定性作用的肿瘤干细胞,而不是大部分无增殖、分化能力的肿瘤实体细胞。这可能促进肿瘤治疗学发生革命性改变。家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)是常见的遗传性大肠肿瘤之一,约占总大肠肿瘤的1%,可分为经典型家族性腺瘤性息肉病(Classical FAP,CFAP),衰减型家族性腺瘤性息肉病(Attenuated FAP,AFAP)和MYH相关性息肉病(MYH associatedFAP,MAP)三种,其临床表现为青春发育期发生结直肠腺瘤,后逐渐增多,甚至满布所有结直肠粘膜,如不及时干预治疗,将发生癌变。肿瘤干细胞理论提示FAP可能也来源于腺瘤干细胞(Adenomastem cells)。分离、培养、鉴定腺瘤干细胞并研究其性质具有重要意义。肿瘤干细胞研究的难点在于确定其特异性表面标志或表面标志的组合方案。应用有针对性的抗原去检测细胞亚群的表面标志将节省大量的时间和研究经费,并有望形成高效的分选方案。我们通过一种新的干细胞标记物微管相关蛋白DCAMKL-1来鉴定FAP腺瘤干细胞,并通过研究干细胞中某些蛋白的表达,研究腺瘤发生发展及转归的机制。第一章家族性腺瘤性息肉病腺瘤干细胞的鉴定、分离、培养以及相关蛋白的研究目的:通过一种新的胃肠干细胞标志物DCAMKL-1对FAP患者腺瘤干细胞进行鉴定,并建立腺瘤细胞分离和培养的办法。研究PCNA、β-catenin、COX-2等蛋白在腺瘤细胞中的表达及这些蛋白与腺瘤发生发展的关系。方法:采用免疫组化的方法,对正常结肠粘膜,FAP腺瘤上皮及癌变的腺瘤上皮进行DCAMKL-1染色,观察DCAMKL-1阳性表达细胞的位置。对比MSI-1阳性表达细胞的位置,对腺瘤细胞进行Hoechst染色。观察PCNA、β-catenin、COX-2等蛋白在腺瘤细胞的表达,并探讨β-catenin、COX-2在正常结肠粘膜,FAP腺瘤上皮及癌变的腺瘤上皮中表达的相关性。结果:在正常结肠粘膜,FAP腺瘤上皮及癌变的腺瘤上皮中,DCAMKL-1阳性表达细胞主要位于结肠隐窝的底部,与用于标记肠干细胞的MSI—1阳性表达位置一致,且随着病变的发展,在腺瘤中逐渐呈现表达增多的现象。DCAMKL-1阳性表达细胞细胞核不能为Hoechst33342染色。PCNA、β-catenin、COX-2的表达均与腺瘤病变的发展相关。结论:DCAMKL-1是一种新奇的胃肠干细胞的标记物,其阳性表达的细胞可能为家族性腺瘤性息肉病及正常结肠粘膜的干细胞。对腺瘤的治疗关键在于切除腺瘤干细胞,以及抑制与腺瘤病情发展呈正相关的COX-2蛋白等。第二章高频电切腺瘤性息肉对家族性腺瘤性息肉病裸鼠病程的影响及转归目的:建立FAPmin裸鼠模型并考察高频电切治疗对FAPmin裸鼠结肠腺瘤发生、发展及转归的影响。方法:采用N-二乙基亚硝胺灌胃诱变的方法建立了FAPmin裸鼠模型;与高频电切治疗后1、3、6、9、12个月,通过免疫组织化学染色技术检测APC、Lgr5、Musashi-1及PTEN蛋白阳性表达率的变化;半定量RT-PCR技术检测APC、Lgr5、Musashi-1及PTEN mRNA表达水平的变化;原位末端转移酶标记技术检测腺瘤细胞凋亡指数的变化。结果:高频电切治疗后1、3、6、9、12个月,模型组裸鼠结肠组织中APC、Lgr5、Musashi-1蛋白及基因表达水平显著升高,而电切组裸鼠上述分子的表达水平则显著下降,两组的变化存在差异,具有统计学意义(p＜0.01);两组腺瘤细胞凋亡指数的变化存在差异,模型组腺瘤细胞凋亡指数从1月到12月的变化范围为(8.29±0.98)%～(7.76±1.34)%,对照组的变化范围为(20.08±1.21)%～(32.79±3.62)%,两组间及组内在各个时间点上存在的差异具有统计学意义(p＜0.01)。结论:采用N-二乙基亚硝胺灌胃诱变的方法能够建立FAPmin裸鼠模型;经高频电切治疗FAP,裸鼠的病情得到了控制,并且随着治疗后时间的延长,FAPmin裸鼠能够逐渐恢复健康。第三章舒林酸联合内镜下高频电切治疗家族性腺瘤性息肉病的临床研究目的:探讨舒林酸联合内镜下高频电切治疗家族性腺瘤性息肉病的新方法及临床疗效。方法:20例经结肠镜检查和组织病理学检查确诊的家族性腺瘤性息肉病患者,随机分为两组,每组10例:舒林酸联合内镜组、舒林酸组。舒林酸联合内镜组患者,行内镜下高频电凝、电切及热活检钳治疗结束后即口服舒林酸400 mg/d,疗程为12个月;舒林酸组患者,仅口服舒林酸400 mg/d,疗程为12个月。两组患者均为每4个月结肠镜复查一次,观察腺瘤的数目、类型、异型增生程度以及治疗后腺瘤细胞凋亡指数变化。结果:舒林酸联合内镜组于治疗后4、8、12个月腺瘤消退率分别为90.96%、95.66%和98.81%,而舒林酸组分别为80.08%、85.35%和89.78%,两组比较差异有统计学意义(p＜0.05)。舒林酸联合内镜组患者,经治疗后腺瘤Ⅲ级异型增生程度明显降低。舒林酸联合内镜组于治疗后4、8、12个月腺瘤细胞凋亡指数分别为(20.08±1.21)%、(25.67±2.07)%、(32.79±3.62)%,舒林酸组分别为(13.29±0.98)%、(19.83±1.56)%、(25.18±2.31)%,两组比较差异有统计学意义(p＜0.05)。结论:通过对以上两种方法的疗效比较可知,舒林酸联合内镜下高频电切治疗家族性腺瘤性息肉病是有效的,且优于单纯服用舒林酸的传统治疗方法。更多还原

【Abstract】 BACKGROUDIt is always a focus in research on the carcinogenesis,development, invasion,metastasis and therapeutics of cancer.Generally it has been believed that tumor cells are mutated from mature cells and have homogeneous potential of proliferation.All tumor cells have the abilities to proliferate infinitely.Accordingly,it is an accepted standard for the curative effect in clinical tumor therapy that tumor volume could grown downwards or even vanish.However,some tumors recurred or even distantly metastasized and could not been cured completely after tumor removal by surgery or some others treatment and had been stable for some periods.Recent researches had discovered that there were some stem-like subpopulations in tumor tissues which had the potential to proliferate infinitely and played much crucial role in initiating carcinogenesis and develoment,while the other cells would die after transitory differentiation. It was thought that the tumor growth resulted from the proliferation of some tumor stem cells with special surface markers.For this reason,the tumor treatment must aim directly at the tumor stem cells but not at the bulk of tumor cells with little ability to proliferate and differentiate.This theory might bring about some revolutionary in tumor therapeutics.Familial adenomatous polyposis(FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer and characterized by the occurrence of hundreds to thousands of colorectal polys.FAP accounts for about 1%of total colorectal cancers and was classified into three types,one is classical FAP(CFAP),the other two are attenuated FAP(AFAP) and MYH associated FAP(MAP).Under the guide of this theory,we believed that FAP possibly also originated from the adenoma stem cells.There were be great importance to study on identification,isolation,culture and the basic characteristics ofthem.However,the key point of the research on adenoma stem cells was how to establish the special surface marker of them.However,it was hope to achieve an efficient sorting scheme of adenoma stem cells by detecting the surface markers with some directing antigens,which would save a lots of time and research funds. ChapterⅠIdentification、Isolation and Cultivation of FAP stem cells by a novel stem cell marker DCAMKL-LObjective:Identification、Isolation and Cultivation of FAP stem cells by a novel stem cell marker DCAMKL-1.Some proteins that expressed in adenoma such as PCNA、β-catenin and COX-2 were studied.And the relationship between the proteins and development of FAP was studied.Methods:Immunohistochemistry was used to identify DCAMKL-1、PCNA、β-catenin and COX-2 expression in normal epithelium、FAP crypts and cancer cell.To study the effects of PCNA、β-catenin、COX-2 on the development of FAP.The cell position that expressed DCAMKL-1 was compared with the cell position 4 that express MSI-1.The cell expressed DCAMKL-1 was stained Hoechst.Results:In normal intestine、FAP intestine and malignant adenoma, the DCAMK-1 was immunoreactive in the cells in the cell position 4 that was considered as a stem cell position.And it showed a trend toward increaseed expression on villi.The nuclei of the cells positive for DCAMK-L could not been stained with Hoechst 33342.Conclusion:DCAMKL-1 is a noval putative stem cell marker.The cell expressed DCAMKL-1 could be gastrointestinal stem cell and adenoma stem cell.The key point to treat the FAP is to cut the adenoma especially the adenoma stem cell.And and selective inhibition of COX-2 is expected to be an effective target point for preventing and curing colorectal cancers and FAP. ChapterⅡClinical study on familial adenomatous polyposis treated by high frequency electric cutting under endoscope in the BALB/c mice modelsObjective:The goal of this study is to prepare the BALB/c mice models with FAP and investigate the therapeutic effects of high frequency electric cutting to FAP.Methods:The BALB/c mice models with FAP were prepared by intragastric administration of N-Nitroso-diethylamine.1 month,3 months, 6 months,9 months and 12 months after being treated by high frequency electric cutting,the expression levels of APC,Lgr5,Musashi-1 and PTEN in the colon tissues of the FAPmin mice have been detected by immunohistochemical assay and semi-quantitive RT-PCR analysis. Additionally,In situ terminal transferase labeling technique has been used to detect the apoptosis index of adenoma cells in different groups.Results:1 month,3 months,6 months,9 months and 12 months after being treated by high frequency electric cutting,the expression levels of APC,Lgr5,Musashi-1 in model group have been increased, while that decreased in treatment group.The differences between these two groups have statistic significance(p<0.01).Moreover,the apoptosis index of adenoma cells in treatment group also increased from (20.08±1.21)%to(32.79±3.62)%,while in model group decreased from (8.29±0.98)%to(7.76±1.34)%.The differences between these two groups have statistic significance(p<0.01)Conclusion:These results indicate that the intragastric administration of N-Nitroso- diethylamine can prepare the BALB/c mice models with FAP successfully.The treatment of high frequency electric cutting on FAP is effective in the reduction of numbers and atypia degree of adenomas,which is the experimental basis of clinical application in the future. ChapterⅢClinical study on familial adenomatous polyposis treated by high frequency electric cutting under endoscope combined with sulindacObjective:To investigate the therapeutic effects of sulindac combined with endoscopy on familial adenomatous polyposis.Methods:20 patients diagnosed as familial adenomatous polyposis were randomly divided into two groups.The sulindac+endoscopy group took 400 mg of sulindac daily after endoscopy treatment;the sulindac group took 400 mg of sulindac daily only.In both group,the changes of number,histopathological subtype and degree of atypia of adenomas and apoptotic index(AI) in adenomas cells were compared before and after treatment.Results:In the sulindac+ endoscopy group,the elimination rates in the adenomas after the treatment in 4,8,12 months were 90.96%,95.66 and 98.81%;in the sulindac group were 80.08%,85.35%and 89.78%, respectively,the difference was statistically significant(p＜0.05).In the sulindac+ endoscopy group,the percentages of the degreeⅢof atypia of adenomas after the treatment decreased dramatically.AI in adenomas cells in the sulindac+ endoscopy group after treatment in 4,8,12 months were(20.08±1.21)%,(25.67±2.07)%and(32.79±3.62)%;in the sulindac group,they are(13.29±0.98)%,(19.83±1.56)%and(25.18±2.31)%, respectively,the difference was statistically significant(p＜0.05).Conclusion:The treatment of sulindac combined with endoscopy on familial adenomatous polyposis is effective in the reduction of numbers and atypia degree of adenomas in patients.更多还原