论文总字数：24426字

摘 要

结肠癌(colon cancer, CC)是一种目前常见的消化系统恶性肿瘤，临床表现主要包括黏液便或黏液脓性血便、肿瘤溃烂、低位肠梗阻、黄疸、腹腔积液、水肿等，是目前致死率第二高的癌症相关疾病。结肠癌高危险性的原因主要在于治疗后的复发、转移，其转移后治疗难度增大，致死率明显升高。癌干细胞(Cancer Stem Cells, CSCs)是癌细胞中一小部分具有自我更新、无限增殖和多向分化潜能的细胞，结肠癌干细胞(colon cancer stem cells, CCSCs)是CC发生、发展、转移、复发的根源，也是结肠癌根治的重点。但由于CCSCs的高活性、特殊免疫原性及强耐药机制，常规治疗方法对其效果不佳。免疫治疗具有精确、灵敏、高杀伤性的特点，被认为是进行靶向CCSCs治疗CC的最佳手段之一。但由于CCSCs表面抗原组成复杂，目前尚不明确CCSCs制备的疫苗的作用机制，以及是否能诱导机体产生比非CCSCs疫苗有更效免疫反应，进而发挥抗肿瘤作用，为此我们开展了CCSCs疫苗的动物实验。

为了制备CCSCs疫苗，本研究以CD133为标记，从鼠源性CT26结肠癌细胞系中分选出CCSCs，并以BalB/c小鼠为实验动物，构建了不同种类结肠癌细胞的疫苗免疫模型。通过对比其肿瘤抑制效果、体内免疫细胞杀伤性和细胞因子表达水平推测疫苗的作用机制。

目的：

初步探讨鼠源性CCSCs疫苗在体内的免疫效应及机制，为CCSCs疫苗的临床实验提供参考信息。

方法：

1. 结肠癌干细胞的分选

以CD133为标记物，通过免疫磁珠分选法从鼠源结肠癌细胞系中分选出结肠癌干细胞

1. 体外结肠癌干细胞生物学性质的研究

以Non-CSC和CT26细胞为对照组，研究结肠癌干细胞的增殖、迁移、侵袭、克隆形成方面的生物学性质

1. 结肠癌干细胞疫苗效果的研究

以BalB/c小鼠为实验动物，对比Non-CSC疫苗、CT26疫苗、CSC疫苗免疫后引发的抗肿瘤能力的差别。

1. 结肠癌干细胞疫苗作用机制的研究

对比不同种疫苗免疫效果好坏和不同免疫小鼠脾NK细胞杀伤性强弱；

IFN-γ、TGF-β含量多少，初步探讨CCSCs疫苗的作用机制。

结果：

1. 培养获得大量鼠源结肠癌CT26细胞并分选出结肠癌干细胞
2. 研究发现，与Non-CSC、CT26细胞相比，CSC的增殖能力、迁移能力、侵袭能力、克隆形成能力有远强于前两者。
3. 比对发现，CSC疫苗具有更好的抗肿瘤效果，经CSC疫苗免疫的小鼠肿瘤发生情况更轻，其效果包括肿瘤发生最晚、肿瘤生长速度最慢、成瘤大小最小，差异具有显著性统计学意义(Plt;0.05)
4. 对比CSC疫苗免疫组、CT26细胞疫苗免疫组、Non-CSC疫苗免疫组小鼠体内脾NK细胞杀伤性，血清中IFN-γ、TGF-β含量，总结得出CSC疫苗在抗肿瘤过程中会激发脾NK细胞的杀伤性，刺激IFN-γ的表达，抑制TGF-β的表达。

结论：

结肠癌干细胞疫苗具有较好的抗肿瘤能力，在本实验中探明的作用机制包括：激活和增加脾NK细胞的活性与杀伤能力；促进IFN-γ的分泌，增加其含量，以此增强机体免疫效应，辅助激活体内多种免疫机制；抑制TGF-β的表达，降低其含量，从而减低TGF-β对免疫系统的抑制，使体内免疫系统保持对结肠癌细胞转移、侵袭的抑制作用，有效减少结肠癌干细胞的侵袭转移。

关键词：结肠癌；癌干细胞；癌干细胞疫苗；免疫治疗Study of colon cancer stem cells vaccine’s mechanism of action in mice

No.41112115 Student: Xu Ao

Supervisor: Jun Dou

Laboratory of Pathogen Biology and Immunology, Ministry of Education Southeast University

Abstract

Colon cancer (CC) is a common gastrointestinal cancer, clinical manifestations include mucous or purulent mucus blood, tumor ulceration, low intestinal obstruction, jaundice, abdominal cavity effusion, edema and so on, which is currently the second high fatality rate of cancer related diseases. The cause of the high risk of colon cancer mainly lies in the recurrence and metastasis after treatment, treatment is harder after the metastasis, and at the same time the fatality rate increased significantly. Cancer stem cells (CSCs) are cancer cells in a small number which has particularity include self-renewal, unlimited proliferation and multi-directional differentiation potentiality, colon cancer stem cells (CCSCs) is the root cause of the occurrence, development, metastasis and recurrence of colon cancer, and is the key point of colon cancer’s complete treatment, too. But as a result of CCSCs’ highly active, special immunogenicity and strong resistance mechanism, conventional treatment’s effect is not fine. Immunotherapy has the characteristics of accurate, sensitive, high destruction, which is considered to be one of the best means of CCSCs’ therapy. However, because of the complex composition of CCSCs’ surface antigen, anti-tumor mechanisms of CCSCs’ vaccine is not yet clear in detail, and as a result its experimental vaccine is still limited to animal experiment.

To further understand CCSCs’ vaccine’s mechanism of action, this research isolate colon cancer steam cells from CT26 cancer cell lines marked with CD133, and construct vaccine model of different kinds of colon cancer cells with the experiment body of BalB/c mice. Presume mechanism of the vaccine by comparing tumor inhibitory effect, immune cell destruction, expression level of cytokines.

Objective

Preliminary discussion on colon cancer stem cell vaccine immune mechanisms in the body, provide the reference information for vaccine’s clinical trial.

Methods and Contents

1. Colon cancer stem cells’ sorting

Marked with CD133,isolate colon cancer steam cells from mice source cancer cell lines by magnetic bead separation method.

1. Study of colon cancer stem cells’ biological characteristics in vitro

Study the biological characteristics of colon cancer stem cells compared with Non-CSC and CT26 cells.

3.Study of the effect of colon cancer stem cells’ vaccine

BalB/c mice as experimental animals, compared tumor resistance difference after immunity by Non - CSC vaccine, CT26 vaccine and CSC vaccine.

1. Study of colon cancer stem cells vaccine’s mechanism of action

Comparing difference between different kinds of vaccines’ affection and different immune cells’ activity, cytokine content, preliminary discussion on the mechanism of action of CCSCs vaccine and treatment strategies for colon cancer stem cells.

Result

1. Cultivate a large number of mice sourced colon cancer cells.
2. Separate colon cancer stem cells from colon cancer cells
3. As the result of comparison ,CSC vaccine has better antitumor effect .Status of tumor of mice immunized by CSC vaccine is lighter, including the latest tumor appearance, the slowest tumor growth, the smallest tumor volume, whose difference is statistically significant(Plt;0.05)
4. Comparing the affection to NK cell, IFN-γ and TGF-β by colon cancer stem cells vaccine

Conclusions:

Colon cancer stem cell vaccine has better anti-tumor ability, its mechanism of action known from this research include :stimulate activity of NK cells, make NK cells have more destruction; promote the secretion of IFN-γ, increase the content of IFN-γ, enhance the immune effect, assist to activate a variety of immune mechanism; inhibit the expression of TGF-β,reduce the content of TGF-βto avoid TGF-β’s inhibition of the immune system, and restrain colon cancer stem cell’s invasion and metastasis.

Key words: Colon cancer; Cancer stem cell; Cancer stem cell vaccine; Immunotherapy目录

第一章 绪论 8

1.1研究背景 8

1.2研究目的和内容 8

1.3技术路线图 9

1.4结肠癌干细胞概述 9

1.5结肠癌治疗现状 10

1.6结肠癌干细胞疫苗研究现状 11

第二章 材料和方法 12

2.1实验材料和试剂 12

2.2实验方法 15

第三章 实验结果与讨论 21

3.1细胞增殖实验 21

3.2细胞迁移实验 21

3.3 Transwell侵袭实验 22

3.4软琼脂克隆实验 23

3.5 以CD133为标记筛选出的结肠癌CSC的鉴定 24

3.6 CT26各组细胞疫苗免疫鼠致瘤后的抗肿瘤效应 24

3.7 CT26细胞攻击后肿瘤的生长情况 25

3.8免疫鼠受CT26细胞攻击后最终成瘤情况 25

3.9 CT26细胞攻击瘤苗免疫鼠后脾NK细胞杀伤活力检测 26

3.10 瘤苗免疫组血清细胞因子IFN-γ和TGF-β水平检测 27

3.11讨论 27

参考文献 29

致谢 32

第一章 绪论

1.1研究背景

结肠癌(colon cancer,CC)是恶性肿瘤疾病的一种，目前世界范围内最常见的消化系统恶性肿瘤之一，同时其致死率在与癌症有关联的疾病中高居第二^[1]，是目前对人类生命健康威胁最大的癌症之一。结肠癌致死率极高的原因，主要在于其愈后极易复发^[2-4]、极易转移的特性，以及随着其癌细胞远端转移而逐步增加的恶化情况、逐步提高的治愈难度和逐步降低的存活率^[5-6]。随着近些年对肿瘤的研究不断深入，目前普遍认为在肿瘤细胞中占极小比例的肿瘤干细胞(cancer stem cells, CSCs)是肿瘤发生、发展的源头，同时其免疫逃避能力与高度的细胞活性是癌症变异、复发重要原因^[7]。随着结肠癌细胞系中结肠癌干细胞(colon cancer stem cells, CCSCs)存在的确认，以结肠癌干细胞为入手点治疗结肠癌成为了当今结肠癌治疗的热点。

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