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研究生:蔡介生
研究生(外文):Tsai, Chien-Sheng
論文名稱:腫瘤內的巨噬細胞接受放射線照射後的反應
論文名稱(外文):The response of Tumor-Associated-Microphage to Irradiation
指導教授:洪志宏洪志宏引用關係
指導教授(外文):Hong, Ji-Hong
學位類別:碩士
校院名稱:長庚大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:43
中文關鍵詞:放射線腫瘤內巨噬細胞
外文關鍵詞:RadiationTumor-associated macrophagesArg-IiNOSCOX-2
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在活體生長的腫瘤是由許多成份共同組成的整合體。人類或老鼠的腫瘤內,巨噬細胞是主要的組成分子。

本研究的目的是要探討腫瘤細胞及腫瘤內巨噬細胞在接受放射線照射後的反應,並說明巨噬細胞對腫瘤生長的影響。

我們使用老鼠的前列腺細胞株 (TRAMP-C1),打入C57B1/6J的老鼠大腿肌肉,待腫瘤長大後接受一次25 Gray,或是分次照射(15次,共60 Gray)的放射線。在特定的時間點上,將老鼠腿上的腫瘤切除,和同樣大小但未曾照射放射線的老鼠腫瘤比較,觀察腫瘤內巨噬細胞的數目﹑活性和其他特定的功能標誌。

不論是單次或是分次照射,腫瘤內的Arg-1﹑COX-2及iNOS的蛋白質量在照射後皆有增加,而這些反應和腫瘤內的巨噬細胞有關。

我們將巨噬細胞和TRAMP-C1細胞混合後,再一起注射入老鼠大腿肌肉,以評估巨噬細胞對腫瘤細胞生長的影響。經過放射線處理過後的巨噬細胞可促使TRAMP-C1腫瘤生長的更快更大。

綜合上述觀察,腫瘤內的巨噬細胞在經過放射線照射過後,可表現出較高的Arg-1﹑COX-2及iNOS﹐並可以促進腫瘤在活體內生長。
Increasing evidence showed that tumor grown in vivo is an integrated structure. Reactions to the radiation damage by environmental components other than tumor cells per se, such as vascular damage and inflammatory responses, might play an important role in tumor killing and growth. It has been known for many years that tumor-associated macrophages (TAMs) are a major cellular component of human and murine cancers. The purpose of this study is to investigate the effects of radiation on tumors and TAMs, and to elucidate the potential of TAMs to influence tumor growth.

A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The irradiated tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status and function, as compared to the un-irradiated tumors at the same size.

Either after single or fractionated irradiation, increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1–2 weeks after tumor irradiation.

Function of TAMs was compared by mixing them with TRAMP-C1 cells and injecting them into mice. When mixing with TAMs from irradiated tumors, TRAMP-C1 cells grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone.

In conclusions, TAMs in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo.
CHAPTER I Introduction………………………………………. 1
1.1 Background and Review of Related Studies…………… 1
1.1.1 The importance of tumor associated macrophage (TAM) and tumor microenvironment………………...................
1
1.1.2 Lack of knowledge on the responses of microenvironment during fractionated radiotherapy……
3
1.2 Objective of the Study………………………………….. 5

CHAPTER II Experimental Setup……………………………… 6
2.1 Animals model and tumor irradiation………………....... 6
2.2 Histological and immunohistological (IHC) examination…………………………………………......
7
2,3 Flow cytometry…………………………………………. 7
2.4 Separation of TAMs……………………………………. 8
2.5 RNA isolation and RNase protection assay…………….. 8
2.6 Western blot…………………………………………...... 9
2.7 Statistical analysis of the data………………………....... 9

CHAPTER III Experiment Results………………………………. 11
3.1 Growth delay of TRAMP-C1 tumors after irradiation………………………………………………..
11
3.2 Histological and Cellular responses in the irradiated tumors……………………………………………….......
11
3.3 iNOS, arginase-I, and COX-2 mRNA expression in TRAMP-C1 tumors after a single dose of 25 Gy……….
11
3.4 Pro-inflammatory cytokine gene expression in tumors irradiated with a 25-Gy single dose……………………..
12
3.5 iNOS, Arg-I, and COX-2 mRNA expression in TRAMP-C1 tumors irradiated with fractionated 60 Gy radiation doses…………………………………………..

13
3.6 Expression of iNOS, Arg-I, and COX-2 protein in CD11b+ TAMs………………………………………..
14
3.7 Macrophages from irradiated tumors promote tumor growth……………………………………………….......
15

CHAPTER IV Discussions and Conclusions…………………….. 17

References ………………………………………………………....
23
Figures …………………………………………………………….. 28
Appendix ………………………………………………………….. 44
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