小鼠胃癌模型建立及其调节性T细胞（Treg）水平变化的研究
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摘要
目的利用3-甲基胆蒽(3-methylcholanthrene,3-MCA)及N-甲基-N-亚硝基脲(N-methyl-N-nitrosourea, MNU)诱导建立小鼠胃癌模型。分别于12周及20周检测小鼠外周血及脾细胞白细胞计数、CD3+CD4+CD8+T细胞亚群及CD4+ CD25+调节性T细胞(CD4+CD25+regulatory T cells, Treg)在小鼠胃癌模型建立过程中不同时期的水平变化特征。
方法本实验采用六周龄、雄性、BALB/c小鼠以3-MCA挂线法于小鼠腺胃挂含有3-MCA线结联合MNU灌胃诱导小鼠胃癌。分别于12周及20周取血及脾细胞检测其白细胞计数、并通过流式细胞术(Flow Cytometries, FCM)检测其T细胞亚群及Treg水平,同时取胃标本切片行病理学检查胃癌发展情况。通过统计学方法分析所得数据。
结果
1.12周时25%小鼠出现腺胃部位的黏膜增厚及慢性炎症。20周时37.5%小鼠成功诱导出小鼠胃癌。对照组小鼠均未见黏膜增厚,慢性炎症或胃癌。
2.实验组小鼠12周及20周时体重变化的降低与对照组相比均具有统计学意义(P<0.05)。
3.实验组小鼠脾白细胞计数在20周的升高具有统计学意义(P<0.05)。
4.实验组小鼠外周血Treg在20周时的升高有统计学意义(P<0.05)。脾Treg比例在12周及20周时的升高均有统计学意义(P<0.05)。脾Treg绝对值在12周时的降低、在20周时的升高均具有统计学意义(P<0.05)。
5.实验组小鼠脾CD3+细胞在20周时的升高具有统计学意义(P<0.05)。脾CD4+细胞在20周时的降低具有统计学意义(P<0.05)。脾CD8+细胞在20周时的降低有统计学意义(P<0.05)。脾CD4+/CD8+比值在20周时的升高有统计学意义(P<0.05)。
6.实验组小鼠脾CD3+细胞绝对值在20周时的升高具有统计学意义(P<0.05)。脾CD4+细胞绝对值在20周时的升高具有统计学意义(P<0.05)。
结论
1.MCA挂线法联合MNU灌胃法成功诱导小鼠胃癌。
2.长期接触致癌物后可导致体重减轻,在停止致癌物攻击一段时间后体重的增长略有恢复,但无法恢复正常水平。如因长期接触致癌物后导致肿瘤可引起体重下降复。
3.长期接触致癌物后可引起外周血白细胞计数的降低,即使停止致癌物攻击后外周血白细胞计数水平仍无法恢复正常水平,但脾白细胞计数呈现升高的趋势、原因可能与免疫系统淋巴细胞反应性增生有关。
4.Treg在自身免疫调节中起作用,且在肿瘤的发生、发展过程中起重要作用。其过量表达在炎症时预防自身过度免疫反应造成自身组织损伤,而在肿瘤形成的过程中则抑制机体产生有效的抗肿瘤免疫应答、导致肿瘤免疫逃逸。
5.T细胞亚群在胃癌诱导早期因炎症反应T细胞总数及CD4+细胞水平均有所升高,而因为Treg高表达预防过度免疫同时引起CD8+细胞减少。在胃癌诱导晚期部份成瘤小鼠因免疫功能受到影响导致T细胞总数及CD8+细胞减少,但仍因为炎症反应或肿瘤影响导致CD4+细胞的升高。
Objective To induce gastric cancer mouse model using 3-methylcholanthrene (3-MCA) and N-methyl-N-nitrosourea (MNU). To assess the characteristics of the changes in levels of white blood count (WBC), CD3+CD4+CD8+T-cell subsets, and CD4+CD25+regulatory T cell (Treg) of both peripheral blood and splenocytes during week 12 and 20 of the construction of the model.
Methods Six week-old male BALB/c mice were utilized to establish gastric cancer model. Glandular stomach of the mice was sutured with 3-MCA-containing silk suture, and subsequent intragastric administration of MNU to induce gastric cancer. On week 12 and 20, peripheral blood and splenocytes were tested for WBC count, and CD3+CD4+CD8+T-cell subsets and Treg were assessed by flow cytometries (FCM) with concurrent collection of mice stomachs for pathologic study. Data were then analyzed statistically for changes in level of WBC, T-cell subsets, and Treg.
Results
1. On week 12,25% of mice showed partial thickening of the gastric mucosa and chronic inflammation. On week 20,37.5% of mice were successfully induced with gastric carcinoma, while the control groups showed no signs of thickening of the gastric mucosa, chronic inflammation or gastric carcinoma.
2. Growths of mice were significantly decreased on week 12 and week 20 when compared with the control group (P<0.05).
3. Splenocyte WBC counts signigicantly increased on week 20 (P<0.05).
4. Treg percentages in the peripheral blood significantly increased on week 20 (P<0.05). Splenocyte Treg percentages showed significant increased on week 12 and week 20 (P<0.05), while the absolute count of Treg showed significant decreased on week 12 (P<0.05) and significant increased on week 20 (P＜0.05).
5. In the splenocyte, CD3+cells significantly increased on week 20 (P<0.05). CD4+cells significantly decreased on week 20 (P＜0.05). CD8+cells significantly decreased on week 20 (P<0.05). CD4+/CD8+ratio significantly increased on week 20 (P<0.05).
6. In the absolute counts for splenocyte, CD3+cells significantly increased on week 20 (P<0.05). CD4+cells significantly increased on week 20(P<0.05).
Conclusions
1. Silk suture with 3-MCA-containing silk suture on to the glandular stomach and subsequent intragastric administration of MNU successfully induce gastric carcinoma in BALB/c mice.
2. Long-term exposure to carcinogens can cause lose of weight or slowing of growths amount mice, even when exposure to carcinogens stops, weight or growths are still lower than normal. If Long-term exposure to carcinogen cause tumor growth might also lead to lose of weight.
3. Long-term exposure to carcinogens can cause decrease in peripheral WBC amount mice, even when exposure to carcinogens stops peripheral WBC are still lower than normal. In contras, the increase of spleen WBC level might be the result of lymphocyte reactive hyperplasia.
4. Treg is important for its role in immune regulation during inflammation, and its crucial suppressing ability during tumor growth. High level of Treg during inflammation prevent autoimmunity self tissue damage, while during tumor formation it inhibited the body to produce effective anti-tumor immune response, which leads to tumor immune escape.
5. On early phase of gastric modle constraction, there were an increased in total number of T-cell and Th cell, and due to the suppression effect of Treg, there was an decreased in Tc cell. Later on when carcinomas were successfully induced, the homeostasis of the immune system was disturbed, and showed an decrease in total number of T-cell and Tc, but because of the effects of chronic inflammation and tumor Th level increased.
方法本实验采用六周龄、雄性、BALB/c小鼠以3-MCA挂线法于小鼠腺胃挂含有3-MCA线结联合MNU灌胃诱导小鼠胃癌。分别于12周及20周取血及脾细胞检测其白细胞计数、并通过流式细胞术(Flow Cytometries, FCM)检测其T细胞亚群及Treg水平,同时取胃标本切片行病理学检查胃癌发展情况。通过统计学方法分析所得数据。
结果
1.12周时25%小鼠出现腺胃部位的黏膜增厚及慢性炎症。20周时37.5%小鼠成功诱导出小鼠胃癌。对照组小鼠均未见黏膜增厚,慢性炎症或胃癌。
2.实验组小鼠12周及20周时体重变化的降低与对照组相比均具有统计学意义(P<0.05)。
3.实验组小鼠脾白细胞计数在20周的升高具有统计学意义(P<0.05)。
4.实验组小鼠外周血Treg在20周时的升高有统计学意义(P<0.05)。脾Treg比例在12周及20周时的升高均有统计学意义(P<0.05)。脾Treg绝对值在12周时的降低、在20周时的升高均具有统计学意义(P<0.05)。
5.实验组小鼠脾CD3+细胞在20周时的升高具有统计学意义(P<0.05)。脾CD4+细胞在20周时的降低具有统计学意义(P<0.05)。脾CD8+细胞在20周时的降低有统计学意义(P<0.05)。脾CD4+/CD8+比值在20周时的升高有统计学意义(P<0.05)。
6.实验组小鼠脾CD3+细胞绝对值在20周时的升高具有统计学意义(P<0.05)。脾CD4+细胞绝对值在20周时的升高具有统计学意义(P<0.05)。
结论
1.MCA挂线法联合MNU灌胃法成功诱导小鼠胃癌。
2.长期接触致癌物后可导致体重减轻,在停止致癌物攻击一段时间后体重的增长略有恢复,但无法恢复正常水平。如因长期接触致癌物后导致肿瘤可引起体重下降复。
3.长期接触致癌物后可引起外周血白细胞计数的降低,即使停止致癌物攻击后外周血白细胞计数水平仍无法恢复正常水平,但脾白细胞计数呈现升高的趋势、原因可能与免疫系统淋巴细胞反应性增生有关。
4.Treg在自身免疫调节中起作用,且在肿瘤的发生、发展过程中起重要作用。其过量表达在炎症时预防自身过度免疫反应造成自身组织损伤,而在肿瘤形成的过程中则抑制机体产生有效的抗肿瘤免疫应答、导致肿瘤免疫逃逸。
5.T细胞亚群在胃癌诱导早期因炎症反应T细胞总数及CD4+细胞水平均有所升高,而因为Treg高表达预防过度免疫同时引起CD8+细胞减少。在胃癌诱导晚期部份成瘤小鼠因免疫功能受到影响导致T细胞总数及CD8+细胞减少,但仍因为炎症反应或肿瘤影响导致CD4+细胞的升高。
Objective To induce gastric cancer mouse model using 3-methylcholanthrene (3-MCA) and N-methyl-N-nitrosourea (MNU). To assess the characteristics of the changes in levels of white blood count (WBC), CD3+CD4+CD8+T-cell subsets, and CD4+CD25+regulatory T cell (Treg) of both peripheral blood and splenocytes during week 12 and 20 of the construction of the model.
Methods Six week-old male BALB/c mice were utilized to establish gastric cancer model. Glandular stomach of the mice was sutured with 3-MCA-containing silk suture, and subsequent intragastric administration of MNU to induce gastric cancer. On week 12 and 20, peripheral blood and splenocytes were tested for WBC count, and CD3+CD4+CD8+T-cell subsets and Treg were assessed by flow cytometries (FCM) with concurrent collection of mice stomachs for pathologic study. Data were then analyzed statistically for changes in level of WBC, T-cell subsets, and Treg.
Results
1. On week 12,25% of mice showed partial thickening of the gastric mucosa and chronic inflammation. On week 20,37.5% of mice were successfully induced with gastric carcinoma, while the control groups showed no signs of thickening of the gastric mucosa, chronic inflammation or gastric carcinoma.
2. Growths of mice were significantly decreased on week 12 and week 20 when compared with the control group (P<0.05).
3. Splenocyte WBC counts signigicantly increased on week 20 (P<0.05).
4. Treg percentages in the peripheral blood significantly increased on week 20 (P<0.05). Splenocyte Treg percentages showed significant increased on week 12 and week 20 (P<0.05), while the absolute count of Treg showed significant decreased on week 12 (P<0.05) and significant increased on week 20 (P＜0.05).
5. In the splenocyte, CD3+cells significantly increased on week 20 (P<0.05). CD4+cells significantly decreased on week 20 (P＜0.05). CD8+cells significantly decreased on week 20 (P<0.05). CD4+/CD8+ratio significantly increased on week 20 (P<0.05).
6. In the absolute counts for splenocyte, CD3+cells significantly increased on week 20 (P<0.05). CD4+cells significantly increased on week 20(P<0.05).
Conclusions
1. Silk suture with 3-MCA-containing silk suture on to the glandular stomach and subsequent intragastric administration of MNU successfully induce gastric carcinoma in BALB/c mice.
2. Long-term exposure to carcinogens can cause lose of weight or slowing of growths amount mice, even when exposure to carcinogens stops, weight or growths are still lower than normal. If Long-term exposure to carcinogen cause tumor growth might also lead to lose of weight.
3. Long-term exposure to carcinogens can cause decrease in peripheral WBC amount mice, even when exposure to carcinogens stops peripheral WBC are still lower than normal. In contras, the increase of spleen WBC level might be the result of lymphocyte reactive hyperplasia.
4. Treg is important for its role in immune regulation during inflammation, and its crucial suppressing ability during tumor growth. High level of Treg during inflammation prevent autoimmunity self tissue damage, while during tumor formation it inhibited the body to produce effective anti-tumor immune response, which leads to tumor immune escape.
5. On early phase of gastric modle constraction, there were an increased in total number of T-cell and Th cell, and due to the suppression effect of Treg, there was an decreased in Tc cell. Later on when carcinomas were successfully induced, the homeostasis of the immune system was disturbed, and showed an decrease in total number of T-cell and Tc, but because of the effects of chronic inflammation and tumor Th level increased.
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[53]Margolis KL, Rodabough RJ, Thomson CA, et al. Prospective study of leukocyte count as a predictor of incident breast, colorectal, endometrial, and lung cancer and mortality in postmenopausal women [J]. Arch Intern Med,2007,167 (17):1822-4.
[54]刘洁凡,赵亚新,曾谦,等.恶性肿瘤患者晚期白细胞计数改变与预后的关系[J].现代中西医结合杂志,2006,15(3)：291-292.
[55]Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease [J]. Nat Immunol,2005,6 (4):353-60.
[56]张莹,姚咏明.调节性T细胞与慢性感染性疾病[J].中华老年多器官疾病杂志,2007,6(5)：365-368.
[57]王剑,卢剑,袁向亮,等.胃癌外周血CD4+-CD25high-Foxp3+-调节性T细胞异常增高与TGF-β1、PGE2的相关性分析[J].诊断学理论与实践,2008,7(3)：308-312.
[58]张育超,王惠英,吕永添,等.胃癌患者围手术期CD4+CD25+调节性T细胞的变化及其意义[J].中国普通外科杂志,2008,17(5)：504-505.
[59]Sasada T, Kimura M, Yoshida Y, et al. CD4+CD25+regulatory T cells in patients with gastrointestinal malignancies:possible involvement of regulatory T cells in disease progression [J]. Cancer,2003,98 (5):1089-99.
[60]Lee CW, Rao VP, Rogers AB, et al. Wild-type and interleukin-10-deficient regulatory T cells reduce effector T-cell-mediated gastroduodenitis in Rag2-/-mice, but only wild-type regulatory T cells suppress Helicobacter pylori gastritis [J]. Infect Immun,2007,75 (6):2699-707. Epub 2007 Mar 12.
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[1]张启瑜.钱礼腹部外科学[M].北京：人民卫生出版社,2006：237.
[2]Parkin DM, Freddie B, Ferlay J, et al. Global Cancer Statistics,2002 [J]. CA Cancer J Clin,2005,55 (2):74-108.
[3]孙秀娣,牧人,周有尚,等.中国1990—1992年胃癌死亡调查分析[J].中华肿瘤杂志,2002,24(1)：4-8.
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[19]王天宝,吴小鹏,张维东,等.重组人内皮抑素对裸鼠胃癌瘤株生长的影响[J].中国普通外科杂志,2004,13(4)：268-271.
[20]王瑜,田晶,李科,等.免疫抑制小鼠肾包膜下胃癌移植瘤模型的建立及微血管观察[J].第四军医大学学报,2004,25(6)：571-573.
[21]张焜和,祝金泉,王崇文,等.改良Steel法建立人胃癌动物模型[J].实用肿癌杂志,1998,13(5)：291-292.
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[2]孙秀娣,牧人,周有尚,等.中国1990—1992年胃癌死亡调查分析[J].中华肿瘤杂志,2002,24(1)：4-8.
[3]周宁新,张国华.胃癌模型的建立与研究[J].中国肿瘤临床,1987,14(6)：364-366.
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[6]Sugimura T, Fujimura S, Baba T. Tumor Production in the Glandular Stomach and Alimentary Tract of the Rat by N-Methyl-N'-nitro-N-nitrosoguanidine[J]. Cancer Res.1970,30 (2):455-65.
[7]徐晶莹,李益民,李玉兰,等MNNG诱导大鼠胃癌模型的建立[J].哈尔滨医科大学学报,2003,37(2)：104-106.
[8]Tatematsu M, Ogawa K, Hoshiya T, et al. Induction of adenocarcinomas in the glandular stomach of BALB/c mice treated with N-methyl-N-nitrosourea [J]. Jpn J Cancer Res,1992,83 (9):915-8.
[9]张黎,王登山,刘继业,等.微量元素Mn、Se对小鼠诱发性胃癌发生的影响[J].甘肃环境研究与监测,1991,2：36-40.
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[13]汤礼军,晏才杰.犬残胃癌模型及其价值的研究[J].重庆医学,1995,24(6)： 336.
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[18]汪芳裕,朱人敏,王琳,等.裸鼠人胃癌原位移植模型的建立[J].癌症,1997,16(5)：343-344.
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[56]张莹,姚咏明.调节性T细胞与慢性感染性疾病[J].中华老年多器官疾病杂志,2007,6(5)：365-368.
[57]王剑,卢剑,袁向亮,等.胃癌外周血CD4+-CD25high-Foxp3+-调节性T细胞异常增高与TGF-β1、PGE2的相关性分析[J].诊断学理论与实践,2008,7(3)：308-312.
[58]张育超,王惠英,吕永添,等.胃癌患者围手术期CD4+CD25+调节性T细胞的变化及其意义[J].中国普通外科杂志,2008,17(5)：504-505.
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[1]张启瑜.钱礼腹部外科学[M].北京：人民卫生出版社,2006：237.
[2]Parkin DM, Freddie B, Ferlay J, et al. Global Cancer Statistics,2002 [J]. CA Cancer J Clin,2005,55 (2):74-108.
[3]孙秀娣,牧人,周有尚,等.中国1990—1992年胃癌死亡调查分析[J].中华肿瘤杂志,2002,24(1)：4-8.
[4]陈孝平主编.外科学[M].北京：人民卫生出版社,2005,569-570.
[5]周本杰,陈蔚文,王建华.胃癌动物模型研究现状与评价[J].广州中医药大学学报,1998,15(2)：156-159.
[6]陈云逸.胃癌动物模型研究进展[J].肿瘤基础与临床,2006,19(1)：75-77.
[7]周宁新,张国华.胃癌模型的建立与研究[J].中国肿瘤临床,1987,14(6)：364-366.
[8]Tsukamoto T, Mizoshita T, Tatematsu M. Animal Models of Stomach Carcinogenesis [J]. Toxicol Pathol,2007,35 (5):636-48.
[9]宋伯根,李义清,赵桂芬,等.胃粘膜损伤程度与胃癌发生的关系[J].癌症,1993,12(5)：372-374.
[10]Sugimura T, Fujimura S, Baba T. Tumor Production in the Glandular Stomach and Alimentary Tract of the Rat by N-Methyl-N'-nitro-N-nitrosoguanidine [J]. Cancer Res,1970,30 (2):455-65.
[11]徐晶莹,李益民,李玉兰,等MNNG诱导大鼠胃癌模型的建立.[J].哈尔滨医科大学学报,2003,37(2)：104-106.
[12]Tatematsu M, Ogawa K, Hoshiya T, et al. Induction of adenocarcinomas in the glandular stomach of BALB/c mice treated with N-methyl-N-nitrosourea [J]. Jpn J Cancer Res,1992,83 (9):915-8.
[13]张黎,王登山,刘继业,等.微量元素Mn、Se对小鼠诱发性胃癌发生的影响[J].甘肃环境研究与监测,1991,2：36-40.
[14]徐建国,包可杰.甲基胆蒽诱发大、小鼠胃癌的实验观察[J].白求恩医科大学学报,1992,18(5)：466—467.
[15]Sugiyama T, Hige S, Asaka M. Development of an H. pylori-infected animal model and gastric cancer:recent progress and issues [J]. J Gastroenterol,2002,37 Suppl 13:6-9.
[16]周本杰,陈蔚文,王建华,等.硝酸结合MNNG攻击大鼠胃癌模型与评价[J].中国药理学通报,2001,17(1)：111-112.
[17]汤礼军,晏才杰.犬残胃癌模型及其价值的研究[J].重庆医学,1995,24(6)：336.
[18]Sugiyama A, Maruta F, Ikeno T, et al. Helicobacter pylori infection enhances N-methyl-N-nitrosourea-induced stomach carcinogenesis in the Mongolian gerbil [J]. Cancer Res,1998,58 (10):2067-9.
[19]王天宝,吴小鹏,张维东,等.重组人内皮抑素对裸鼠胃癌瘤株生长的影响[J].中国普通外科杂志,2004,13(4)：268-271.
[20]王瑜,田晶,李科,等.免疫抑制小鼠肾包膜下胃癌移植瘤模型的建立及微血管观察[J].第四军医大学学报,2004,25(6)：571-573.
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