健脾理气中药对大鼠肝癌微环境Treg细胞的影响
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摘要
目的：通过观察健脾理气中药对肝癌微环境中调节性T细胞(regulatory T, Treg)的影响,探讨健脾理气中药调节肝癌免疫微环境的Treg细胞的机制。
方法：
1.分组造模：
(1)选6周龄雄性清洁级Wistar大鼠60只,体重140g±10g,随机分为正常空白组、对照组、健脾理气组3组,每组20只。同一组每5只大鼠分为一笼,标记鼠笼。
(2)正常组大鼠正常喂养饲料和饮水；对照组和健脾理气组大鼠一直自由饮用80ppm二乙基亚硝胺(DEN)水溶液。自造模后第16周起进行治疗,健脾理气组用健脾理气中药(由党参10g、茯苓20g、白术20g、淮山20g、陈皮10g、柴胡10g、白芍10g、枳实10g、甘草10g等九味中药组成,采用标准的水煎醇沉工艺加工成含1g生药／ml,4℃冰箱保存备用)混合于颗粒饲料中喂养,大鼠日摄取剂量约为人类公斤体重的10倍。对照组大鼠继续按原方式喂养。
2.标本采集和制作：实验满20周称大鼠体重,脱颈处死大鼠。大鼠处死后剖开腹腔,剪取整个肝脏,记录一般情况及肝内癌结节数及大小,称肝质量,在无菌条件下分别制备正常组大鼠肝脏标本,对照组和健脾理气组大鼠肝脏内癌组织(同时有多个癌结节则取其中最大者)、癌旁组织(距离癌组织3mm内的组织)及癌远端组织(距离癌组织1cm外的组织)标本,分别用10%福尔马林固定,常规石蜡包埋,连续切取4um厚的切片,苏木素衬染色,热水蓝化,吹干后,树脂封片。免疫组化法检测大鼠肝组织中Treg细胞及CD4+T、CD8+T细胞。较好的标本照相留存。
结果：
1.健脾理气中药干预的大鼠肝癌模型在体重下降(P=0<0.01)和肝重增加方面(P=0.021<0.05)均不如对照组大鼠明显,且肝内癌结节个数较对照组大鼠少(P=0.011<0.05),所形成的肿瘤较对照组大鼠小(P=0.026<0.05)。
2.通过免疫组化检测,健脾理气中药组大鼠肝癌微环境中Treg细胞数量较对照组大鼠肝癌微环境中少(P=0.0072<0.01),CD4+T细胞数量较对照组大鼠增加(P=0<0.01),对肝癌微环境中CD8+T细胞数量则无明显影响(P=0.527>0.05)。
结论：健脾理气中药能够延缓肿瘤生长、增殖的机制之一可能是通过减少微环境中Treg细胞数量,提升CD4+T细胞数量,改善微环境中免疫抑制状态来实现。
Objective:To observe the Jianpiliqi Chinese herbal medicines' effect on the liver micro-environment of regulatory T cells (regulatory T, Treg) and explore the effect of Jianpiliqi herb on liver immune regulation of Treg cellular mechanism of micro-environment.
Methods:
1. Group modeling:
(1) select 60 clean grade Wistar male rats with 6-week-old, weighing 140g±10g, divide randomly into blank group, control group, Jianpiliqi group, each of Section 20. Each five rats in the same group were vested in one cage, marking the cages.
(2)The blank group rats were fed the normal stuffs and water; control group and Jianpiliqi group rats were fed 80ppm diethylnitrosamine (DEN) aqueous solution all the time, and start to accept the treatment after 16 weeks. The Jianpiliqi group rats fed with stuffs containing the Chinese Medicines (consisting of Codonopsis 10g, Poria 20g, Atractylodes 20g, Chinese yam 20g, tangerine peel 10g, bupleurum chinense 10g, white peony root 10g, citrus aurantium 10g and licorice 10g nine Chinese herbal medicines, being processed into 1g crude drug per ml with the standard water decoction and alchol sedimentation technics, Reserved in 4℃refrigerator), intake dose is about 10 times to human in terms of body kg/weight. The control group were fed quondamly.
2. Specimen collecting and producting:Weighing the rats and killed them by neck off when the experiment expire to 20 weeks. cutting open the abdominal cavity, clipping the whole liver after the rats being killed, recording general situation and registering the number and size of cancerous nodes, weighing the liver. Prepared respectively the specimens of the normal group rats'hepatic tissue, the control group and Jianpiliqi group rats'hepatic celluar carcinoma tissue (taking the largest one while multiple carcinoma nodes), carcinoma adjacent tissues (within 3mm away cancerous side) and the distal carcinoma tissues (lcm away from cancer organizations) under axenic conditions, then fixed with 10% formalin, paraffin-embedded routinely and cut by 4um thick slices continuously, hematoxylin staining lining, hot Aqua Blue-based, Seal the slices with resin after drying. Detecting the Treg cells and the CD4+ T, CD8+ T cells of the slices by immunohistochemical method. Taking pictures and preserving the preferable samples.
Results:
1. To the Jianpiliqi group rats, in weight lost (P=0<0.01) and liver weight increased (P=0.021<0.05) are less than the control group rats significantly, and the number of hepatic carcinoma nodes are less than the control group rats (P=0.011<0.05), the tumor are smaller than the control group (P=0.026<0.05)
2. By immunohistochemistry, the number of Jianpiliqi group rats'Treg cells in the micro-environment of the hepatic celluar carcinoma are less than the control group rats (P=0.0072<0.01), while CD4+ T cells in the micro-environment of the hepatic celluar carcinoma are increased more than the control group rats (P=0.000<0.01), but the Jianpiliqi Chinese Medcine had no effect on the CD8+ T cells in the micro-environment of the hepatic celluar carcinoma (P=0.527>0.05).
Conclusion:Jianpiliqi Chinese herbal medicines to slow tumor growth and proliferation may be one mechanism of micro-environment by reducing the number of Treg cells enhance CD4+ T cells and improve the micro-environment of immunosuppression to achieve.
方法：
1.分组造模：
(1)选6周龄雄性清洁级Wistar大鼠60只,体重140g±10g,随机分为正常空白组、对照组、健脾理气组3组,每组20只。同一组每5只大鼠分为一笼,标记鼠笼。
(2)正常组大鼠正常喂养饲料和饮水；对照组和健脾理气组大鼠一直自由饮用80ppm二乙基亚硝胺(DEN)水溶液。自造模后第16周起进行治疗,健脾理气组用健脾理气中药(由党参10g、茯苓20g、白术20g、淮山20g、陈皮10g、柴胡10g、白芍10g、枳实10g、甘草10g等九味中药组成,采用标准的水煎醇沉工艺加工成含1g生药／ml,4℃冰箱保存备用)混合于颗粒饲料中喂养,大鼠日摄取剂量约为人类公斤体重的10倍。对照组大鼠继续按原方式喂养。
2.标本采集和制作：实验满20周称大鼠体重,脱颈处死大鼠。大鼠处死后剖开腹腔,剪取整个肝脏,记录一般情况及肝内癌结节数及大小,称肝质量,在无菌条件下分别制备正常组大鼠肝脏标本,对照组和健脾理气组大鼠肝脏内癌组织(同时有多个癌结节则取其中最大者)、癌旁组织(距离癌组织3mm内的组织)及癌远端组织(距离癌组织1cm外的组织)标本,分别用10%福尔马林固定,常规石蜡包埋,连续切取4um厚的切片,苏木素衬染色,热水蓝化,吹干后,树脂封片。免疫组化法检测大鼠肝组织中Treg细胞及CD4+T、CD8+T细胞。较好的标本照相留存。
结果：
1.健脾理气中药干预的大鼠肝癌模型在体重下降(P=0<0.01)和肝重增加方面(P=0.021<0.05)均不如对照组大鼠明显,且肝内癌结节个数较对照组大鼠少(P=0.011<0.05),所形成的肿瘤较对照组大鼠小(P=0.026<0.05)。
2.通过免疫组化检测,健脾理气中药组大鼠肝癌微环境中Treg细胞数量较对照组大鼠肝癌微环境中少(P=0.0072<0.01),CD4+T细胞数量较对照组大鼠增加(P=0<0.01),对肝癌微环境中CD8+T细胞数量则无明显影响(P=0.527>0.05)。
结论：健脾理气中药能够延缓肿瘤生长、增殖的机制之一可能是通过减少微环境中Treg细胞数量,提升CD4+T细胞数量,改善微环境中免疫抑制状态来实现。
Objective:To observe the Jianpiliqi Chinese herbal medicines' effect on the liver micro-environment of regulatory T cells (regulatory T, Treg) and explore the effect of Jianpiliqi herb on liver immune regulation of Treg cellular mechanism of micro-environment.
Methods:
1. Group modeling:
(1) select 60 clean grade Wistar male rats with 6-week-old, weighing 140g±10g, divide randomly into blank group, control group, Jianpiliqi group, each of Section 20. Each five rats in the same group were vested in one cage, marking the cages.
(2)The blank group rats were fed the normal stuffs and water; control group and Jianpiliqi group rats were fed 80ppm diethylnitrosamine (DEN) aqueous solution all the time, and start to accept the treatment after 16 weeks. The Jianpiliqi group rats fed with stuffs containing the Chinese Medicines (consisting of Codonopsis 10g, Poria 20g, Atractylodes 20g, Chinese yam 20g, tangerine peel 10g, bupleurum chinense 10g, white peony root 10g, citrus aurantium 10g and licorice 10g nine Chinese herbal medicines, being processed into 1g crude drug per ml with the standard water decoction and alchol sedimentation technics, Reserved in 4℃refrigerator), intake dose is about 10 times to human in terms of body kg/weight. The control group were fed quondamly.
2. Specimen collecting and producting:Weighing the rats and killed them by neck off when the experiment expire to 20 weeks. cutting open the abdominal cavity, clipping the whole liver after the rats being killed, recording general situation and registering the number and size of cancerous nodes, weighing the liver. Prepared respectively the specimens of the normal group rats'hepatic tissue, the control group and Jianpiliqi group rats'hepatic celluar carcinoma tissue (taking the largest one while multiple carcinoma nodes), carcinoma adjacent tissues (within 3mm away cancerous side) and the distal carcinoma tissues (lcm away from cancer organizations) under axenic conditions, then fixed with 10% formalin, paraffin-embedded routinely and cut by 4um thick slices continuously, hematoxylin staining lining, hot Aqua Blue-based, Seal the slices with resin after drying. Detecting the Treg cells and the CD4+ T, CD8+ T cells of the slices by immunohistochemical method. Taking pictures and preserving the preferable samples.
Results:
1. To the Jianpiliqi group rats, in weight lost (P=0<0.01) and liver weight increased (P=0.021<0.05) are less than the control group rats significantly, and the number of hepatic carcinoma nodes are less than the control group rats (P=0.011<0.05), the tumor are smaller than the control group (P=0.026<0.05)
2. By immunohistochemistry, the number of Jianpiliqi group rats'Treg cells in the micro-environment of the hepatic celluar carcinoma are less than the control group rats (P=0.0072<0.01), while CD4+ T cells in the micro-environment of the hepatic celluar carcinoma are increased more than the control group rats (P=0.000<0.01), but the Jianpiliqi Chinese Medcine had no effect on the CD8+ T cells in the micro-environment of the hepatic celluar carcinoma (P=0.527>0.05).
Conclusion:Jianpiliqi Chinese herbal medicines to slow tumor growth and proliferation may be one mechanism of micro-environment by reducing the number of Treg cells enhance CD4+ T cells and improve the micro-environment of immunosuppression to achieve.
引文
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[1]吴孟超.中医药在肝癌防治中的作用、地位和存在的问题[J].中西医结合学报,2003；1(3)；163～164.
[2]Hede K. Environmental protection:studies highlight importance of tumor microenvironment [J]. J Natl Cancer Inst,2004,96 (15):1120-1121.
[3]Brown JM. Tumor microenvironment and the response to anticancer therapy [J]. Cancer Biol Ther,2002,1 (5):453-458.
[4]陈中,晏建军,倪家连,等.肝癌微环境中CD4+CD25+调节性T细胞的分布及其临床病理意义[J].中华肝胆外科杂志,2007,10(10)：678～681.
[5]Qiang Gao, Shuang-Jian Qiu, Jia Fan, et al. Intratumoral Balance of Regulatory and Cytotoxic T Cells Is Associated With Prognosis of Hepatocellular Carcinoma After Resection[J]. Journal of Clinical Oncology, Vol 25, No 18, 2007:PP.2586～2593.
[6]吴迪.CD4+CD25+调节性T细胞与抗肿瘤免疫[J].国际肿瘤学杂志,2007；34(5)：324～327.
[7]管冬元,方肇勤.健脾理气法防治肝癌的研究进展[J].上海中医药大学学报,2005,19(2)：60～63.
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