益气健脾化瘀方联合5-FU对MFC荷瘤小鼠皮下移植瘤的生长及免疫功能的影响
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摘要
目的:探讨益气健脾化瘀方联合5-氟尿嘧啶(5-FU)对MFC荷瘤615小鼠皮下移植瘤的生长及免疫功能的影响。方法:24只615小鼠随机分为模型组,益气健脾化瘀方(20 g·kg~(-1))组,5-FU(25 mg·kg~(-1))组,联合(益气健脾化瘀方20 g·kg~(-1)+5-FU 25 mg·kg~(-1))组,每组6只,皮下接种法建立小鼠胃癌移植瘤模型。于末次给药后完整剥离移植瘤、脾、胸腺,计算抑瘤率、胸腺、脾指数。流式细胞仪测定外周血、肿瘤组织中髓源抑制性细胞(MDSC)水平及其亚型多形核细胞样(PMNMDSC),单核细胞样(M-MDSC)的水平及外周血中各免疫细胞的水平;实时荧光定量PCR(Real-time PCR)检测瘤组织中精氨酸酶-1(Arg-1),诱导型一氧化氮合酶(i NOS) mRNA表达水平。结果:与模型组比较,益气健脾化瘀方组小鼠体质量增加、瘤重减轻,5-FU组小鼠体质量、瘤重、胸腺、脾指数均下降(P <0. 05,P <0. 01)。与5-FU组比较,联合组体质量增加,瘤重减轻,胸腺、脾指数升高(P <0. 05,P <0. 01)。与模型组比较,联合组肿瘤组织及外周血中总MDSCs水平下降(P <0. 05,P <0. 01),益气健脾化瘀方组,5-FU组及联合组PMN-MDSC水平均下降(P <0. 01);与5-FU组比较,联合组PMN-MDSC水平下降(P <0. 05),M-MDSC水平下降(P <0. 05)。与模型组比较,益气健脾化瘀方组巨噬细胞及其M1型水平增加(P <0. 05),各T细胞群水平均增加(P <0. 05,P <0. 01),5-FU组巨噬细胞,CD3+,CD4+,CD8+T细胞群水平下降(P <0. 05,P <0. 01)。与5-FU组比较,联合组巨噬细胞,CD4+,CD8+T细胞群水平增高(P <0. 01)。与模型组比较,各组i NOS,Arg-1 mRNA表达均下降(P <0. 05,P <0. 01)。与5-FU组比较,联合组i NOS,Arg-1 mRNA表达均下降(P <0. 05,P <0. 01)。结论:益气健脾化瘀方对肿瘤有一定的抑制作用,且与5-FU联合使用能更好地抑制皮下移植瘤的生长,并改善化疗后机体的免疫状态,其机制可能与降低MDSCs的水平及增加T细胞、巨噬细胞的水平有关。
Objective: To discuss the effect of Yiqi Jianpi Huayu recipe( YQJPHY) combined with5-fluorouracil( 5-FU) on the growth and immune function of subcutaneous transplanted tumor in MFC tumor bearing 615 mice. Method: Twenty-four mice were inoculated subcutaneously to establish the transplanted tumor model of gastric cancer in mice,and then randomly divided into model control group,YQJPHY( 20 g·kg~(-1))group,5-FU( 25 mg·kg~(-1)) group and( YQJPHY + 5-FU) combined group,with 6 rats in each group. After the last administration,the transplanted tumor,spleen and thymus were stripped completely. The tumor inhibition rate,thymus and spleen index were calculated. Flow cytometry was used to determine the content of myeloidderived suppressor cells( MDSCs) and its subtype polykaryotype cells( PMN-MDSC),single karyotype cells( M-MDSC) in both peripheral blood and tumor tissue,and macrophages and their M1 type,M2 type,T lymphocyte,B lymphocyte,and natural killer cells( NK cells) in peripheral blood. Expressions of arginase-1( Arg-1) and inducible nitric oxide synthesis( i NOS) gene in tumor tissues were detected by Real-time PCR.Result: Compared with model control group,the weight of mice in YQJPHY group increased,whereas the weight of tumor,the weight of tumor,the index of thymus and spleen decreased in 5-FU group( P < 0. 05,P < 0. 01).Compared with 5-FU group,combined group showed gained weight,decreased tumor weight,and increased thymus and spleen index( P < 0. 05,P < 0. 01). Compared with model control group,the total MDSCs content in tumor tissue and peripheral blood of the combined group was decreased,and the PMN-MDSC content in 5-FU group and the combined group were decreased( P < 0. 05,P < 0. 01). Compared with 5-FU group,the contents of total MDSCs,PMN-MDSC,M-MDSC in combined group were all decreased( P < 0. 05,P < 0. 01). Compared with model control group,the number of macrophages and their M1 type in YQJPHY group were higher than those in 5-FU group,while the contents of CD3+,CD4+,CD8+T cell group decreased( P < 0. 05,P < 0. 01). Compared with model control group,gene expressions of i NOS,Arg-1 decreased in all groups( P < 0. 05,P < 0. 01).Compared with 5-FU group,gene expressions of i NOS,Arg-1 decreased in combined group( P < 0. 05,P <0. 01). Conclusion: YQJPHY can better inhibit the growth of subcutaneous transplanted tumor when combined with 5-FU,and improve immune status after chemotherapy. The mechanism may be related to the decrease of MDSCs content and the increase of T cell and macrophages content.
Objective: To discuss the effect of Yiqi Jianpi Huayu recipe( YQJPHY) combined with5-fluorouracil( 5-FU) on the growth and immune function of subcutaneous transplanted tumor in MFC tumor bearing 615 mice. Method: Twenty-four mice were inoculated subcutaneously to establish the transplanted tumor model of gastric cancer in mice,and then randomly divided into model control group,YQJPHY( 20 g·kg~(-1))group,5-FU( 25 mg·kg~(-1)) group and( YQJPHY + 5-FU) combined group,with 6 rats in each group. After the last administration,the transplanted tumor,spleen and thymus were stripped completely. The tumor inhibition rate,thymus and spleen index were calculated. Flow cytometry was used to determine the content of myeloidderived suppressor cells( MDSCs) and its subtype polykaryotype cells( PMN-MDSC),single karyotype cells( M-MDSC) in both peripheral blood and tumor tissue,and macrophages and their M1 type,M2 type,T lymphocyte,B lymphocyte,and natural killer cells( NK cells) in peripheral blood. Expressions of arginase-1( Arg-1) and inducible nitric oxide synthesis( i NOS) gene in tumor tissues were detected by Real-time PCR.Result: Compared with model control group,the weight of mice in YQJPHY group increased,whereas the weight of tumor,the weight of tumor,the index of thymus and spleen decreased in 5-FU group( P < 0. 05,P < 0. 01).Compared with 5-FU group,combined group showed gained weight,decreased tumor weight,and increased thymus and spleen index( P < 0. 05,P < 0. 01). Compared with model control group,the total MDSCs content in tumor tissue and peripheral blood of the combined group was decreased,and the PMN-MDSC content in 5-FU group and the combined group were decreased( P < 0. 05,P < 0. 01). Compared with 5-FU group,the contents of total MDSCs,PMN-MDSC,M-MDSC in combined group were all decreased( P < 0. 05,P < 0. 01). Compared with model control group,the number of macrophages and their M1 type in YQJPHY group were higher than those in 5-FU group,while the contents of CD3+,CD4+,CD8+T cell group decreased( P < 0. 05,P < 0. 01). Compared with model control group,gene expressions of i NOS,Arg-1 decreased in all groups( P < 0. 05,P < 0. 01).Compared with 5-FU group,gene expressions of i NOS,Arg-1 decreased in combined group( P < 0. 05,P <0. 01). Conclusion: YQJPHY can better inhibit the growth of subcutaneous transplanted tumor when combined with 5-FU,and improve immune status after chemotherapy. The mechanism may be related to the decrease of MDSCs content and the increase of T cell and macrophages content.
引文
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[23]Kitamura T,Qian B Z,Soong D,et al.CCl2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages[J].J Cell Biol,2015,209(6):2096-117.
[24]郝文斌,相芬芬,刘巧丽,等.髓源抑制细胞在肿瘤微环境中的研究进展[J].免疫学杂志,2017,33(8):729-732.
[25]杨继乐,张莉,王莉.单核-巨噬细胞的分化和功能研究进展[J].细胞与分子免疫学杂志,2014,30(11):1213-1216.
[26]CAO Z X,CHEN X P,WU Z D.Changes of immune function in patients with liver cirrhosis after splenectomy combined with resection of hepatocellular carcinoma[J].Hepatobiliary Pancreat Dis Int,2003,2(4):562-565.
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[28]赵冬耕,孙佳,王明艳,等.抗癌扶正方对人肝癌细胞SMMC-7721 PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2012,18(11):136-139.
[29]谢淮冰,王瑞平,胡守友,等.健脾疏肝抗毒汤对裸鼠MDA-MB-231-pAcGFP异种移植瘤生长及转移能力的影响[J].中国实验方剂学杂志,2017,23(8):149-153.
[2]LI Z,LEI H,LUO M,et al.DNA methylation downregulated mir-10b acts as a tumor suppressor in gastric cancer[J].Gastric Cancer,2015,18(1):43-54.
[3]Ko J S,Bukowski R M,Fincke J H.Myeloid-derived suppressor cells:a novel therapeutic target[J].Curr Oncol Rep,2009,11(2):87-93.
[4]邓晓霞,李清宋,陈中,等.黄芪抗肿瘤作用机制的研究进展[J].中药新药与临床药理,2016,27(2):307-312.
[5]孙政华,邵晶,郭玫.党参化学成分及药理作用研究进展[J].安徽农业科学,2015,43(33):174-176.
[6]黄臣虎,陆茵,孙志广,等.莪术抗癌作用机制研究进展[J].中草药,2010,41(10):1745-1747.
[7]冯娅茹,张文婷,李二文,等.三棱化学成分及药理作用研究进展[J].中草药,2017,48(22):4804-4818.
[8]Louvet C,Andre T,Tigaud J M,et al.PhaseⅡstudy of oxaliplatin,fluorouracil and folinic acid in locally advanced or metastatic gastric cancer patients[J].JClin Oncol,2002,20(23):4543-4548.
[9]Frickhofen N,Beck F J,Jung B,et al.Capecitabine can induce acute coronary syndrome similar to 5-fluorouracil[J].Ann Oncol,2002,13(5):797-801.
[10]李雪青,乔钦增,焦建民,等.中西医结合治疗中晚期原发性肝癌的临床疗效分析[J].中国药物与临床,2011,11(7):820-821.
[11]丁照黎.中西医结合对肿瘤放化疗患者的临床影响观察与分析[J].中国实用医药,2011,6(7):185-186.
[12]李惠,沈凯凯.中药抗肿瘤联合用药研究进展[J].上海中医药大学学报,2017,31(3):90-94.
[13]ZHANG X,DING J,GOU C,et al.Qingchangligan formula attenuates the inflammatory response to protect the liver from acute failure induced by d-galactosamine/lipopolysaccharide in mice[J].J Ethnopharmacol,2017,201(6):108-116.
[14]杨芳,伍菲凡,王晓燕,等.Ficoll和Percoll法分离结直肠癌相关巨噬细胞的比较[J].实用医学杂志,2015,31(9):1394-1398.
[15]Serafini P,Mgebroff S,Noonan K et al.Myeloid-derived suppressor cells promote cross tolerance in B-cell lymphoma by expanding regulatory T cells[J].Cancer Res,2008,68(13):5439-5449.
[16]张锡纯.医学衷中参西录[M].太原:山西科学技术出版社,2009:168.
[17]付海尔,李建民,刘玉红.左归丸对肾阴虚模型大鼠神经-内分泌-免疫功能的影响[J].中国实验方剂学杂志,2017,23(22):155-159.
[18]俞琦,蔡琨,田维毅.合欢皮总皂苷对H22荷瘤小鼠细胞免疫功能的影响[J].中国实验方剂学杂志,2016,22(15):143-148.
[19]Ueha S,Shand F,Matsusshima K,et al.Myeloid cell population dynamics in healthy and tumor-bearing mice[J].Int Immunopharmacol,2011,11(7):783-788.
[20]Lavin Y,Winter D,Blecher-Gonen R,et al.Tissueresident macrophage enhancer landscapes are shaped by the local microenvironment[J].Cell,2014,159(6):1312-1326.
[21]Solito S,Marigo I,Pinton L,et al.Myeloid-derived suppressor cell heterogeneity in human cancers[J].Ann N Y Acad Sci,2014,1319(1):47-65.
[22]Sceneay J,Chow M T,Chen A,et al.Primary tumor hypoxia recruits CD11b+/Ly6Cmed/Ly6G+immune suppressor cellsand compromises NK cell cytotoxicity in the premetastatic niche[J].Cancer Res,2012,72(16):3906-3911.
[23]Kitamura T,Qian B Z,Soong D,et al.CCl2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages[J].J Cell Biol,2015,209(6):2096-117.
[24]郝文斌,相芬芬,刘巧丽,等.髓源抑制细胞在肿瘤微环境中的研究进展[J].免疫学杂志,2017,33(8):729-732.
[25]杨继乐,张莉,王莉.单核-巨噬细胞的分化和功能研究进展[J].细胞与分子免疫学杂志,2014,30(11):1213-1216.
[26]CAO Z X,CHEN X P,WU Z D.Changes of immune function in patients with liver cirrhosis after splenectomy combined with resection of hepatocellular carcinoma[J].Hepatobiliary Pancreat Dis Int,2003,2(4):562-565.
[27]Cools N,Ponsaerts P,Van Tendeloo V F,et al.Regulatory T cells and human disease[J].Clin Dev Immunol,2007,doi:10.1155/2007/89195.
[28]赵冬耕,孙佳,王明艳,等.抗癌扶正方对人肝癌细胞SMMC-7721 PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2012,18(11):136-139.
[29]谢淮冰,王瑞平,胡守友,等.健脾疏肝抗毒汤对裸鼠MDA-MB-231-pAcGFP异种移植瘤生长及转移能力的影响[J].中国实验方剂学杂志,2017,23(8):149-153.