摘要
研究背景
原发性肝癌是全球高发的恶性肿瘤,据国际癌症研究中心估计,2000年全球肝癌发病人数56.4万,肝癌死亡54.9万;其中中国肝癌发病人数30.6万,死亡30.0万,中国的发病(死亡)数占全世界肝癌的一半,由此可见,原发性肝癌是威胁人类健康的主要疾病之一。我国现有癌症病人约250万人,平均每死亡5个人中就有一个死于肝癌,每年因治疗而支付的医疗费用达300多亿元,间接损失更高达上千亿元。目前临床上的治疗一般视手术、放疗、化疗为三大支柱,但由于肝癌起病隐匿,恶性程度高,发展变化快,患者就诊时往往已进入中晚期,失去了最佳手术时机,而大多数化疗药物虽可抑制肿瘤细胞过度增生,但毒副作用大,对正常组织细胞也有很强的杀伤作用,部分病人因身体极度虚弱已不能耐受西医西药的治疗。而中医药在治疗肝癌方面因其独特的优势而被临床所广泛采纳,在治疗局部的同时,重视全身治疗,改善和提高病人的生活质量,延长生命周期,并且副作用小,因此,发挥我国中医药的优势,为肝癌的治疗寻求高效低毒、价廉质优的中药复方,并探讨其作用机理,对于提高肝癌患者生存质量,降低肝癌治疗费用,扩展中药复方应用范围,具有十分重要的社会效益,显著的经济效益和学术价值。
中医理论认为,肝癌的发病是在内外因共同作用下,机体阴阳失调,脏腑功能障碍,经络阻塞,气血运行失常,气滞血瘀,湿聚痰凝,郁久化火,终致气、血、湿、热、瘀、毒互结而发病。关于本病的辩证施治,临床治疗常选用活血化瘀、清热祛湿以及养阴柔肝等治法组方。但本病病机复杂,病症危重,往往多型并见,治疗本病较为棘手。目前研究认为,中药复方治疗肝癌机理主要体现在以下几个方面:直接杀伤肿瘤细胞、抑制肿瘤血管生成、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡分化、调节机体免疫功能以及影响癌基因表达等,但对以上三种不同治法中药复方治疗肝癌机理的比较研究还少有报道。
目的
通过本课题的研究,探讨中医活血化瘀、清热祛湿、养阴柔肝三种治法治疗肝癌的作用机制,比较其不同作用靶点,为中药复方治疗肝癌提供理论依据。
方法
1.无菌抽取H_(22)瘤细胞(浓度为1×10~7 mL~(-1)),小鼠右侧腋下消毒后接种0.2 mL。
2.以活血化瘀、清热祛湿、养阴柔肝三方最佳浓度(由预实验结果确定)中药水煎液,灌胃H_(22)荷瘤小鼠,观察其一般情况(包括毛发色泽、饮食情况、活动度等)。
3.称量各组瘤体重量,计算抑瘤率,分析三种治法方药对肿瘤细胞生长的抑制作用。
4.显微镜下观察瘤体组织细胞形态学变化。
5.称量各组脾脏重量,计算脾指数,分析三种治法方药对荷瘤小鼠免疫功能的影响。
6.应用TUNEL技术,检测瘤体细胞凋亡情况,计算凋亡指数。
7.应用免疫组化技术,对细胞内细胞周期及凋亡细胞相关蛋白(CyclinD1、PCNA、Bcl-2及Bax)表达水平进行分析,探讨三种治法方药抑制瘤体细胞增殖、诱导细胞凋亡的不同作用机制。
结果
1.小鼠移植性肝癌(H_(22))模型复制成功,4天后肉眼可见皮下实体瘤,成功率100%。
2.活血化瘀、清热祛湿、养阴柔肝组抑瘤率分别为:43.64%、50.91%、45.45%。
3.活血化瘀、清热祛湿、养阴柔肝方对H_(22)小鼠脾重无显著影响(P>0.05)。
4.活血化瘀、清热祛湿、养阴柔肝方对肝癌细胞增殖影响显著(P<0.05)。
5.活血化瘀、清热祛湿、养阴柔肝方对肝癌细胞凋亡影响显著(P<0.05)。
6.免疫组化显示:活血化瘀、清热祛湿、养阴柔肝方作用后,肝癌细胞PCNA、CyclinD1、Bcl-2表达量减少,Bax表达量增多,与模型对照组比较有显著性差异(P<0.05)。
结论
1.三种治法均具有抑制肝癌瘤体生长的作用,其中清热祛湿方的抑瘤作用最明显。
2.三种治法均可以降低PCNA在H_(22)肝癌细胞内的表达量,抑制细胞内DNA合成进程,进而抑制肝癌细胞的增殖。
3.三种治法方药均可以明显抑制肝癌细胞CyclinD1表达,推断三种治法能够抑制肝癌细胞增殖活性的分子机制,可能是通过降低肝癌细胞内CyclinD1的阳性表达,阻断CyclinD1-CDK4-Rb通路,降低细胞周期转换速度实现的。
4.三种治法均可诱导肿瘤细胞发生凋亡;相关蛋白Bcl-2、Bax可能是三种治法诱导肝癌细胞凋亡的重要调控因子。
5.活血化瘀方法主要通过抑制肝癌细胞增殖发挥抑癌作用。
6.清热祛湿、养阴柔肝方法主要通过诱导肿瘤细胞凋亡发挥抑癌作用,二者作用结果相似。
Background
Primary hepatic carcinoma(PHC) is a high occured rate disease all over the world,IARC estimated that there are 564,000 people suffer from PHC and 549,000 are died;In China,there are 306,000 patients,and 300,000 are died,which is a half of total caicinomatous and death number,so PHC is one of the main diseases that threatens human's life in our country.There are 2,500,000 cancer patients in China,and one fifth is died from it,the medical care expense on cancer amounts to over¥30 billions annually,the indirect loss is higher than hundreds of billion yuan.Now the main therapies are opration,radiotherapy and chemo,but lots of patient don't suit for opration at advanced stage when they go to hospital as the hide,malignant and growth nature of PHC,most chemotherapy drugs can restrain excess of proliferation,but side effects are so severe that patients can't afford by their weak bodys.However,Chinese patent medicine are extensive absorbed by advantages of comprehensive therapy,enhance the quality and prolong life and little side effect in clinic.Therefor,it is overwhelmingly significant in social benefit,economic benefit and especially academical values to exert the advantage of Chinese patent medicine,seek for some kind of high-effective and low-cytotoxic Chinese patent medicine,study the mechanism of it,to improve survival probability,reduce therapy expence and widen the range of Chinese patent medicine's application.
PHC is a diease which is under mutual effect of intra and external factors,can lead to imbalance of body's Yin and Yang,disfunction of viscera,choken meridian,abnormal operation of Qi and blood,stagnation of Qi and blood,accumulation of wetness and phlegm, reduction evil fire,finally to fall ill combined with Qi,blood,wetness,heat,Liver Qi depression and pathogenic fire and toxin,we always choose promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy to treat the disease in clinic.But PHC is a thorny problem in clinic work since complicated pathogenesis,critical symptoms and contemporaneous syndrome types. The modern medicine shows that the mechanism of Chinese patent medicine cured PHC are various,embodied by:kill the cancer cells directly,restrain to form of cancer's blood vessel, restrain to proliferation of cancer cell,inducement apoptosis of cancer cell,regulate the function of body's immunity and influence gene's expression,but we haven't found the mechanism and relative study of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy yet.
Objective
To study the therapy mechanism of PHC by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy,compare the function targets,provide experimental evidence on the treatment of PHC with Chinese patent medicine.
Methods
1.Exsuct asepsis H_(22) tumor cell which concentrated to 1×10~7mL~(-1),hypodermic inject 0.2mL at right oxter of mice after antisepsis.
2.Repectively fed H_(22) tumor-bearing mice by the water-solubility liquid of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy,whose best concentration of Chinese patent medicine have been confirmed by fore-experiment,observed their common situations(included by fur's colour,appetite situation,activity limit and so on).
3.Weighed tumor up,accounted rate of anti-tumor,analysed inhibiting of growth of tumor cell treated with Chinese patent medicine.
4.Observed shape's change of tumor cells by microscope.
5.Weighed spleen up,accounted rate of its exponential,analysed immunity effect of tumor-bearing mice treated with Chinese patent medicine.
6.TUNEL was applied to test the situation of apoptosis.
7.Immunohistochemistry was applied to analyze the expression level of cell cycle and apoptosis cell of CyclinD1、PCNA、Bcl-2 and Bax proteins in the cell,to research the relationship between the mechanism of proliferating,apoptosis,the regulation and control of gene expression.
Results
1.H_(22) tumor-bearing mice model was established successfully,,visible hypodermal tumor after 4 days,the rate of successful model is 100%.
2.Rate of anti-tumor of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy groups are 43.64%、50.91%and 45.45%.
3.The effect of splenic exponential was not obviously treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy(P>0.05).
4.The effect of proliferating was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy(P<0.05).
5.The effect of apoptosis was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy(P<0.05).
6.The data of immunohistochemistry indicated:compared with the model group,the expression of PCNA、CyclinD1 and Bcl-2 were reduced,Bax was increased remarkably after treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy (p<0.05).
Conclusion
1.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,especial for heat clearing and wetness removing therapy.
2.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can reduce cell's expression of PCNA,resist compose progress of DNA,then resist proliferating of carcinoma cell.
3.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,which mechanism may reduced expression of CyclinD1,blocked the way of CyclinD1-CDK4-Rb.
4.The apoptosis-associated proteins Bcl-2 and Bax were probably significant regulation factors of apoptosis-induced by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy.
5.The result of experiment indicated that promoting blood circulation and removing blood stasis therapy can resist the growth of PHC mostly by resisted proliferating of carcinoma cell.
6.The result of experiment indicated that heat clearing and wetness removing therapy and nourishing Yin of liver and kidney therapy can resist the growth of PHC mostly by abducted apoptosis of carcinoma cell.
引文
1.叶维法,钟振义.临床肝胆病学[M].天津:天津科学技术出版社,1998:35-38
2.谢慧珺.中药复方治疗肝癌机理的实验研究现状及展望[J].中医研究,2009,22(1):62-64
3.俞顺章.原发性肝癌流行病学研究进展[J].实用肿瘤杂志,1998,13(3):130
4.吴孟超.原发性肝癌外科综合治疗的现状和展望[J].中华外科杂志,2004,42(1):13-15
5.邹移海,徐志伟,苏钢强.实验动物学[M].北京:科学出版社,2004:154
6.李海燕,方肇勤,梁尚华.小鼠移植性肝癌(H_(22))模型的研究及在中医药抗肿瘤中的应用[J].中国中医基础医学杂志,2000,6(1):27-29
7.王永炎.中医内科学[M].上海:上海科学技术出版社.2003:78
8.黄承钰,曾令福,徐亚林,等.小鼠实体型肝癌H22移植实验条件探讨[J].临床与实验病理学杂志,1997,13(2):188-189
9.苗明三.实验动物与动物实验方法[M].上海:上海科学技术出版社,2005:67
10.张绪慧,陈达理,罗荣城.鳖甲煎丸对H22荷瘤小鼠的抑瘤作用及对增殖核抗原表达的影响[J].南方医科大学学报,2006,26(12):1791-1793
11.陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993:67
12.陈清勇,吴玉泉,何永生,等.复方抗瘤冲剂抑制小鼠肉瘤S_(180)的生长及对p53和增殖细胞核抗原表达的影响[J].解放军医学杂志,2001,26(11):853-855.
13.水若鸿.套细胞淋巴瘤石蜡包埋组织中细胞周期蛋白D1和t(11;14)易位检测的临床病理意义[J].中华病理学杂志,2003,8,32:337-341.
14.杜标炎,王慧峰,谭宇蕙等.六味地黄丸对小鼠移植性肝癌自杀基因治疗增效作用的病理学研究[J].广州中医药大学学报,2007,24(5):386-391.
15.许良中,杨文涛.免疫组织化学反应结果的判断标准[J].中国癌症杂志,1996,6(4):229-231
16.周振华,宋明志,于尔辛等.健脾理气方对小鼠HAC肝癌细胞凋亡和bax基因蛋白表达影响的实验研究[J].中国中西医结合脾胃杂志,2000,8(2):78-82
17.申杰.中医统计学[M].北京:人民军医出版社,2003
18.贺石静,王键,王净净.中医科研设计与统计学[M].长沙:湖南科学技术出版社,2001
19.陈平雁.SPSS13.0统计软件应用教程[M].北京:人民卫生出版社,2006
20.曾普华,潘敏求.中医药治疗原发性肝癌概述[J].湖南中医杂志,2003,19(4):58-59
21.段富津.方剂学[M].上海:上海科学技术出版社,2002
22.雷载权.中药学[M].上海:上海科学技术出版社,2001
23.刘明华,卿晨.刘吉开,等.伍氏百叶散对荷瘤小鼠肿瘤生长及免疫功能的影响[J].中国新药杂志.2006,15(3):201-204
24.Kastelan Z,Lukac 3.DerezicD,etal.Lymphocyte subsets,lym-phocyte reactivity tomitogens,NK cell activity and neutrophil andmonocyte phagocytic functions in patients with bladder carcinoma[J].AnticancerRes,2003,23(6D):5185-5189.
25.Van Sandick JW,BoermeesterM A,Gisbertz S S,et al.Lym-phocyte subsets and T(h) 1/T(h)2 immune responses in patientswith adenocarcinoma of the oesophagusor oesophagogastric junction:relation to pTNM stage and clinicaloutcome[J].CancerIm-munol Immunother,2003,52(10):617-624.
26.Hadden JW.Immunodeficiencyandcancer:prospects for correction[J].Int Immunopharmacol.2003.3(8):1061-1071.
27.邹仲之.组织学与胚胎学[M].北京:人民卫生出版社,第六版,2005
28.尤其,韩世愈,黄明莉.蛤蚧对S_(180)核肉瘤小鼠的抑瘤作用及对免疫系统的影响[J].哈尔滨医科大学学报.2005,39(5):402-404
29.Antonio Garcia-Espana.Edgard Salazar,Tung-Tien Sun,et al.Differertial Expression of Cell Cycle Regulators in Phenotypic Variants of Transgenically Induced Bladder Tumors:Implication for Tumor Behavior[J].Cancer Res,2005,65(4):1150-1157
30.Gary K,Schwartz,Manish A,et al.Targeting the Cell Cycle:A New Approach to Cancer Therapy[J].Journal of Clinical Oncology,2005,23(12):9408-9421
31.Boehm T,Folkman J,Browder T,et al.Antingiogenic therapy of experimental cancer does not induce acquired drug resistance[J].Nature,1997,390(6658):404-407
32.温进坤,韩梅.医学分子生物学理论与研究技术[M].北京:科学出版社.第二版.2002
33.Kubuki Y,Suzuki M,Sasaki H,et al.Telomerase activity and telomere length as prognostic factors of adult T-cell leukenia[J].Leuk Lymphona,2005,46(3):393-399
34.Micheal BM.Expression of nomal and tumor-associated antigen in human breast carcinoma[J].Nature,1984,309(9):374
35.Stanislav N,Naryzhny,Helen Zhao,et al.Proliferating Cell Nuclear Antigen (PCNA)May Function as a Double Homotrimer Complex in the Mammalian Cell[J].The Journal of Biological Chemistry.2005,280(14):13888-13894
36.Utpal K,Mukhopadyay,Adrian M,et al.RNA Silencing of Checkpoint Regulators Senditizes p53-Defective Prostate Cancer Cell to Chemotherapy while Sparing Normal Cell[J].Cancer Research,2005,65(3):2872-2881
37.Wildemann B,Schmidmaier G,Ordel S,et al.Cell proliferation and differentiation during fracture healing are influenced by locally applied IGF-I and TGF-β:Comparison of two proliferation markers,PCNA and BrdU[J].Bromed Mater Res,2003,65B(1):150-156
38.Bo Chen,Haiyan Pan,Liyin Zhu,et al.Progesterone Inhibits the Estrogen-Induced Phosphoinositide 3-Kinase→AKT→GSK-3β→Cyclin Dl→pRB Pathway to Block Uterine Epithelial Cell Proliferation[J].Molecular Endocrinology,2004,19(8):1978-1990
39.冯作化.医学分子生物学[M].北京:人民卫生出版社,2005
40.陈意生.肿瘤分子细胞生物学[M].北京:人民军医出版社.2004,144-162
41.王振义.肿瘤的诱导分化和凋亡疗法[M].上海:上海科学技术出版社,1998:143-173
42.Gavrieli Y,Sherman Y,Ben-Sasson SA.Identification of programmed cell death in situ via specific labeling of nuclear DNAfragmentation[J].Journal of Cell Biology,1992,119(3):493
43.刘文兰,张颖.细胞凋亡检测方法[J].解剖科学进展,1999,5(4):330-332
44.Inik Chang,Sunshin Kim,Ja Young Kim,et al.Nuclear Factor β Protects Pancreatic β-Cells From Tumor Necrosis Factor-α-Mediated Apoptosis[J].Diabetes,2003,52(5):1169-1175
45.Weiqin Jiang,Zongjian Zhu,Neehar Bhatia,et al.Mechanisms of Energy Restriction:Effects of Corticosterone on Cell Growth,Cell Cycle Mzchinery,and Apoptosis[J].Cancer Research,2002,62(15):5280-5287
46.龚守良.细胞凋亡分子机理的研究现状[J].中国实验诊断学.2000.4(3):147-148
47.Adams JM,Cory S.The bcl-2 protein family:arbiters of cell survival[J].Science.1998,281(5381):1322-1326
48.Humlova Z.Protooncogene Bcl-2 in process of apoptosis[J].Sb Lek,2002.103:419-425
49.Deveraux QL,Schendel SL,Reed JC.Antiapoptotic proteins:The Bcl-2 andinhibitor of apoptosis protein families[J].Cardiol Clin.2001,19:57-74
50.Klasa RJ,Gillum AM,Klein RE,Frankel SR.Oblimersen Bcl-2 antisense:facilitating apoptosis in anticancer treatment[J].Antisense Nucleic Acid Drug Dev.2002,12:193-213
51.季红斌,翟琦巍,郑仲承.Bcl-2基因的转录调控[J].生物化学与生物物理学报.2000.32(2):95-99
[1]于莉莉,李佩文,郝迎旭.平肺方抗癌作用的机实验研究[J].中国医药学报,1996,11(2):42.
[2]白绍槐.张旋波,沈文律.中药扶正消痞冲剂对人肝癌裸鼠移植瘤的抗癌作用[J].华西医科大学学报,1995,26(2):180-182.
[3]高俊峰.王长进,徐德新.等.中药消结灵抗小鼠肝癌细胞机理的研究[J].中国医科大学学报,997.26(2):126-128.
[4]李偃,邱培伦,任英云.等.新星1号抗肿瘤作用的实验研究[J].陕西中医,1996,17(10):473-475
[5]韦艾凌,唐健.癌痛消胶囊调节小鼠荷H22移植性肝癌细胞VEGF表达的实验研究[J].广西中医药,2004.27(4):47-49.
[6]张春荣,姜伟,齐元富.六神丸对鼠S_(180)生长的抑制作用与抑制血管生成的关系[J].中国预防医学杂志.2005,6(4):327-330.
[7]李炜,赵旭涛,孙莉.等.六神丸抗肿瘤血管生成的实验研究[J].中医药学报,2006,34(4):25-29.
[8]贾英杰,张莹,史福敏.复方苦参注射液对小鼠移植性S180肉瘤血管形成抑制作用[J].天津中医学院学报.2006.25(1):25-26.
[9]Rizzo MG,Ottavio L,Travali S.et al.The promoterof the human proliferating cell nuclear antigen(PCNA)gene is bidirecuional[J].Exp Cell Res,1990,188:286-293.
[10]张绪慧,陈达理,罗荣城.鳖甲煎丸对H22荷瘤小鼠的抑瘤作用及对增殖细胞核抗原表达的影响[J].南方医科大学学报.2006.26(12):1791-1793.
[11]杨大国.邓欣,李知玉,等.正肝方药物血清诱导人肝癌细胞分化的体外实验研究[J].中国中西医结合杂志,2004.24(2):140-142.
[12]刘平,周建锋,胡义扬,等.益气养阴诱导SMMC-7721肝癌细胞分化作用与意义[J].中国中医基础医学杂志.2000,6(8):29-31.
[13]戴恩来,赵健雄,朱玉真.等.扶正抑瘤汤对肿瘤细胞周期及端粒酶影响的实验研究[J].中国中西医结合杂志.2001.21(10):760-762.
[14]Thomberry NA,Rano TA.Peterson EP.et al.A combinatorial approach defines specificities of members of the caspase family and granzyme B.Functional relationships established for key mediators of apoptosis[J].JbiolChem,1997,272(29):17907-17911.
[15]张晓晖,姚天明,黄高舁.等.细胞凋亡的最新研究进展[J].第四军医大学学报,2002,23(Suppl):42-44.
[16]赵建雄,郭芙莲,白德成.等.扶正抑瘤颗粒对小鼠肝癌H22细胞凋亡率及线粒体膜电位的影响[J].中西医结合学报,2006,4(3):271-274.
[17]张天娥,骆永珍,闫智勇.克癌临治疗移植性肝癌(H22)的实验研究[J].华西医学,1998,13(4):451-453.
[18]刘华,毛小玲,钟海波.晚癌康2号对原发性肝癌细胞凋亡基因bcl-2,bax和Fas表达的影响[J].中医药学刊,2005.23(8):1409-1410.
[19]林小东,林礼务,薛恩生,等.复方中药99-克星瘤内注射治疗裸鼠肝癌的实验研究[J].中华医学超声杂志(电子版).2005.2(3):131-135.
[20]Kastelan Z,Lukac J.Derezic D.et a.1 Lymphocyte subsets,lymphocytereactivity tomitogens,NK cellactivity and neutrophilandmonocyte phagocytic functions in patients with bladder carcinoma.Anticancer Res,2003;23(6D):5185-5189
[21]van Sandick JW,BoermeesterMA,Gisbertz SS,etal.Lymphocyte subsets andT(h)1/T(h)2immune responses in patientswith adenocarcinoma of the oesophagusoroesophagogastric junction:relation to pTNM stage and clinical outcome.Cancer Immunol Immunother.2003;52(10):617-624
[22]周阿高,张勇.孔德云.等.大黄蜇虫丸对荷瘤小鼠免疫作用的研究[J].中成药.2006.28(12):1772-1774.
[23]蒋时红.李成海,王雪萍.等.益肝化毒胶囊对S180荷瘤小鼠IL-2、TNF-α的影响[J].中医研究.2004,17(6):16-17.
[24]邓毅峰,谢湘林,张大方.扶正消积胶囊对荷瘤动物免疫功能的影响[J].长春中医药大学学报.2007,23(1):19-20.
[25]周阿高,张勇,蒉纲,等.不同治法中药复方对H22肝癌小鼠瘤质量和体质量及免疫功能的影响[J].中西医结合学报.2008,6(1):77-82.
[26]朱小玉,陈运贤.钟雪云.等.参芪扶正注射液对化疗后免疫功能的保护作用研究[J].中国免疫学杂志.2006,22(10):925-928.
[27]郑王巧,宋丽华.李海菊,等.参芪扶正注射液对肝癌小鼠5-FU化疗后免疫功能的影响[J].长治医学院学报.2008,22(4):256-258.
[28]石世得,李任先,周岱翰.等.参桃软肝丸对荷肝癌小鼠的抑瘤作用及提高JL-2、NK活性的实验研究[J].中药新药与临床药理,2001,12(3):216-218.
[29]陈建业,陈俊杰,李瑞祥,等.护生宝口服液对H22肝癌小鼠抑癌作用的实验研究[J].中草药,2002,33(10):929-931.
[30]王鹏,陈震,黄雯霞.等.生脉注射液对H22肝癌小鼠的免疫调节作用[J].中药药理与临床.2005,21(6):1-3.
[31]曹治云,杜建,陈立武.等.中药复方对肝癌小鼠移植瘤体液免疫的影响[J].福建中医学院学报.2007,17(2):33-34