摘要
研究目的:
1.临床部分
纳入Ⅲb-Ⅳ期、肝郁脾虚型原发性肝癌患者,治疗组采用新健脾理气方联合体外高频热疗,对照组采用单纯新健脾理气方,观察新健脾理气方联合热疗治疗晚期肝癌的临床疗效,探讨其在晚期原发性肝癌综合治疗中的作用,从而为临床治疗晚期肝癌提供新的治疗方案。
2.实验部分
采用人肝癌细胞株HepG2进行体外实验,探讨:①新健脾理气方联合热疗治疗肝癌的作用及机制;②新健脾理气方联合热疗是否能达到协同增效的效果。
研究方法:
1.临床部分
根据纳入标准选入41例肝功能Child-Pugh A或B级、Ⅲb-Ⅳ期(AJCC分期)、肝郁脾虚型原发性肝癌患者,分成治疗组和对照组。治疗组采用新健脾理气方联合体外高频热疗(新健脾理气方250ml bid po, d1-30;体外高频热疗采用珠海和佳公司生产的HG-2000型体外高频热疗机(电磁波频率为13.56MHZ),设置温度为42.5℃-43℃,每次60min,每周至少3次)。对照组单纯口服新健脾理气方治疗。30天为1周期,治疗连续3周期。在首3个月治疗,每月复查一次,之后每2个月复查一次,以评估两组的疾病控制率(DCR=CR+PR+SD)、疾病进展时间(TTP)、总体生存时间(OS)、毒副反应。所有数据采用SPSS13.0统计软件进行统计分析,患者的基本特征、近期疗效、毒副反应评价采用描述性统计,TTP、OS采用Kaplan-Meier分析;临床特征的组间比较采用方差分析和Pearsorn χ2检验;近期疗效比较采用两个独立样本Mann-Whitney U Test。双侧检验P<0.05为差异有统计学意义。
2.实验部分
选取人肝癌细胞株HepG2作为研究对象,细胞生长于含10%胎牛血清、100U/ml青霉素和100U/ml链霉素的DMEMH培养基中,细胞传代24h进入对数生长期后进行各种干预措施。新健脾理气方组加入0.25mg/ml、0.5mg/ml、lmg/ml、1.5mg/ml、2mg/ml浓度新健脾理气方溶液的完全培养基200ul;热疗组细胞置于43.0℃恒温循环水浴箱中孵育1小时;新健脾理气方+热疗组加入上述各浓度新健脾理气方溶液的完全培养基后,再放置于43℃恒温水浴箱中孵育1小时;对照1组加入等体积完全培养基,对照2组加入与2mg/ml新健脾理气方溶液等体积PBS的完全培养基,空白组加入不含细胞、与对照组等体积的培养基。处理后的各组细胞放置于37℃,5%CO2培养箱中继续培养,分别于24、48、72小时按实验要求进行。MTT法测定新健脾理气方联合热疗对肝癌细胞增殖的抑制作用;荧光倒置显微镜下观察新健脾理气方联合热疗作用后的人肝癌细胞株HepG2的形态学变化;流式细胞仪检测新健脾理气方联合热疗对人肝癌细胞株HepG2的细胞凋亡的影响;Western Blotting法检测Bax、Bcl-2蛋白的表达。
研究结果:
1.临床部分
(1)临床特征
患者于2010年9月至2012年12月纳入研究。治疗前两组基线资料,性别、年龄、分期、体力状态评分、肝功能分级、肝炎状态等均无显著性差异,组间分布均衡(P>0.05)。对照组1例因未遵循治疗方案而出组。实际可评估疗效病例40例,已死亡31例,其余9例正在随访中,41例患者的临床特征见表1-1。其中95%为男性患者,年龄32-79岁,中位年龄60岁。体力状态为1分、2分的患者分别为19例(46.3%)、15例(37.5%),3分的患者有7例(17.1%)。乙型肝炎背景的患者33例(80.5%),丙型肝炎患者3例(7.3%)。肝功能Child-Pugh分级中以A级患者为多,共29例(70.7%),B级的有12例(29.3%)。所有患者在入组时均属晚期肝癌,其中分期为Ⅲb/Ⅲc期和Ⅳ期的患者分别为20例(48.8%)和21例(51.2%)。
(2)中位疾病进展时间
治疗组和对照组中位疾病进展时间(mTTP)分别为4.5个月和3.1个月,治疗组的mTTP延长了1.4个月,与对照组比较差异有统计学意义(P<0.01)。
(3)中位总体生存时间
治疗组和对照组中位总生存时间(mOS)分别为6.6个月和5.5个月,治疗组的mOS延长了1.1个月,与对照组比较差异有统计学意义(P<0.01)。
(4)疾病控制率
治疗组和对照组的疾病控制率(DCR=CR+PR+SD)分别为25%和15%,治疗组的DCR略高于对照组,但两组比较差异无统计学意义。
(5)毒副反应
治疗组和对照组治疗前后血常规、肝肾功能、心电图检查均无明显变化,无明显血液学毒性反应,无呕吐、腹泻等胃肠道毒性反应,无过敏、脱发及皮肤毒性反应。
2.实验部分
(1)MTT法检测新健脾理气方联合热疗对人肝癌细胞株HepG2增殖的研究
新健脾理气方及新健脾理气方联合热疗作用不同时间对人肝癌细胞株HepG2增殖均有抑制作用,且随药物浓度的增大和作用时间的延长而增强,呈一定的时间和剂量依赖关系。经热疗处理后不同时间对人肝癌细胞株HepG2均有一定程度的抑制,增殖抑制率随作用时间的延长而增强,呈一定的时间依赖关系,其中以热疗后72h对人肝癌细胞株HepG2增殖抑制率为最高。新健脾理气方与热疗联合应用后的增殖抑制率高于单独应用热疗及单独应用新健脾理气方的增殖抑制率,具有协同效应,且以二者联合应用作用24h后对人肝癌细胞株HepG2抑制的协同效应最强。
(2)荧光显微镜及流式细胞仪检测新健脾理气方联合热疗对人肝癌细胞株HepG2凋亡的研究
新健脾理气方、热疗及新健脾理气方联合热疗作用于人肝癌细胞株HepG224h后,荧光显微镜下可观察到肝癌细胞出现凋亡相关的形态学改变。新健脾理气方联合热疗作用于人肝癌细胞株HepG2后,其细胞的凋亡形态学改变更为明显。新健脾理气方、热疗作用于人肝癌细胞株HepG224h后,肝癌细胞的早期凋亡率均有升高,具有一定的剂量依赖性。新健脾理气方联合热疗的凋亡率升高,二者联合应用的促凋亡作用增强。
(3)Western Blotting检测新健脾理气方联合热疗对人肝癌细胞株HepG2Bax. Bcl-2蛋白表达的研究
新健脾理气方、热疗及新健脾理气方联合热疗作用24h后出现Bax蛋白表达增强、Bcl-2蛋白表达减弱和Bax/Bcl-2升高,其中以终浓度为0.5mg/ml的新健脾理气方联合热疗时Bax/Bcl-2升高最为明显。选定终浓度为0.5mg/ml的新健脾理气方、热疗及二者联合作用不同时间,亦出现Bax蛋白表达增强,Bcl-2蛋白表达减弱及Bax/Bcl-2升高。二者联合作用24h的Bax蛋白表达增强最为明显,作用48h的Bcl-2蛋白表达减弱最明显,作用48h的Bax/Bcl-2升高最明显。
研究结论:
1.临床部分
本研究通过前瞻性随机对照试验,新健脾理气方联合体外高频热疗可有效延长总生存时间和疾病进展时间,且较单纯新健脾理气方中药治疗效果好,为不能接受手术、介入及靶向药物治疗的晚期原发性患者提供了新的中西医结合治疗方案。
2.实验部分
(1)新健脾理气方与热疗单独及联合应用对人肝癌细胞株HepG2均具有抑制增殖的作用,二者联合应用的增殖抑制率升高,具有协同效应。
(2)新健脾理气方与热疗单独及联合应用对人肝癌细胞株HepG2均具有诱导细胞凋亡的作用,二者联合应用的促凋亡作用增强。
(3)新健脾理气方与热疗单独及联合应用均上调人肝癌细胞株HepG2Bax蛋白表达水平,下调Bcl-2蛋白表达水平,二者联合应用时Bax蛋白表达增强更明显,Bcl-2蛋白表达减弱更明显。
Objective
1. Clinical study:To include advanced Hepatocellular Carcinoma patients, and divided into two groups. In the treatment group, Xin JianPi LiQi decoction (250ml, po, bid) combined with extracorporeal High Frequency Thermotherapy was given, while Xin JianPi LiQi decoction (250ml, po, bid) was given only in the control group. The purpose is to investigate the efficacy of the combination of these two treatments, to provide direction for clinical treatment, and providing ground and guideline for the use of the two treatments.
2. Experimental study:To use Human hepatocellular carcinoma HepG-2cell line to further investigate the effect and mechanism of XJPLQF combined with hyperthermia and whether it has synergic effect when they are used together in vitro.
Methods
1.Clinical study:Forty-one patients of advanced-stage hepatocellular carcinoma were recruited according to the inclusion criteria, and were divided into treatment and control group randomly. Patients were given Xin JianPi LiQi decoction (250ml, po, bid) combined with extracorporeal High Frequency Thermotherapy in the treatment group. Patients were given Xin JianPi LiQi decoction (250ml, po, bid) only in the control group.30days was a treatment period. After3cycles of treatment, the disease control rate (DCR), time to progression (TTP), overall survival (OS), and safety were assessed between two groups. SPSS13.0was used for statistical analysis. Descriptive analysis was used for baseline characteristics, short-term efficacy and safty. Kaplan-Meier analysis was performed for the comparison of TTP and OS. Variance analysis was conducted for the clinical characteristics between groups. All reported P values were two sided.
2. Experimental study:Hepatocellular carcinoma HepG-2cell line was selected in this study. It was cultivated in DMEM-H. The HepG2cells were divided into four groups:control group(normal cultured HepG2cells), JPLQF group(concentration of0.25mg/ml,0.5mg/ml,1.0mg/ml,1.5mg/ml,2. Omg/ml), hyperthermia group (cultured in the water bath43℃for1h and XJPLQF combined with hyperthermia group. The inhibition of cellular proliferation was detected by MTT method. The morphological alteration was observed by Hoechst33258staining. The apoptosis rates were analyzed by flow cytometry. The levels of Bax and Bcl-2proteins were detected by Western blotting analysis.
Results
1. Clinical study
(1) Baseline characteristics
41cases were recruited from the In-patient Oncology Department of Guangdong Provincial Hospital of TCM from September2010to December2012. The baseline characteristics, such as age, sex, staging, Eastern Cooperative Oncology Group (ECOG) performance status score, Child-Pugh liver function class and Chronic hepatitis virus infection, had no significant difference and was balanced between groups. One patient withdrew in control group because of change in self-decision. There were40evaluable cases,31cases deceased, while follow-up was conducting.
(2) Time to Progression (TTP)
By the cut-off date, patients in the treatment group had median time to progression of4.5months, as compared with3.1months in the control group. The TTP was prolonged for1.4months, indicating significant difference between two groups(P<0.01).
(3) Overall Survival (OS)
By the cut-off date, patients in the treatment group had median overall survival of6.6months, as compared with5.5months in the control group. The OS was prolonged for1.1months, indicating significant difference between two groups(P<0.05).
(4) Short-term Efficacy
Short-term efficacy was evaluated after3cycles of treatment. Disease control rate (DCR) was25%and15%in the treatment and control group. There was no significant difference between two groups.
(5) Safety
Safety evaluation indicated that there were no significant differences before and after treatment on blood routine, liver and Kidney function and ECG examination. There were no hepatotoxicity, gastrointestinal symptoms, alopecia or severe cardio, renal or hepatotoxicity.
2. Experimental study
(1) Effect of Xin JianPi LiQi decoction combined with hyperthmia on cell proliferation by MTT method.
Xin JianPi LiQi decoction administrated alone and combined with hyperthmia can inhibit the proliferation of human hepatocellular carcinoma HepG-2cells. The inhibition ratio was gradually enhanced with the rise of medicine concentration and time, that is they present time and dose dependent relation. Hyperthmia can also suppress the proliferation of human hepatocellular carcinoma HepG-2cells. The effect is in a time-dependent way and the72-hour's anti-proliferation rate is the highest one. The inhibitory effect on proliferation was promoted obviously when they were used together. The synergetic anti-proliferation rate was highest when they used together for24h.
(2) Effect of Xin JianPi LiQi decoction combined with on morphological alteration by Hoechst33258staining, and apoptosis rates by flow cytometry method.
Cells in each group been drug or hyperthmia administrated for24h without exception appeared a change on apoptosis morphology. The change of apoptosis morphology in electron microscope was most obvious in the group of Xin JianPi LiQi decoction combined with hyperthmia. The apoptotic rate was higher in each group compared with control group. The effect was in a dose dependent way. The apoptotic rate of was higher in the group of Xin JianPi LiQi decoction combined with hyperthmia than that of single XJPLQF or hyperthermia.
(3) Effect of Xin JianPi LiQi decoction combined with hyperthmia on Bax and Bcl-2proteins levels by Western blotting analysis
Xin JianPi LiQi decoction and hyperthmia administrated alone and together for24h can raise the expression of Bax proteins and the level of Bax/Bcl-2, but degrade the expression of Bcl-2proteins. The level of Bax/Bcl-2was up regulated most obviously after cells treated for24h by Xin JianPi LiQi decoction with concentration of0.5mg/ml, combined with hyperthmia. When Xin JianPi LiQi decoction and hyperthmia was administrated alone and together for different time by the concentration of Xin JianPi LiQi decoction of0.5mg/ml, the protein level of Bax and level of Bax/Bcl-2were also up regulated, the protein level of Bcl-2was also down regulated.
When Xin JianPi LiQi decoction and hyperthmia used together, the24-hour's expression of Bax proteins and the48-hour's level of Bax/Bcl-2was the highest, while the48-hour's expression of Bcl-2proteins was the lowest.
Conclusions
1.Clinical study
The time to progression and overall survival were siganificantly longer in Xin JianPi LiQi decoction combined with hyperthmia group than in the control group. Therefore, Xin JianPi LiQi decoction combined with hyperthmia can improve the survival and effectively prolong the TTP and OS.
2. Experimental study
(1) XJPLQF combined with hyperthermia has anti-proliferation effect on Human hepatocellular carcinoma HepG-2cells, of which anti-proliferation was higher than that of single XJPLQF or hyperthermia.
(2) XJPLQF combined with hyperthermia has apoptotic effect on Human hepatocellular carcinoma HepG-2cells, of which the apoptotic rate was higher than that of single XJPLQF or hyperthermia.
(3) XJPLQF combined with hyperthermia can promote the expression of Bax proteins and reduce the expression of Bcl-2proteins.
引文
[1]Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin,2011, Mar-Apr;61 (2):69-90. Epub 2011 Feb 4. Erratum in:CA Cancer J Clin,2011, Mar-Apr;61(2):134.
[2]Jemal A., Bray F., Center M. M., et al. Global cancer statistics [J]. CA:a cancer journal for clinicians,2011,61(2):69-90.
[3]卫生部.中国卫生统计年鉴[M].中国协和医科大学,2011,287-338.
[4]Ferlay J., Shin HR, Bray F., et al. Globcan 2008, Cancer Incidence and Mortality Worldwide:IARC Cancer Base No.10 (Internet). International Agency for Research on Cancer, Lyon, France[J]. globocan iarc fr,2010,98(15):1031.
[5]Thoas M. B., Zhu A. X. Hepatocellular carcinoma:the need for progress [J]. Journal of clinical oncology,2005,23(13):2892.
[6]Llovet J, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma. New Engl J Med,2008,359(4):378-390.
[7]Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma:a phase Ⅲ randomised, double-blind, placebo-controlled trial. Lancet Oncol,2009,10(1):25-34.
[8]Lau WY, Lai ECH. Hepatocellular carcinoma:current management and recent advances. Hepatobiliary and Pancreatic Diseases International,2008,7(3):237-257.
[9]叶显道.原发性肝癌外科治疗进展.铜陵职业技术学院学报,2010,3:25-27.
[10]樊嘉,王征.原发性肝癌的外科治疗进展[J]消化外科,2006,5(6):39724001.
[11]樊嘉,吴志全,汤钊猷等.不同治疗模式对不能切除的肝癌二期手术预后的影响[J]中华外科杂志,2001,39(10):745281.
[12]樊嘉,汤钊猷,吴志全,等.门静脉癌栓和肉眼癌栓对肝癌患者术后生存的影响[J].中华外科杂志,2005,43(7):433-435.
[13]Mazzaferro V, Regalia E, Doci R, et all. Liver transplantation for the treat2mnet of small hepatocellular carcinomas in patients with cirrhosis[J] N Engl Med,1996, 334(11):693291.
[14]Yao FY, Ferrell L, Bass NM, et a l1L iver transp lantation for hepatocellularcarcinoma: comparison of the p roposed UCSF criteria with the Milan crite2ria and the Pittsburgh modified TNM criteria[J]. Liver Transplant,2002,8(9):7652741
[15]Mazzaferro V, Regalia E, Doci R, et a l1L iver transp lantation for the treat2ment of small hepatocellular carcinomas in patients with cirhosis[J]. N Engl Med,1996, 334(11):693291.
[16]樊嘉,周俭,徐泱,等.肝癌肝移植适应证的选择:上海复旦标准[J].中华医学杂志,2006,86(18):12272311.
[17]樊嘉,杨广顺,傅志仁,等.肝癌肝移植适应证标准一验证及再思考[J].器官移植,2010,1(3):155-157
[18]刘芳,张阳,周金平,等.伊立替康联合化疗方案治疗原发性肝癌的临床研究[J].大连医科大学学报,2008,30(1):44-47.
[19]于芳,柳江,阿依夏木.奥沙利铂联合卡培他滨治疗晚期原发性肝癌[J].中国癌症杂志,2006,16(10):880.
[20]秦叔逵,曹梦苒,钱军等.奥沙利铂为主的FOLFOX方案治疗晚期原发性肝癌[J].临床肿瘤学杂志,2005,10(1):58-60.
[21]彭春雷,徐爱兵,苏小琴.吉两他滨联合奥沙利铂治疗中晚期原发性肝癌的临床对照研究[J].南通大学学报:医学版,2008,28(4):281-283.
[22]张俊凯,潘佩玲,吴颖猛,等.FOLFOX6方案治疗晚期原发性肝癌的临床观察[J].临床肿瘤学杂志,2010,15(1):70-74.
[23]Shim JH, Park JW, Nam BH, et al. Efficacy of combination chemotherapy with capecitabine plus cisplatin in patients with unresectable hepatocellular carcinoma. Cancer Chemother Pharmacol.2009,63 (3):459-67.
[24]Lee JO, Lee KW, Oh DY, et al. Combination chemotherapy with capecitabine and cisplatin for patients with metastatic hepatocellular carcinoma. Ann Oncol.2009,20 (8):1402-7.
[25]杨朝旭,秦叔逵.奥沙利铂治疗原发性肝癌的临床研究进展[J].临床肿瘤学杂志,2010,(09)
[26]S. Qin, Y. J. Wang, Q. Wu, et al. Phase III study of oxaliplatin plus 5-fluorouracil/leucovorin (FOLFOX4) versus doxorubicin as palliative systemic chemotherapy in advanced HCC in Asian patients.2010 American Society of Clinical Oncology.
[27]Uka K, Aikata H, Takaki S, et al. Systemic gemcitabine combined with intra-arterial low-dose cisplatin and 5-f luorouracil for advanced hepatocellular carcinoma:seven cases. World J Gastroenterol.2008,28; 14 (16):2602-89.
[28]Y. Z. Patt.PhaseⅡtrial of gemcitabine (G), oxaliplatin (0), and erlotinib (T) for the treatment of hepatocellular carcinoma (HCC) and biliary tree cancers (BTC) 2010 American Society of Clinical Oncology.
[29]亚砷酸注射液治疗原发性肝癌的Ⅱ期多中心临床研究[J].中华肿瘤杂志,2011,33(9):697-701.
[30]石洁,程树群.放射治疗肝癌门静脉癌栓的现状及展望[J].中国微创外科杂志,2005,5:125-126.
[31]Zhao JD, Xu ZY, Zhu J, Qiu JJ, Hu WG, Cheng LF, Zhang XJ, Jiang GL. Application of active breathing control in 3-dimensional conformal radiation therapy for hepatocellular carcinoma:the feasibility and benefit. Radiother Oncol,2008,87:439-444.
[32]苏学峰,戴建平.肝动脉化疗栓塞加三维适形放疗治疗肝癌38例临床观察[J].中国药物与临床,2008,8:318-319.
[33]Llovet J, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma. New Engl J Med,2008,359(4):378-390.
[34]Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma:a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol,2009,10(1):25-34
[35]Han GH, Yang JJ, Shao GL, et al. Evaluation of transcatheter arterial chemoembolization (TACE) plus sorafenib in Chinese patients with unresectable hepatocellular carcinoma: A subgroup analysis of START trial. J Clin Oncol 30,2012 (suppl; abstr e14605)
[36]Schwartz M, Roayaie S, Konstadoulakis M. Strategies for the management of hepatocellular carcinoma. Nat Clin Pract Oncol.2007; 4(7):424-432.
[37]Sun W, Sohal D, Hailer DG, et al. Phase 2 trial of bevacizumah, capecitabine, and oxaliplatin in treatment of advan ced hepatocellular carcinoma. Cancer,2011,117(14): 3187-3192.
[38]Ang SF, Tan SH, Toh HC, et al. Activity of thalidomide and capecitabine in patients with advanced hepatocellular carcinoma[J]. Am J Clin Oncol,2011 Mar 2.
[39]李雪梅.经肝动脉化疗栓塞术治疗肝癌的临床观察[J].实用肝脏病杂志,2006,(03).
[40]Varela M, RealM I, BurrelM, et al. Chemoembolization of hepatocellular carcinoma with drug eluting beads:efficacy and doxorubicin pharmaco2kinetics [J]. Hepatol,2007; 46(3):474281.
[41]Takayasu K, A rii S, lkai I, et a l1Prospective cohort study of trans arterial chemoembolization for unresectable hepatocellular carcinoma in 8510patients[J]. Gastroenterology,2006; 131(2):461291.
[42]郭晓华,何建荣,金红来,等.TACE联合经皮无水乙醇注射在原发性肝癌治疗中的价值[J].医学影像学杂志,2009,19(5):555-557.
[43]蔡联明,谢旭平,蓝小林,等.TACE联合PEI治疗原发性肝癌42例分析[J].实用临床医学,2009,10(2):49-51.
[44]张磊,卞建民.不同肝癌治疗中超声引导微波热凝的临床分析.实用医学杂志[J],2008,24:3546-3548.
[45]吕明德,徐辉雄,谢晓燕,等.经皮微波和射频消融治疗肝细胞性肝癌的临床比较研究[J].中华肝胆外科杂志2007;,13:528.
[46]陈敏山,张耀军,李锦清,等.射频消融治疗肝脏恶性肿瘤的八年经验总结(附803例报道)[J].中华外科杂志,2007,45(21):1469-1471.
[47]Lau WY, Lai EC. The current role of radiofrequency ablation in the management of hepatocellular carcinoma:a systematic review. Ann Surg,2009,249:20-25.
[48]Bruix J, Sherman M. AASLD PRACTICE GUIDELINE:Management of Hepatocellular Carcinoma: An Update (2010). J Hepatol,2010,53 (3):1020-1022.
[49]Cormier JN, Thomas KT, Chari RS, et al. Management of hepatocellular carcinoma [J]. Gastrointest Surg 2006; 10:761-780.
[50]周霖,杨永平,冯永毅,陆荫英,王春平,王新真,安林静,张昕,王福生.氩氦刀冷冻治疗原发性肝癌的初步临床研究[J].癌症,2009,(01).
[51]印春涛,黄健,徐高峰.氩氦刀冷冻消融联合肝动脉化疗栓塞治疗原发性肝癌[J].中国介入影像与治疗学,2011,8(2):117-120.
[52]汪少华,李丹,张郁馨,等.Exosomes瘤苗对小鼠肝癌的免疫治疗作用和免疫保护效应[J]广东医学,2010,31(增刊):31-33.
[53]Palmer DH, Midgley RS, Mirza N,Torr EE, et al. A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma,2009 Jan; 49(1):124-32.
[54]Bertelli R, Neri F, Tsivian M, et al. Endolymphatic immunotherapy in inoperable hepatocellular carcinoma[J]. Transplant Proc.2008 Jul-Aug; 40(6):1913-5.
[55]Hao MZ, Lin HL, Chen Q, et al. Efficacy of transcatheter arterial chemoembolization combined with cytokine-induced killer cell therapy on hepatocellular carcinoma:a comparative study[J]. Chin J Cancer,2010 Feb; 29(2):172-7.
[56]Li M, Lu C, Cheng J, et al. Combination therapy with transarterial chemoembolization and interferon-alpha compared with transarterial chemoembolization alone for hepatitis B virus related unresectable hepatocellular carcinoma[J]. J Gastroenterol Hepatol.2009 Aug; 24(8):1437-1444.
[57]史守良,董秀敏,胡万宁,等.P53基因治疗原发性肝癌的临床研究[J].现代中两医结合杂志,2012,21(10):1036-1037.
[58]Won YS, Lee SW. Targeted retardation of hepatocarcinoma cells by specific replacement of alpha-fetoprotein RNA. J Biotechnol 2007; 129:614-619.
[59]Glazer ES, Curley SA. Radiofrequency field-induced thermal cytotoxicity in cancer cells treated with fluorescent nanoparticles. Cancer.2010;116(13):3285-3293.
[60]FukamiT, Nakasu S, Baba K, et al. Hyperthermia induces translocation of apoptosis-inducing faetor(AIF) an apoptosis in human glioma cell lines[J].J Neurooncol,2004,70(3):319-331.
[61]Hildebrandt B, Wust P, Ahters 0, et al. The cellular and molecular basis of hyperthermia[J]. Crit Rev Oncol Hematol,2002; 43(1):33-56.
[62]OhnishiT. The role of the p53 molecule in cancer therapies with radiation and/or hyperthermia [J]. J Cancer Res Ther, 2005,1 (3):147-150.
[63]彭楠,赵彼得.临床肿瘤热疗学[M].北京:人民军医出版社,2002,92-95.
[64]Sawaji Y, Sato T, Takeuchi A, et al. Anti-angiogenic action of hyperthemia by suppressing gene expression and prodoction of tumour-derived vascular endothelial growth factor in vivo and in vitro[J].Br J Cancer,2002.86(10):1597-1603.
[65]丁向东,赵鑫,刘萍等.41.8℃全身热疗结合化疗治疗耐药性晚期非小细胞肺癌的临床应用[J].中国老年学杂志,2008,28(1):77-78.
[66]Othman T, Goto S,Lee JB, et al. Hyperthemic enhancement of the apoptotic and antiproliferative activities of paclitaxel[J]. Pharmacology,2001,62(4):208-212.
[67]康艳霞,张东伟,闵婕等.热疗联合长春瑞滨对肺癌细胞株作用的实验研究[J].临床肿瘤学杂志,2009,(csco年会专刊):260-261.
[68]潘博.潘敏求主任医师治疗肝癌经验[J].湖南中医杂志,2011,3(27):46-48.
[69]周岱翰.临床中医肿瘤学[M].北京:人民卫生出版社,2003:175.9.
[70]李朝军.刘嘉湘教授治疗肝癌经验[J].山西中医,2009,12(25):9-10.
[71]郑心婷.林丽珠教授治疗原发性肝癌经验介绍[J].2009,2(41):11-12.
[72]李永健,方肇勤,唐辰龙,等.2060例原发性肝癌中医证候分布规律的临床流行病学调查研究[J].中国医药学报,2003,3(18):144-146.
[73]司富春,岳静宇,刘紫阳.近30年临床原发性肝癌中医证型和用药规律分析.世界中西医结合杂志,2011,1(6):8-10.
[74]林丽珠,蓝韶清.原发性肝癌中医证型与免疫功能关系的研究[J].浙江中两医结合杂志,2001,11(7):408-410.
[75]万茜,章永红.原发性肝癌中医证候特征及与生存质量的关系[J].贵阳中医学院学报,2008,30(4):27-28.
[76]王泽民,丁霞,王婧.原发性肝癌的证素初探[J].中国中医基础医学杂志,2007,13(10)739-740.
[77]潘敏求,曾普华,潘博.中医药治疗中晚期原发性肝癌的规律探析[J].中医药学刊,2003,21(10):1 641.
[78]方肇勤,李永健,唐辰龙,等.2060例原发性肝癌患者证候特点分析[J].中医杂志,2004,45(1):53-54.
[79]李永健,方肇勤,唐辰龙,等.2060例原发性肝癌中医证候分布规律的临床流行病学调查研究[J].中国医药学报,2003,18(3):144-146.
[80]尹桂平.肝癌证治的中医文献研究.硕十论文.山东中医药大学.2005.
[81]陈德溯,方肇勤.中药治疗原发性肝癌中晚期的药味频率分析[J].辽宁中医杂志,2002,29(4):187-189.
[82]王吕俊,胡艳,付婷婷,等.广州地区原发性肝癌证候规律研究[J].中华中医药学刊,2010,29(11):2314-2318.
[83]廖桂雅.原发性肝癌病因与中医证型分析探讨.硕十论文,广州中医药大学,2008.
[84]刘鲁明主编.于尔辛肝癌经验集—健脾理气法则治疗肝癌的研究.人民卫生出版社,2004年4月,第1版:265-302.
[85]李瑞燕,高建平.党参粗多糖抗S180腹水瘤小鼠肿瘤的初步研究[J].长治医学院学报,2011,25(2):94-96.
[86]朱庆均,郑广娟,王江东.白术对S180荷瘤小鼠瘤组织P53基因表达的影响[J].山东中医药大学学报,2004,38(5):387.
[87]郑广娟.白术对小鼠S180肉瘤的抑瘤作用及肿瘤凋亡相关基因bc1-2表达的影响[J].生物医学工程研究,2003,22(3):48.
[88]孙向阳,邓毅.甘草多糖的主要药理作用概述.中国社区医师:医学专业.2011,24:179-180.
[89]覃玉桃,韦长元,李挺.慧苡仁总提取物注射液对晚期原发性肝癌患者免疫功能影响的研究[J].肿瘤防治杂志,2001,8(1):23.
[90]王佾先,孟旭晖,亢寿海,等.川陈皮素的抗癌作及机理研究[J].中华临床医学实践杂志,2004,3(3):261-263.
[91]钱十辉,王佾先,亢寿海,等.陈皮提取物体外抗肿瘤作用的研究[J].中药材,2003,26(10):744-745.
[92]赵浩如,李瑞,林以宁,等.白花蛇舌草不同提取工艺对抗肿瘤活性的影响[J].中国药科大学学报.2002,33(6):510-513.
[93]胡玲,罗晓韵,唐纯志.白花蛇舌草对H22肝癌细胞荷瘤组织bcl-2和bax蛋白表达的影响.中国中两医结合消化杂志,2011,6(19):378-381.
[94]吴万垠,郭伟剑,常钢.莪术油微球释放莪术油对人肝癌SMMC27721细胞的作用[J].世界华人消化杂志,2003,11(3):260-263.
[95]吴万垠,邓嵘,区勇全.经肝动脉灌注莪术油微球对大鼠移植性肝癌的治疗作用[J]中华肝脏病杂志2000,8(1):24-26.
[96]白雪,关宝生,魏晓东,等.八月札水提物对H22荷瘤鼠血清总抗氧化能力、超氧化物歧化酶活性和丙二醛含量的影响.社区医学杂志,2010,11(8):4-5.
[97]Shi XF, Du DJ, Xie DC, et all Effect of steroidal saponins in Rhizom a Parid is on H22 animal transplant tumor [J] J Chin Med Mater,1992,15 2:33-36.
[98]刘成,夏子禹,刘平,等.健脾理气方预防肝硬化及癌变的实验研究[J].中西医结合肝病杂志,1998,8(1):20-23.
[99]吴万垠,钱耕荪,于尔辛,等.健脾理气合剂抑制小鼠HBV协同AFB1致癌的机制[J].华人 消化杂志,1998,6(7):594-596.
[100]肖正明,宋景贵,徐晖,等.黄芪水提取物对体外培养人肝癌细胞增殖及代谢的影响[J].世界华人消化杂志,2000,8(1):4.
[101]于尔辛.健脾理气合剂对荷瘤脾虚模型小鼠免疫功能影响分析[J].肿瘤,1983,3(4):168-171.
[102]吉利,蔡玉文.辽宁中医杂志,2009,6(36):1041-1042.
[103]林丽珠.中医药在原发性肝癌综合治疗中对生存质量的维护作用[J].中华肿瘤杂志.2003,25(2):202-203.
[104]许得盛,沈白尹,王文健,等.右归饮、四君子汤、桃红四物汤调节肾虚、脾虚、血瘀证患者免疫功能的观察[J].中国中西医结合杂志,1999,19(12):712-714.
[105]郭伟剑,于尔辛,郑颂国,等.健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究[J].世界华人消化杂志,2000,8(1):52.
[106]盂志强,郭伟剑,于尔辛,等.健脾理气方药物血清对肝癌细胞端粒酶活性及凋亡的影响[J].世界华人消化杂志,2000,8(8):879.
[107]蔡玉文,邢晓静,王旭,等.中国中西医结合消化杂志,2009,4(17):211-213.
[108]吴洪斌,徐立涛,孟志强.健脾理气颗粒对H22肝癌小鼠抑瘤机制的实验研究[J].中华中医药杂志(原中国医药学报)2011,1(26):75-79.
[109]周振华,宋明志,于尔辛,等.健脾理气方药对小鼠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.
[110]孟志强,于尔辛,宋明志.健脾理气中药对肝癌端粒酶活性的影响.中国中医基础医学杂志,2000,1(6):23-26.
[111]陈震,于尔辛,宋明志,等.健脾理气中药抗肿瘤肝转移及其机理初步研究.中国临床医学,2002,1(9):46-48.
[112]陈颖,陈震,屠红,等.C57BL/6小鼠的血循环DNA定量研究[J].肿瘤,2005,5(25):427-429.
[113]陈震,黄雯霞,程琳.健脾理气中药上nm23表达[J].中国癌症杂志,2002,12(2):120-122.
[114]张书岭,尹桂山,王桂香,等.益气健脾汤对大鼠血浆、脾脏环核苷酸及其酶系的调整作用[J].中西医结合杂志,1990;10(11):672-674.
[115]于尔辛,刘鲁明,宋明志等.全肝移动条野放射结合中药治疗大肝癌的临床研究[J].中华肿瘤杂志,1992,14(1):57-60.
[116]郭伟剑,宋明志,于尔辛.原发性肝癌术后复发的外放射综合治疗-附23例临床病例分析[J].中国癌症杂志,1996,6(3):181-183.
[117]刘鲁明,于尔辛.放射结合中药治疗大肝癌预后的多因素分析[J].中国癌症杂志,1996,6(3):181-183.
[118]周振华,刘鲁明,黄雯霞等.中药联合肝动脉介入化疗和三维适形放疗治疗肝癌27例疗效观察[J].新中医,2003,35(10):38-40.
[119]赵增虎,杜欣,刘秀芳.中国中医药信息杂,2006,13(1):62-63.
[120]任华萍,宋明志,程琳.健脾理气方减少肝癌肝动脉化疗栓塞后毒副作用疗效观察.上海中医药杂志,2000,34(3).
[121]吴万垠,柴小妹,黄鹂等.亚砷酸联合健脾理气中药治疗32例晚期肝细胞癌.第十一届全国中两医结合肿瘤学术大会论文集,157-166.
[122]陈闯,张作军,陈蔚等.中国中两医结合消化杂志,2006,14(1):51-52.
[123]Ahmedin Jemal, Freddie Bray, Melissa M, et al. Global Cancer Statistics. CA CANCER J CLIN 2011,61:69-90.
[124]Yau T, Chan P, Ng KK, et al. Phase 2 open-lable study of single-agent sorafenib in treating advanced hepatocellular carcinama in a hepatitis B-endemic Asian population: presence of lung metastasis predicts poor response, Cancer.2009,115(2):428-436.
[125]Bolondi L, Caspary W, Bennouna J, et al. Clinical benefit of sorafenib in hepatitis C patients with hepatocelllular carcinoma (HCC):Subgroup analysis of the SHARP trial. Gastrointestinal Cancers Symposium, Orlando, Florida; 2008:abstr 129.
[126]Raoul J, Santoro A, Beaugrand M, et al. Efficacy and safety of sorafenib i in patients with advanced hepatocellular carcinama according to ECOG performance status:A subanalysis from the SHARP trial. Clin Oncol.2008,26(May 20 Suppl):abstr 4587.
[127]江明性主编.药理学,第三版,北京:人民卫生出版社,1989;35.
[128]杨藻寰主编.医用药理学,第二版,北京:人民卫生出版社,1982;23.
[129]金正均.合并用药中的相加.中国药理学报,1980,1(2):70-76.
[130]新健脾理气方对H22小鼠肝癌及EGFR-STAT3信号通路的影响.博十论文.广州中医药大学,2012.
[131]Hartwell LH and Weinert TA. Checkpoints:controls that ensure the order of cell cycle events [J]. Science,1989,246:629-634.
[132]Wyllie, AH, Kerr JFR and Currie AR. Cell death:The significance of apoptosis [J]. Int Rev Cytol,1980,68:251-306.
[133]Wyllie AH. Apoptosis:and overview. Br Med Bull,1997,53:451-465.
[134]Kroemer G., Galluzzi L., Vandenabeele P., et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009[J]. Cell Death & Differentiation,2008,16(1):3-11.
[135]Hildebrandt b, Wust P, Ahlers D, et al. The cellular and molecular basis of hyperthermia [J]. Crit Rev Oncol Hematol 2002,43:33-56.
[136]Wstermann AM, Grosen EA, Katschilllki DM, et al. A pilotstudy of wholebody hyperthermia and carboplatin in plalinuin resistant ovarian caneerr [J]. Eur J Caneer, 2001,37(9):1111-1117.
[137]0 Neill KL, Fairbairn DW, Smith MJ, Poe BS. Critical parameters ifluencing hyperthermia induced apoptosis in human lymphoid cell lines[J]. Apoptosis,1998,3:369-373.
[138]Beerheide W, Tan Y J, Teng E, et al. Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas [J]. Biochem Biophys Res Commun,2000,273 (1):54-61.
[139]O'Neill JW, Hockenbery DM. Bcl-2- related proteins as drug targets [J]. Curr Med Chem,2003,10 (16):1553-1562.
[140]Cory S, Adam s JM. Killing cancer cells by flipping the Bcl-2/Bax switch [J]. C ancer Cell,2005,8 (1):5-6.
[141]Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death [J]. Cell,1993,74(4):609-619.
[142]Stefanak i C, Antoniou C, S tefanaki K, et al. Bcl-2 and Bax incongen ital naevi [J].Br J Dermatol,2006,154 (6):1175-1179.
[143]单乐群,裘秀春,马保安,等.中低温度热疗诱导骨肉瘤细胞株凋亡[J].第四军医大学学报,2007,28(16):1482-1484.