新健脾理气方联合热疗治疗晚期原发性肝癌的临床与实验研究
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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.
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.
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