清肝化瘀方药对原发性肝癌患者的疗效及肝癌患者含药血清对肝癌细胞增殖的抑制作用
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摘要
目的:原发性肝癌是我国常见的恶性肿瘤之一,死亡率占第二位。由于肝癌起病隐匿,早期常无明显症状,一旦发现多为中晚期,失去了手术机会。目前多采用肝动脉插管化疗栓塞(TACE)、超声引导无水乙醇瘤内注射(PEI)、多电极射频、超声聚焦刀等局部治疗,以及中医治疗。姚树坤教授根据中医传统理论和近十年的临床研究,以清热解毒、破瘀散结、健脾益气为治法,针对大多数患者的中医证型,研制成清肝化瘀方药,从临床和基础实验证实对肝癌前病变及癌变有明显的阻断作用。从癌基因、抑癌基因、抑制血管形成、细胞凋亡、细胞周期和免疫机制等多角度综合研究清肝化瘀方药的作用机理,表明清肝化瘀方药多环节、多靶点分子网络的作用机理,丰富与完善了中药抗癌研究的理论体系。本实验观察清肝化瘀方药对原发性肝癌患者的疗效及肝癌患者含药血清经丙酮或乙醇处理后对肝癌细胞增殖的抑制作用。
方法:参照2001年全国肝癌学术会议修订的原发性肝癌的诊断标准和临床分期标准,选择Ⅱb期或虽为Ⅲ期(没有远处转移、肝功能Child-Pugh B级),不能手术及肝动脉插管化疗栓塞的,肝胆湿热伴有血瘀证原发性肝癌患者20名,所有病例不同程度地伴有腹痛、纳差、黄疸、消瘦、乏力等。其中男性13例,女性7例,年龄在46-75岁之间,平均年龄(53.2±5.5)岁。Ⅱb期患者9例,Ⅲa期11例。
1患者给予清肝化瘀方药20ml,每日2次,饭后服用,42天为一疗程。服药期间忌食生冷、油腻、辛辣食品。每周观察并记录患者的临床表现,每两周检查肝功能及甲胎蛋白,于用药结束后复查CT,观察肿瘤缓解情况。
2患者分别于服药前、服药2周、4周、6周服药后2小时静脉采血10ml,分离血清。预处理(向血清内加入4倍量的丙酮混均,3000r/min离心,取上清温水浴蒸发,除去丙酮)。采用四唑盐比色法(简称MTT)比较经丙酮处理后含药血清对肝癌细胞SMMC-7721增殖的抑制作用。
3患者分别于服药前、服药2周、4周、6周服药后2小时静脉采血10ml,分离血清。预处理(向血清内加入4倍量的乙醇混均,3000r/min离心,取上清温水浴蒸发,除去乙醇)。采用MTT法比较经乙醇处理后含药血清对肝癌细胞HepG2增殖的抑制作用。
结果:1患者于用药后腹胀、肝区疼痛、纳差、食欲不振各单项积分较用药前明显减少,且都有统计学意义。有效率分别为:70.59%;76.92%;84.62%;83.33%。各症状平均消失时间为分别为17.4、19.5、14.8、15.2天,且缓解后未再次出现。各项生化指标及AFP较用药前均有明显下降,经统计学分析各指标与用药前相比有明显差异(P<0.05)。患者服用清肝化瘀方药后完全缓解0例,部分缓解0例,稳定12例,稳定率为60%。KPS评分改善者15例,占75%,稳定3例,降低2例。
2未经丙酮处理的用药6周后肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞增殖即体现出较强的抑制作用,抑制率可达28.6%,P=0.032,作用48小时,抑制作用更加明显,抑制率可达34.5%,P=0.035。经过丙酮处理的肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞SMMC-7721的抑制作用较未经丙酮处理含药血清对肝癌细胞SMMC-7721抑制作用稍弱,用药6周患者含药血清培养24小时抑制率可达26.8%,P=0.029。作用48小时后抑制率增强,抑制率可达33.1%,P=0.024,抑制效应呈时间依赖性,随着肝癌患者用药时间延长和对肝癌细胞作用时间的延长,抑制率增强。
3未经乙醇处理的用药6周后肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞增殖即体现出较强的抑制作用,抑制率可达29.2%,P=0.043,作用48小时,抑制作用更加明显,抑制率可达36.2%,P=0.029,并且对肝癌细胞的抑制率随患者用药时间延长和细胞培养时间延长而增加。经过乙醇处理的肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞HepG2的抑制作用较未经乙醇处理含药血清对肝癌细胞HepG2抑制作用稍弱,用药6周患者含药血清抑制率可达24.9%,P=0.022。作用48小时后抑制率增强,用药6周患者含药血清对肝癌细胞HepG2的抑制率最高可达33.2%,P=0.028。抑制效应同样呈时间依赖性,随着肝癌患者用药时间延长和对肝癌细胞作用时间的延长,抑制率增强。
结论:1清肝化瘀方药能明显缓解原发性肝癌患者的临床症状,降低各项生化指标及AFP值,改善肝功能分级,肿瘤稳定率为60%。
2经丙酮处理后肝癌患者含药血清对肝癌细胞SMMC-7721抑制作用较未处理含药血清弱。说明经丙酮处理后含药血清去除了非药物因素对实验结果的干扰,突出了中药的药理学作用,也可能经过处理,使药物作用于机体后产生的内生性有效成分失活而失去或减少其原有疗效。
3经乙醇处理后清肝化瘀方药含药血清对肝癌细胞HepG2的抑制作用较未处理含药血清弱。说明经过处理,使药物作用于机体后产生的内生性有效成分失活而失去或减少其原有疗效。
Objective: Primary liver cancer(PLC) is one of the commonest malignant diseases in our country. The mortality rate is the second of all diseases. Because of onset occult, it is often no obvious symptoms in the early period, most of them lose the opportunity of surgical resection when detected. So most patients receive transcatheter arterial chemoembolization (TACE), percutaneous ethano injection(PEI), radio frequency- ablation(RFA), focused ultrasound(FUS) or Chinese traditional medicine(TCM). According to the theory of TCM and clinical study over ten years, professor YaoShukun had developed Qinggan Huayu recipe with the therapeutic principles of clearing away heat and toxic material, removing blood stasis and resolving lumps, strengthening the spleen and replenishing qi. Researches had proved that Qinggan Huayu recipe had obvious repressive effect on the genesis of PLC. The mechanisms of oncogene, antioncogene, anti-angiogenesis, apoptosis, cell cycle and immune function on Qinggan Huayu recipe had been investigated which showed the mult-targets and integral regulation of TCM. This research was to observe the effect of Qinggan Huayu Recipe on primary liver cancer patients and the inhibition of drug serum of liver cancer patients treated by acetone or ethanol on hepatoma cells.
Methods: According to a prognostic and clinical pathological criterion which mended by Society of Liver Cancer(CACA) in the 2001 Chinese liver cancer conference, 20 PLC patients were chosen , who had damp -heat of liver and gall with blood stasis syndrome pathological stageⅡb orⅢ(no faraway organ metastasis, liver function rank Child-Pugh B)PLC patients which had lost an operation chance and transcatheter arterial chemoembolization, all the patients company with varying degrees of abdominal pain、dyspepsia、jaundice、emaciation、hypodynamia ect. There were 13 males and 7 females, at the age between 46-75 years old, the average age was 53.2±5.5,Ⅱb patients with nine cases,Ⅲwere 11 cases.
1 20ml Qinggan Huayu Recipe was taken by Patients, twice perday, aftermeals. 42 days was one treatment course. Anything raw, cold, greasy or spicy was avoid ate while taking medicatio(during treatment). The patients' clinical manifestation was observed and recorded everyweek, liver function and alpha-fetoprotein was checked every two weeks, CT was reexaminationed after use, the remission of tumor was observed .
2 10ml venous blood of the patients were collected on weeks 0, 2, 4, 6 of the treatment, 2 hours after drug taken. The serum was joined four times acetone, well mixing, centrifuged at 3000r/min for the supernatants and then acetone was evaporated by warm bath as pretreatment. Tetrazolium bromide colorimetric assay(MTT) was taken to evaluate the inhibition effect of medicated serum pretreated by acetone on proliferation of SMMC-7721.
3 10ml venous blood of the patients were collected on weeks 0, 2, 4, 6 of the treatment, 2 hours after drug taken. The serum was joined four times ethanol, well mixing, centrifuged at 3000r/min for the supernatants and then ethanol was evaporated by warm bath as pretreatment. Tetrazolium bromide colorimetric assay(MTT) was taken to evaluate the inhibition effect of medicated serum pretreated by ethanol on proliferation of HepG2.
Result:1 The single points of abdominal distension、liver pain、dyspepsia、inappetence could be obviously reduced than those pre-administration. The respectively effective rate was 70.59%;76.92%;84.62%;83.33%. The average time for symptoms disappeared was 17.4、19.5、14.8、15.2, and not appear again after ease. The biochemical indicators and AFP were obviously decreased than those pre-administration, and by statistical analysis of each index in therapy mean have obvious difference. 0 was obtained complete relief by taking Qinggan Huayu Recipe, 0 was obtained partial relief, 12 cases stable, the stable rate was 60%. KPS score was improved in 15 cases, accounting for 75%, stabilized in 3 cases, lowed in 2 cases.
2 Strong inhibitory effect was showed on multiplication of hepatocellular carcinoma cell which incubated for 24 hours with the Qinggan Huayu Recipe serum no-acetone-treated from PLC patients taking drug for 6 weeks. The 24 hours’inhibition rate was 28.6%,P=0.032.The 48 hours’inhibition rate was more obvious , up to 34.5%,P=0.035. A little weaker inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum acetone-treated from PLC patients taking drug for 6 weeks,which was 26.8% , P=0.029, compared with the no-acetone-treated serum of PLC patients, was up to 33.1%,P=0.024 for the 48 hours’inhibition rate. Parallel with the increasing time of patients’drug taking and incubation of hepatocellular carcinoma cell, the inhibitory effect was increased and was time-dependent.
3 Strong inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum no-ethanol-treated from PLC patients taking drug for 6 weeks. The 24 hours’inhibition rate was 29.2%,P=0.043. More obvious was the 48 hours’inhibition rate,up to 36.2%,P=0.029. A little weaker inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum ethanol-treated from PLC patients taking drug for 6 weeks, which was 24.9%, P=0.022, compared with the no-ethanol-treated serum of PLC patients, and was up to 33.2%,P=0.028 for the 48 hours’inhibition rate. Parallel with the increasing time of patients’drug taking and incubation of hepatocellular carcinoma cell, the inhibitory effect was increased and was time-dependent.
Conclusions: The clinical symptoms in patients with primary liver cancer could be significantly alleviated by taken Qinggan Huayu Recipe, the biochemical indexes and AFP could be reduced, liver function grade could be improved, and the tumor stable rate was 60%.
The inhibition to SMMC-7721 of drug serum of PLC which was treated by acetone was weaker than that without acetone treated drug serum. It was demonstrated that after treated by acetone the interference of non-drug factors on the experimental was removed, then the pharmacology role of Chinese medicine was highlighting. However, it was probably that after treatment, the drug effects was arised after the endogenous inactivation was deactivated and then its original efficacy was reduced.
The inhibition to HepG2 of drug serum of PLC which was treated by ethanol was weaker than that without ethanol treated drug serum.It was probably that after treatment, the drug effects was arised after the endogenous inactivation was deactivated and then its original efficacy was reduced .
方法:参照2001年全国肝癌学术会议修订的原发性肝癌的诊断标准和临床分期标准,选择Ⅱb期或虽为Ⅲ期(没有远处转移、肝功能Child-Pugh B级),不能手术及肝动脉插管化疗栓塞的,肝胆湿热伴有血瘀证原发性肝癌患者20名,所有病例不同程度地伴有腹痛、纳差、黄疸、消瘦、乏力等。其中男性13例,女性7例,年龄在46-75岁之间,平均年龄(53.2±5.5)岁。Ⅱb期患者9例,Ⅲa期11例。
1患者给予清肝化瘀方药20ml,每日2次,饭后服用,42天为一疗程。服药期间忌食生冷、油腻、辛辣食品。每周观察并记录患者的临床表现,每两周检查肝功能及甲胎蛋白,于用药结束后复查CT,观察肿瘤缓解情况。
2患者分别于服药前、服药2周、4周、6周服药后2小时静脉采血10ml,分离血清。预处理(向血清内加入4倍量的丙酮混均,3000r/min离心,取上清温水浴蒸发,除去丙酮)。采用四唑盐比色法(简称MTT)比较经丙酮处理后含药血清对肝癌细胞SMMC-7721增殖的抑制作用。
3患者分别于服药前、服药2周、4周、6周服药后2小时静脉采血10ml,分离血清。预处理(向血清内加入4倍量的乙醇混均,3000r/min离心,取上清温水浴蒸发,除去乙醇)。采用MTT法比较经乙醇处理后含药血清对肝癌细胞HepG2增殖的抑制作用。
结果:1患者于用药后腹胀、肝区疼痛、纳差、食欲不振各单项积分较用药前明显减少,且都有统计学意义。有效率分别为:70.59%;76.92%;84.62%;83.33%。各症状平均消失时间为分别为17.4、19.5、14.8、15.2天,且缓解后未再次出现。各项生化指标及AFP较用药前均有明显下降,经统计学分析各指标与用药前相比有明显差异(P<0.05)。患者服用清肝化瘀方药后完全缓解0例,部分缓解0例,稳定12例,稳定率为60%。KPS评分改善者15例,占75%,稳定3例,降低2例。
2未经丙酮处理的用药6周后肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞增殖即体现出较强的抑制作用,抑制率可达28.6%,P=0.032,作用48小时,抑制作用更加明显,抑制率可达34.5%,P=0.035。经过丙酮处理的肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞SMMC-7721的抑制作用较未经丙酮处理含药血清对肝癌细胞SMMC-7721抑制作用稍弱,用药6周患者含药血清培养24小时抑制率可达26.8%,P=0.029。作用48小时后抑制率增强,抑制率可达33.1%,P=0.024,抑制效应呈时间依赖性,随着肝癌患者用药时间延长和对肝癌细胞作用时间的延长,抑制率增强。
3未经乙醇处理的用药6周后肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞增殖即体现出较强的抑制作用,抑制率可达29.2%,P=0.043,作用48小时,抑制作用更加明显,抑制率可达36.2%,P=0.029,并且对肝癌细胞的抑制率随患者用药时间延长和细胞培养时间延长而增加。经过乙醇处理的肝癌患者清肝化瘀方药含药血清作用24小时对肝癌细胞HepG2的抑制作用较未经乙醇处理含药血清对肝癌细胞HepG2抑制作用稍弱,用药6周患者含药血清抑制率可达24.9%,P=0.022。作用48小时后抑制率增强,用药6周患者含药血清对肝癌细胞HepG2的抑制率最高可达33.2%,P=0.028。抑制效应同样呈时间依赖性,随着肝癌患者用药时间延长和对肝癌细胞作用时间的延长,抑制率增强。
结论:1清肝化瘀方药能明显缓解原发性肝癌患者的临床症状,降低各项生化指标及AFP值,改善肝功能分级,肿瘤稳定率为60%。
2经丙酮处理后肝癌患者含药血清对肝癌细胞SMMC-7721抑制作用较未处理含药血清弱。说明经丙酮处理后含药血清去除了非药物因素对实验结果的干扰,突出了中药的药理学作用,也可能经过处理,使药物作用于机体后产生的内生性有效成分失活而失去或减少其原有疗效。
3经乙醇处理后清肝化瘀方药含药血清对肝癌细胞HepG2的抑制作用较未处理含药血清弱。说明经过处理,使药物作用于机体后产生的内生性有效成分失活而失去或减少其原有疗效。
Objective: Primary liver cancer(PLC) is one of the commonest malignant diseases in our country. The mortality rate is the second of all diseases. Because of onset occult, it is often no obvious symptoms in the early period, most of them lose the opportunity of surgical resection when detected. So most patients receive transcatheter arterial chemoembolization (TACE), percutaneous ethano injection(PEI), radio frequency- ablation(RFA), focused ultrasound(FUS) or Chinese traditional medicine(TCM). According to the theory of TCM and clinical study over ten years, professor YaoShukun had developed Qinggan Huayu recipe with the therapeutic principles of clearing away heat and toxic material, removing blood stasis and resolving lumps, strengthening the spleen and replenishing qi. Researches had proved that Qinggan Huayu recipe had obvious repressive effect on the genesis of PLC. The mechanisms of oncogene, antioncogene, anti-angiogenesis, apoptosis, cell cycle and immune function on Qinggan Huayu recipe had been investigated which showed the mult-targets and integral regulation of TCM. This research was to observe the effect of Qinggan Huayu Recipe on primary liver cancer patients and the inhibition of drug serum of liver cancer patients treated by acetone or ethanol on hepatoma cells.
Methods: According to a prognostic and clinical pathological criterion which mended by Society of Liver Cancer(CACA) in the 2001 Chinese liver cancer conference, 20 PLC patients were chosen , who had damp -heat of liver and gall with blood stasis syndrome pathological stageⅡb orⅢ(no faraway organ metastasis, liver function rank Child-Pugh B)PLC patients which had lost an operation chance and transcatheter arterial chemoembolization, all the patients company with varying degrees of abdominal pain、dyspepsia、jaundice、emaciation、hypodynamia ect. There were 13 males and 7 females, at the age between 46-75 years old, the average age was 53.2±5.5,Ⅱb patients with nine cases,Ⅲwere 11 cases.
1 20ml Qinggan Huayu Recipe was taken by Patients, twice perday, aftermeals. 42 days was one treatment course. Anything raw, cold, greasy or spicy was avoid ate while taking medicatio(during treatment). The patients' clinical manifestation was observed and recorded everyweek, liver function and alpha-fetoprotein was checked every two weeks, CT was reexaminationed after use, the remission of tumor was observed .
2 10ml venous blood of the patients were collected on weeks 0, 2, 4, 6 of the treatment, 2 hours after drug taken. The serum was joined four times acetone, well mixing, centrifuged at 3000r/min for the supernatants and then acetone was evaporated by warm bath as pretreatment. Tetrazolium bromide colorimetric assay(MTT) was taken to evaluate the inhibition effect of medicated serum pretreated by acetone on proliferation of SMMC-7721.
3 10ml venous blood of the patients were collected on weeks 0, 2, 4, 6 of the treatment, 2 hours after drug taken. The serum was joined four times ethanol, well mixing, centrifuged at 3000r/min for the supernatants and then ethanol was evaporated by warm bath as pretreatment. Tetrazolium bromide colorimetric assay(MTT) was taken to evaluate the inhibition effect of medicated serum pretreated by ethanol on proliferation of HepG2.
Result:1 The single points of abdominal distension、liver pain、dyspepsia、inappetence could be obviously reduced than those pre-administration. The respectively effective rate was 70.59%;76.92%;84.62%;83.33%. The average time for symptoms disappeared was 17.4、19.5、14.8、15.2, and not appear again after ease. The biochemical indicators and AFP were obviously decreased than those pre-administration, and by statistical analysis of each index in therapy mean have obvious difference. 0 was obtained complete relief by taking Qinggan Huayu Recipe, 0 was obtained partial relief, 12 cases stable, the stable rate was 60%. KPS score was improved in 15 cases, accounting for 75%, stabilized in 3 cases, lowed in 2 cases.
2 Strong inhibitory effect was showed on multiplication of hepatocellular carcinoma cell which incubated for 24 hours with the Qinggan Huayu Recipe serum no-acetone-treated from PLC patients taking drug for 6 weeks. The 24 hours’inhibition rate was 28.6%,P=0.032.The 48 hours’inhibition rate was more obvious , up to 34.5%,P=0.035. A little weaker inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum acetone-treated from PLC patients taking drug for 6 weeks,which was 26.8% , P=0.029, compared with the no-acetone-treated serum of PLC patients, was up to 33.1%,P=0.024 for the 48 hours’inhibition rate. Parallel with the increasing time of patients’drug taking and incubation of hepatocellular carcinoma cell, the inhibitory effect was increased and was time-dependent.
3 Strong inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum no-ethanol-treated from PLC patients taking drug for 6 weeks. The 24 hours’inhibition rate was 29.2%,P=0.043. More obvious was the 48 hours’inhibition rate,up to 36.2%,P=0.029. A little weaker inhibitory effect was showed on multiplication of hepatocellular carcinoma cell incubated for 24 hours with the Qinggan Huayu Recipe serum ethanol-treated from PLC patients taking drug for 6 weeks, which was 24.9%, P=0.022, compared with the no-ethanol-treated serum of PLC patients, and was up to 33.2%,P=0.028 for the 48 hours’inhibition rate. Parallel with the increasing time of patients’drug taking and incubation of hepatocellular carcinoma cell, the inhibitory effect was increased and was time-dependent.
Conclusions: The clinical symptoms in patients with primary liver cancer could be significantly alleviated by taken Qinggan Huayu Recipe, the biochemical indexes and AFP could be reduced, liver function grade could be improved, and the tumor stable rate was 60%.
The inhibition to SMMC-7721 of drug serum of PLC which was treated by acetone was weaker than that without acetone treated drug serum. It was demonstrated that after treated by acetone the interference of non-drug factors on the experimental was removed, then the pharmacology role of Chinese medicine was highlighting. However, it was probably that after treatment, the drug effects was arised after the endogenous inactivation was deactivated and then its original efficacy was reduced.
The inhibition to HepG2 of drug serum of PLC which was treated by ethanol was weaker than that without ethanol treated drug serum.It was probably that after treatment, the drug effects was arised after the endogenous inactivation was deactivated and then its original efficacy was reduced .
引文
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