摘要
目的:
原发性肝癌起病隐匿,恶性程度高,病情变化快,死亡率高,是我国最常见的恶性肿瘤之一,目前临床上缺乏有效的治疗方法。因此,寻找疗效明显、毒副反应少、延长带瘤生存时间和提高带瘤生活质量的肝癌治疗方法是目前临床肿瘤学的研究难点。
扶正抗癌方是笔者临床上用于治疗肝癌的中药验方;静脉注射维生素C(VC)是近年来在肿瘤基础研究和临床应用方面有潜在价值的研究热点之一。在前期临床研究结果及参考国外相关研究资料的基础上,本研究以Walker256移植性大鼠肝癌模型、二乙基亚硝胺(DEN)诱发性大鼠肝癌模型,及用体外培养的大鼠肝癌细胞(CBRH7919)和大鼠正常肝细胞(BRL)为研究载体,观察扶正抗癌方、VC及两者联用对大鼠肝癌发生、发展过程的干预作用,并进一步探讨其可能机制。
方法:
1.采用Walker256癌肉瘤细胞株接种SD大鼠制作原位移植肝癌模型,分为正常组、假手术组、模型组、中药低剂量组(扶正抗癌方水煎剂12.1g/kg灌胃)、中药中剂量组(扶正抗癌方水煎剂24.2g/kg灌胃)、中药高剂量组(扶正抗癌方水煎剂48.4g/kg灌胃)。定时记录体重、进食及饮水等一般状况;实验w1、w2、w3取材,检测血清总蛋白(STP)、白蛋白(ALB)、球蛋白(GLB)、白球比值(A/G)、谷丙转氨酶(ALT)、谷草转氨酶(AST)及肿瘤诊断指示酶碱性磷酸酶(ALP)、γ-谷氨酰转移酶(GGT)和岩藻糖苷酶(AFU);称瘤重,测量瘤块长短径;取瘤组织行病理观察;记录肿瘤转移情况;观察各组大鼠的生存时间。研究不同剂量的扶正抗癌方对移植性大鼠肝癌的抑制作用,并探索最佳量效关系。
2.制备Walker256移植性肝癌模型,分为正常组、假手术组、模型组、VC低剂量组(维生素C钠2.83g/kg静脉注射)、VC高剂量组(维生素C钠5.65g/kg静脉注射)。定时记录各组大鼠的一般状况;检测肝功能(STP、ALB、GLB、A/G、ALT、AST、ALP、GGT、AFU);称瘤重、测量瘤块长短径;取瘤组织行病理观察。研究静脉注射不同剂量VC对移植性大鼠肝癌的抑制作用。
3.制备DEN诱发大鼠肝癌模型,分别给予扶正抗癌方48.4g/kg、维生素C钠2.83g/kg、扶正抗癌方48.4g/kg与维生素C钠2.83g/kg联用、化疗药优福定(UFT)0.09g/kg治疗。造模后w8、w14、w20取材,定时记录各组大鼠体重、进食饮水等一般状况;检测STP、ALB、GLB、A/G、ALT、AST、ALP、GGT、AFU,观察各组大鼠肝功能的变化;检测外周血中CD~(4+)/CD~(8+)比值和白细胞介素2(IL-2)表达水平,了解机体的免疫功能状态;检测肝组织匀浆超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、丙二醛(MDA)表达量,了解大鼠肝脏氧化损伤和抗自由基能力;HE染色观察大鼠肝脏病理学变化;免疫组化法检测肝癌标记物甲胎蛋白(AFP)及肝癌组织中血管内皮生长因子(VEGF)的表达;运用PCR及实时定量PCR技术,分别检测p53基因突变情况和c-myc、cyclinD1的表达量;记录各组大鼠的生存时间。
4.利用血清药理学方法制备扶正抗癌方、VC、扶正抗癌方与VC联用、UFT的大鼠含药血清,分别给予2.5%、5%、10%三个浓度的上述含药血清,应用CCK8法检测扶正抗癌方、VC、扶正抗癌方与VC联用、UFT含药血清培养不同时间(24h、48h、72h)对BRL细胞和CBRH7919细胞增殖的影响;选择对CBRH7919细胞有选择性杀伤作用的最佳含药血清浓度及血清孵育时间,运用流式细胞术进一步观察各组CBRH7919细胞周期及凋亡率的变化,并运用化学比色法检测各组CBRH7919细胞凋亡相关蛋白Caspase-3表达量的变化。
结果:
1.扶正抗癌方对大鼠Walker256移植性肝癌的抑制作用
1.1不同剂量的扶正抗癌方在不同时间段保肝护肝作用肝功能结果显示:与模型组比较,实验w1,中药高剂量组AST降低(P＜0.05)。实验w2,低剂量组ALB、A/G升高(P＜0.05),GGT降低(P＜0.05),ALT、ALP显著降低(P＜0.01);中剂量组ALB、A/G升高(P＜0.05),ALT、ALP、GGT则显著降低(P＜0.01);高剂量组ALB升高(P＜0.05),A/G亦显著升高(P＜0.01),ALT、ALP、GGT则显著低于模型组(P＜0.01)。实验w3,中药低剂量组ALT、AST、GGT降低(P＜0.05),ALP则显著降低(P＜0.01);中剂量组ALB较模型组升高(P＜0.05),ALT、AST、ALP、GGT显著降低(P＜0.01);高剂量组A/G升高(P＜0.05),ALB亦明显高于模型组(P＜0.01),ALT、AST、ALP、GGT、AFU则显著降低(P＜0.01)。
1.2不同剂量的扶正抗癌方在不同时间段的抑癌作用实验w1-w3,中药各剂量组均能抑制肿瘤生长,其中高剂量组于实验w3时的肿瘤重量抑制率高于低剂量组(P＜0.05)。病理结果示,高剂量扶正抗癌方在肝癌早、中期(w1、w2)可抑制癌细胞向周围浸润,在肝癌晚期(w3)则能促进癌细胞坏死。实验w3,中药低、中、高剂量组肿瘤转移评分均低于模型组(P＜0.05)。
1.3不同剂量的扶正抗癌方对大鼠生存时间的影响中药高剂量组大鼠的生存时间延长,与模型组比较有差异(P＜0.05)。
2.维生素C对大鼠Walker256移植性肝癌的抑制作用
2.1维生素C的保肝护肝作用VC低剂量组血清A/G高于模型组(P＜0.05),ALT明显低于模型组(P＜0.01),GGT亦低于模型组(P＜0.05);VC高剂量组血清ALT亦低于模型组(P＜0.05)。
2.2维生素C的抑癌作用VC抑制肿瘤体积和重量的效果不明显(P＞0.05),但病理结果显示VC可促进肿瘤细胞坏死,其中VC低剂量组肝癌的坏死程度高于模型组(P＜0.05)。
3.扶正抗癌方、维生素C及两者联用对DEN诱发大鼠肝癌的防治作用
3.1不同时间段各组大鼠肝功能变化实验w8,中药组、VC组血清ALP较模型组降低(P＜0.05),中药与VC联用不仅能显著降低ALP(P＜0.01),还能抑制肝脏AST的释放,与模型组比较有差异(P＜0.05)。实验w14,中药、VC以及两者联用保肝护肝的作用更明显,其中中药组ALT、AST显著低于模型组(P＜0.01),中药还能抑制肝脏GGT的合成,含量较模型组降低(P＜0.05);与模型组比较,VC组、中药+VC组ALT降低(P＜0.05),AST显著降低(P＜0.01),此外还可改善蛋白合成功能,其中VC组STP、GLB回升(P＜0.05),中药+VC组作用更明显,STP、GLB显著高于模型组(P＜0.01)。随着肝癌的进展,实验w20,中药仍能抑制肝脏释放AST,表达量低于模型组(P＜0.05);中药+VC组AST显著低于模型组(P＜0.01),且ALT亦低于模型组(P＜0.05),化疗组各指标与模型组比较无差异(P＞0.05)。
3.2不同时间段各组大鼠肝癌进展的比较病理结果显示中药、VC及两者联用可延缓癌变进展,在诱癌晚期还可以促进癌细胞坏死,UFT亦能促进癌细胞坏死。此外,w8,w14时,中药+VC组AFP低于模型组(P＜0.05),w20时,中药+VC组AFP进一步降低,较模型组差异显著(P＜0.01),且明显低于化疗组(P＜0.05);此时中药+VC组大鼠的体重/肝重比值比模型组回升(P＜0.05)。
3.3不同时间段各组大鼠免疫功能变化肝癌大鼠存在一定的细胞免疫障碍,表现为CD~(4+)/CD~(8+)比值减少,且免疫增强因子IL-2表达降低。实验w8,VC能使肝癌大鼠的CD~(4+)/CD~(8+)比值回升(P＜0.05),中药、中药与VC联用的效果更明显,CD~(4+)/CD~(8+)比值显著高于模型组(P＜0.01);w14时,中药+VC组CD~(4+)/CD~(8+)比值亦明显高于模型组(P＜0.01);实验w20,与模型组比较,中药、中药与VC联用不仅能显著增加CD~(4+)/CD~(8+)比值(P＜0.01),还能使血清IL-2表达增加(P＜0.05)。
3.4不同时间段各组大鼠氧化损伤程度大鼠饮用DEN后,肝脏MDA表达量增加。给予药物治疗后,实验w8,VC组和中药+VC组MDA表达减少(P＜0.05);实验w14,中药组SOD分泌增加(P＜0.05),VC组和中药+VC组SOD显著增加(P＜0.01);实验w20,中药组、VC组、中药+VC组、化疗组MDA均显著低于模型组(P＜0.01),且中药组SOD、GSH-PX表达增加(P＜0.05),VC、中药与VC联用增加SOD、GSH-PX的作用更明显,其表达量显著高于模型组(P＜0.01)。
3.5不同时间段各组大鼠VEGF表达量变化免疫组化方法检测各组肝脏VEGF表达,结果显示,实验w8时VC组、中药+VC组VEGF表达量明显降低,与模型组比较有显著差异(P＜0.01),中药组VEGF表达量亦下降(P＜0.05);实验w14,中药组、VC组、中药+VC组均能显著抑制VEGF表达(P＜0.01);在诱癌晚期(w20)中药+VC组VEGF不仅低于模型组(P＜0.05),亦低于化疗组(P＜0.05)。
3.6不同时间段各组大鼠p53突变率及c-myc、cyclinD1表达量DEN诱发的大鼠肝癌p53突变率高,并存在多处突变点,w14时,中药、VC、中药与VC联用均能减少其突变点(P＜0.01);此外肝癌大鼠的c-myc、cyclinD1基因表达量随着癌变进展逐渐增加,中药、VC、中药与VC联用以及UFT均能减少c-myc、cyclinD1的表达,其中中药与VC联用效果最佳,在实验w20,中药+VC组c-myc、cyclinD1的表达量分别约为模型组的1/18、1/16。
3.7各组大鼠生存时间比较中药组和中药+VC组大鼠的生存时间较模型组延长(P＜0.05),且中药+VC组大鼠生存时间长于化疗组(P＜0.01),中药组大鼠生存时间亦长于化疗组(P＜0.05)。
4.扶正抗癌方、维生素C及两者联用对大鼠肝癌细胞增殖、周期、凋亡的影响5%的中药组、VC组、中药+VC组、化疗组含药血清作用48h能选择性抑制CBRH7919细胞增殖,其中中药+VC组的作用显著优于中药组和化疗组(P＜0.01)。中药组含药血清能使CBRH7919细胞G_1期比率高于空白血清对照组(P＜0.01),使细胞增殖阻滞在G_1期;VC组、中药+VC组含药血清均能使CBRH7919细胞S期比率显著升高(P＜0.01),化疗组含药血清亦能使CBRH7919细胞S期比率升高(P＜0.05),使细胞增殖阻滞在S期。中药组、VC组、中药+VC组和化疗组含药血清使Caspase-3表达增加(P＜0.05),并诱导CBRH7919细胞凋亡,中药+VC组、化疗组凋亡率较空白血清对照组显著升高(P＜0.01),其效果优于中药组(P＜0.01)和VC组(P＜0.05)。
结论:
1.扶正抗癌方、VC及两者联用对大鼠肝癌有防治作用,主要表现在保护肝功能、抑制肝癌细胞的增殖、抑制肝癌的生长、抑制AFP表达、改善大鼠的带瘤生存状态及延长生存时间等方面。
2.扶正抗癌方与VC联用抑制肝癌细胞增殖并诱导其凋亡的效果优于扶正抗癌方、VC单用;与化疗药UFT相比,扶正抗癌方及扶正抗癌方与VC联用在延长大鼠带瘤生存时间方面效果更明显;此外,扶正抗癌方与VC联用能抑制肝脏AFP、VEGF表达,效果亦优于化疗药UFT。
3.扶正抗癌方、VC及两者联用防治肝癌的作用机制可能与以下几方面有关:
3.1提高血清CD~(4+)/CD~(8+)比值,增加IL-2的表达,从而增强机体的细胞免疫力;
3.2增强肝脏SOD、GSH-PX的活力,减少MDA的表达量,从而提高大鼠肝脏的抗自由基能力,减轻肝脏的氧化损伤;
3.3降低肝脏的VEGF表达量,进而抑制肝癌的血管生成;
3.4降低c-myc、cyclin D1的表达,阻滞肝癌细胞生长周期,抑制肝癌细胞的增殖;
3.5抑制抑癌基因p53的突变,增加Caspase-3表达,促进肝癌细胞凋亡。
Objective:
Human primary hepatocellular carcinoma,with its notoriously difficult early detection,high degree of malignancy,aggressive development,and high mortality rate,is one of the most common cancers in China.Presently,there is no effective clinical cure for this disease.Therefore, in the field of clinical oncology,investigation into effective therapies that promise minimal side effects,increased rates of survival,and improved quality of life against this disease has become the subject of popular research.
Anti-Cancer Herbal Immune Booster(ACHIB) is an effective Chinese herbal remedy developed by this researcher for the treatment of hepatocarcinoma.In recent years,concurrent studies in theoretical oncology and clinical applications of intravenous delivery of Vitamin C(VC) have also proved worthwhile for exploration.Based on previous clinical trials and related research studies overseas,this study used Walker256 implanted rat hepatoma model,DEN (diethylnitrosamine)-induced rat hepatoma model,CBRH7919(in vitro rat hepatoma cells),and BRL(in vitro normal rat liver cells).These were used to observe what possible interventional roles were made by ACHIB,VC,and their combinations on the onset and development of hepatocarcinogenesis in rats and to further explore their possible mechanisms.
Methods:
1.The carcinosarcoma Walker 256 was implanted into the rats' livers and the rats were divided into several groups:a normal group,a sham-operated group,a model group,a low-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage,12.1g/kg),a medium-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage,24.2g/kg), and a high-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage, 48.4g/kg).The general conditions,such as weight,eating/drinking data,were recorded at regular intervals.In week 1,week 2,and week 3 of the experiment,the subjects' serum total protein(STP),albumin(ALB),globulin(GLB),ratio of albumin to globulin(A/G),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and the enzyme tumor markers alkaline phosphatase(ALP),γ-glutamyl transferase(GGT) and fucosidase(AFU) were tested; the tumor's weights and sizes by its long and short diameters were measured;the pathological development of tumor tissues and their surrounding tissues were observed;tumor metastasis was recorded;and the survival time of the remaining rats in each group was observed;the inhibiting roles of different doses of ACHIB on implanted liver hepatoma in rats and the most effective dose-result relationship were also investigated.
2.The carcinosarcoma Walker 256 was implanted into the rats' livers and the rats were divided into a normal group,a sham-operated group,a model group,a low-dose VC group(VC intravenous,2.83g/kg),and a high-dose VC group(VC intravenous,5.65g/kg).The general condition of the rats was observed;their liver functions(STP,ALB,GLB,A/G,ALT,AST,ALP, GGT,and AFU) were tested;each subject's tumor weight and size of long and short diameters were measured;and the pathological development of tumor tissue was observed.The inhibiting roles of different doses of VC on implanted liver hepatoma in rats and the most effective dose-result relationship were also investigated.
3.Rats were fed with DEN to create test groups with liver cancer.These groups were given, respectively,extracts of ACHIB 48.4g/kg,VC injection 2.83g/kg,combination of ACHIB 48.4g/kg with VC injection 2.83g/kg,and UFT 0.09g/kg.In weeks 8,14,and 20,rats in each group were randomly selected,their body weight and quality of life were observed;their STP, ALB,GLB,A/G,ALT,AST,ALP,GGT,AFU were tested,the changes in liver function were observed;peripheral blood CD~(4+)/CD~(8+) and Interleukin-2(IL-2) expression level were detected to understand the immune function of rats in each group;liver tissue homogenate superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),malondialdehyde(MDA) expression were investigated to understand the oxidative damage in the rat liver and anti-free radical capacity;HE staining was used to observe pathological picture changes in the rat liver; immunohistochemistry was used to detect liver marker alpha-fetoprotein(AFP) expression and hepatocellular carcinoma vascular endothelial growth factor(VEGF) expression;PCR and real-time quantitative PCR technology were used to detect p53 gene mutations and c-myc, cyclin D1 expression;and the survival time of rats in each group was recorded.
4.Serum pharmacology was used to prepare ACHIB,VC,ACHIB+VC,and UFT containing serum for the rats,with concentrations of 2.5%,5%,and 10%respectively.CCK8 assay was used to observe these serums,cultured at different times(24h,48h,72h),and its effects on normal rat liver cells(BRL) and rat hepatoma cells(CBRH7919) proliferation;the best combination of drug containing serum and incubation time for selective killing of CBRH7919 cells was determined;flow cytometry was used to further observe the groups' CBRH7919 cell cycle and apoptosis rate of change;and chemical colorimetric was used to detect apoptosis CBRH7919 cells in each group and changes of expression of apoptosis-related protein, Caspase-3.
Results:
1.ACHIB's Inhibiting Effect on Implanted Walker256 Liver Cancer in SD Rats
1.1 The protection of liver function by different doses of ACHIB at different times.Liver function results demonstrated that,in comparison with the model group,during week 1 of the experiment,the high-dose ACHIB group's AST decreased(P＜0.05).In week 2,the low-dose group's ALB and A/G increased(P＜0.05),GGT decreased(P＜0.05),and ALT, ALP significantly decreased(P＜0.01);the middle-dose group's ALB and A/G increased(P＜0.05),while ALT,ALP,and GGT significantly decreased(P＜0.01);the high-dose group's ALB increased(P＜0.05),A/G also significantly increased(P＜0.01),while ALT,ALP and GGT were significantly lower than the model group(P＜0.01).In week 3,the low-dose ACHIB group's ALT,AST,GGT decreased(P＜0.05),while ALP significantly decreased(P＜0.01);the middle-dose group's ALB was higher than the model group(P＜0.05),while ALT,AST,ALP,and GGT decreased significantly(P＜0.01);the high-dose group's A/G increased(P＜0.05),ALB was also significantly higher than that in the model group(P＜0.01),while ALT,AST,ALP,GGT,and AFU were significantly reduced(P＜0.01).
1.2 The results of tumor inhibition by different doses of ACHIB at different times.During weeks 1 to 3 of the experiment,different doses of ACHIB groups all demonstrated inhibition of tumor growth.Tumor weight inhibition rate in week 3 of the experiment was higher in the high-dose group than that in the low-dose group(P＜0.05).Pathology results showed that a high-dose ACHIB in the early- and medium-term(week 1,week 2) can inhibit the infiltration of cancer cells to surrounding tissues.In the advanced stage(week 3),ACHIB promoted cancer cell necrosis.The scores of tumor metastasis in week 3 of the experiment for low-,middle-,and high-doses of ACHIB were all lower than that of the model group(P＜0.05).
1.3 The effects of different doses of ACHIB on the survival time of experimental rats.The high-dose ACHIB group's survival time had statistical differences when compared with the model group(P＜0.05).
2.Inhibiting Effects of VC on Implanted Liver Cancer in Rats
2.1 The protective effects of VC on rat liver.Serum A/G in the low-dose VC group was higher than the model group(P＜0.05),while ALT was significantly lower than the model group(P＜0.01),GGT was also lower than the model group(P＜0.05).Serum ALT in the high-dose VC group was lower than that in the model group(P＜0.05).
2.2 The tumor inhibition role of VC on liver cancer.The inhibition effects of VC on tumor volume and weight were not obvious(P＞0.05),but pathology results showed that VC could promote tumor cell necrosis,in which the extent of liver necrosis in the low-dose VC group was higher than that in the model group(P＜0.05).
3.Protective Effect of ACHIB,VC and ACHIB+VC in DEN-Induced Rat Liver Cancer
3.1 Each group's changes in liver function were observed at different times.In week 8 of the experiment,ALP levels were lower in the ACHIB and VC groups than in the model group(P＜0.05).ACHIB+VC not only had a significant reduction in ALP(P＜0.01),but also inhibited AST release from the liver,and showed statistical differences when compared with the model group(P＜0.05).By week 14 of the experiment,ACHIB,VC,and ACHIB+VC showed obvious effects on liver function protection,in which the ACHIB group's ALT and AST were significantly lower than the model group(P＜0.01).ACHIB also inhibited the synthesis of liver GGT,with lower content than the model group(P＜0.05);the VC group's and ACHIB+VC group's ALT decreased(P＜0.05) in comparison with that of model group,while AST decreased significantly(P＜0.01),they also demonstrated improvement of protein synthesis,with STP and GLB recovery in the VC group(P＜0.05). The improvement in the ACHIB+VC group's protein synthesis was even more significant, with STP and GLB significantly higher than the model group's(P＜0.01).By week 20 of the experiment,ACHIB still inhibited the release of AST compared to the model group(P＜0.05);AST was significantly lower in the ACHIB+VC group than in the model group(P＜0.01),and ALT was also lower than the model group(P＜0.05).The treatment results of the chemotherapy group compared with the model group showed no difference(P＞0.05).
3.2 Comparison of the progression of liver cancer in rats at different times.Pathology results showed that ACHIB,VC,and ACHIB+VC all delayed the progress of liver cancer including the induction of cancer cell necrosis in its terminal stage.UFT also promoted cancer cell necrosis.In addition,AFP levels in the ACHIB+VC group were lower than the model group in weeks 8 and 14(P＜0.05).By week 20,ACHIB+VC group showed further reduction of the AFP level compared with the model group(P＜0.01),making it lower than that of the chemotherapy group(P＜0.05);the body weight/liver weight of rats in the ACHIB+VC group recovered in comparison with the model group's(P＜0.05).
3.3 Immune function changes in different medication groups at different times.In rats with liver cancer,there are some barriers to cell-mediated immunity,reflected in CD~(4+)/CD~(8+) reduction,and reduction of immune-enhancing factor IL-2 expression.By week 8,the VC treated rats' CD~(4+)/CD~(8+) had increased(P＜0.05).ACHIB and ACHIB+VC showed more obvious effects,with CD~(4+)/CD~(8+) significantly higher than the model group's(P＜0.01);by week 14,ACHIB+VC group's CD~(4+)/CD~(8+) were also significantly higher than the model group's(P＜0.01);by week 20,compared with the model group,both the ACHIB group and the ACHIB+VC group not only had significant increases in CD~(4+)/CD~(8+)(P＜0.01),but also had an increase in the serum IL-2 expression(P＜0.05).
3.4 The extent of oxidative damage of rats in each group at different times.Rats,after drinking DEN,showed increases in their liver MDA expression.After medical treatment, MDA expression decreased in the VC group,and the ACHIB+VC group by week 8(P＜0.05).By week 14,the SOD levels increased in the ACHIB group(P＜0.05).SOD levels significantly increased in the VC group and the ACHIB+VC group(P＜0.01).By week 20, the MDA levels in the ACHIB group,the VC group,the ACHIB+VC group,and the chemotherapy group were significantly lower than the model group(P＜0.01).There were also increased SOD and GSH-PX expressions in the ACHIB group(P＜0.05).The VC group,the ACHIB group,and the ACHIB+VC group recorded significant increases in the SOD and GSH-PX expressions,which were significantly higher than those of the model group(P＜0.01).
3.5 Changes in VEGF expression in rats at different times.The immunohistochemical method was used to detect VEGF expression in the livers of each group.Results demonstrated that by week 8,VEGF expression showed an obvious decrease in the VC group and the ACHIB+VC group,and was significantly lower when compared with the model group(P＜0.01);the expression of VEGF in the ACHIB group also decreased(P＜0.05).By week 14,VEGF expressions were significantly inhibited(P＜0.01) in the ACHIB group,the VC group,and the ACHIB+VC group;in the terminal stage of cancer by week 20, the VEGF expression in the ACHIB+VC group was not only lower than the model group(P＜0.05),but also lower than the chemotherapy group(P＜0.05).
3.6 The rate of p53 mutation and c-myc,cyclin D1 expression at different times in each group.DEN-induced liver cancer in rats had a high rate of p53 mutation,with the existence of multiple point mutations.By week 14,the mutation points of the ACHIB,VC, and ACHIB+VC groups were significantly reduced(P＜0.01).As the rat's liver cancer progressed,the liver cancer gene expression of c-myc,cyclin D1 gradually increased.But c-myc,cyclin D1 expressions were significantly inhibited by ACHIB,VC,ACHIB+VC,and UFT,with the best effect produced by ACHIB+VC;by week 20,c-myc,cyclin D1 expressions in the ACHIB+VC group were only about 1/18 and 1/16 of the model group.
3.7 Comparison of survival time of rats in each group.The survival times of the ACHIB group and ACHIB+VC group were longer compared with the model group's survival time (P＜0.05).The ACHIB+VC group's survival times were significantly longer than the chemotherapy group's(P＜0.01),while ACHIB group's was also longer than the chemotherapy group's(P＜0.05).
4.The Effect of ACHIB,VC and ACHIB+VC on the Proliferation,the Cell Cycle and the Apoptosis Rat of Rate Hepatoma Cells
Drug Containing Serum 5%respectively of ACHIB,VC,ACHIB+VC and UFT for 48h can Selectively Inhibit the Proliferation in vitro of CBRH7919 rat hepatoma cell line.The result of the ACHIB+VC group was better than that of ACHIB and UFT groups(P＜0.01).The ACHIB serum made the G_1 period:CBRH7919 cell quantity ratio higher than that of the serum control group(P＜0.01) and kept cell proliferation in the G_1 phase;the S period:CBRH7919 cell quantity ratio increased significantly in VC and ACHIB+VC groups (P＜0.01),the S period:CBRH7919 cell quantity ratio also increased in the UFT group(P＜0.05);hence,proliferation of cancer cells was blocked in its S phase.The expression of Caspase-3 increased in the ACHIB,VC,ACHIB+VC and the UFT groups(P＜0.05) resulting in an escalation of CBRH7919 apoptosis.The apoptosis rates of ACHIB+VC group and UFT group increased significantly compared to serum control group(P＜0.01),and better than that of the ACHIB group(P＜0.05) and VC group(P＜0.01).
Conclusion:
1.ACHIB,VC and ACHIB+VC inhibit Walker256 rat implanted liver carcinosarcoma.Their main roles are:liver protection,inhibition of cancer cell proliferation resulting in retardation of liver cancer development,inhibition of AFP expression,improvement in the quality of life and prolongation of the survival times of rats with hepatocarcinoma.
2.ACHIB+VC inhibits the increase of hepatocarcinoma cells and induces their apoptosis better than the mono-therapies of ACHIB and VC.ACHIB and ACHIB+VC significantly prolong the survival times of rats with hepatocarcinoma much better than the chemotherapeutic UFT. Furthermore,ACHIB+VC inhibits the expressions of AFP and VEGF much better than chemotherapeutic UFT.
3.The anti-liver cancer mechanisms of ACHIB,VC and ACHIB+VC are related to the following:
3.1 Increasing the ratio of serum CD~(4+)/CD~(8+),and increasing the expression of IL-2,thereby strengthening the cell's immune capability.
3.2 Increasing the activity of liver's SOD and GSH-PX,and decreasing the expression of MDA, thereby increasing the anti-free radical capability of the liver and reducing the oxidative damage due to free radicals.
3.3 Inhibiting the liver's expression of VEGF thereby inhibiting the cancer tumor's blood vessel growth.
3.4 Lowering the c-myc,cyclin D1 expressions thereby disrupting the cancer cell cycle and inhibiting the increase of cancer cells.
3.5 Inhibiting the mutation of cancer gene p53,and increasing the expression of Caspase-3 thereby speeding up the apoptosis rate of cancer cell.
引文
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