槐耳清膏在肝癌介入治疗中作用机制的实验研究
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摘要
目的
观察槐耳清膏体外抑制肝癌细胞生长的影响；观察槐耳清膏联合经肝动脉化疗栓塞对兔VX2肝癌肝功能、VEGF、MVD、肿瘤生长及转移的影响。观察槐耳清膏联合经肝动脉化疗栓塞对兔VX2肝癌细胞凋亡及其相关蛋白P53、Bax和Bcl-2表达的影响。为槐耳清膏联合TACE治疗肝癌提供理论依据。
材料与方法
人肝癌细胞株Hep-G2采用DMEM培养基(含10％胎牛血清)培养。取对数生长期的肝癌Hep-G2细胞，分别与DMEM(含10％胎牛血清)和含槐耳清膏不同浓度(1mg／ml，2mg／ml，4mg／ml，8mg／ml)的DMEM培养液(含10％胎牛血清)作用24小时、48小时、72小时后，应用MTT比色法检测肝癌细胞的增殖情况，用流式细胞仪检测肝癌细胞的凋亡情况。
将VX2瘤粒直接种植于新西兰大白兔左肝内2周，将36只MRI证实已成功接种VX2的荷瘤兔随机分为3组，每组12只，开腹经肝动脉穿刺分别给予不同处理：A组为生理盐水对照组，经肝动脉注入0.2ml／kg体重生理盐水；B组为TACE组，经肝动脉注入超液态碘化油0.2ml／kg+丝裂霉素0.5mg／kg乳剂；C组为TACE+槐耳清膏灌服组，TACE方法同B组，同时术后每天灌服槐耳清膏500mg／kg。介入治疗前1天及治疗后3天、7天、14天抽血检测血清的ALT和AST。每天观察动物生长情况，术后二周处死动物，测量动物体重、肿瘤体积、肿瘤坏死区面积，计算肿瘤生长率、坏死率；观察肝、肺及腹腔淋巴结转移情况；用免疫组织化学方法检测各组肿瘤细胞Ⅷ因子、VEGF、P53、Bax和Bcl-2蛋白的表达情况，对Ⅷ因子染色阳性血管内皮细胞进行MVD计数；用TUNEL法检测各组肿瘤细胞的凋亡指数。
结果
MTT结果显示：在槐耳清膏浓度为1-8mg／ml，分别处理细胞24小时、48小时、72小时后，与对各自对照组相比(p＜0.05)，其能明显抑制Hep-G2细胞的增殖，随着槐耳清膏药物浓度的增加，细胞存活率降低，A值变小，IR值增大，呈量效关系。
流式细胞仪检测结果显示：槐耳清膏能明显诱导肝癌细胞凋亡，在槐耳清膏浓度为1-8mg／ml时，分别处理细胞24小时、48小时、72小时后，其诱导Hep-G2细胞的凋亡率与对照组相比均有显著性差异(p＜0.05)，随着槐耳清膏药物浓度的增加，时间延长，其诱导Hep-G2细胞的凋亡率逐步增加。
术前1天3组动物体重、肿瘤体积无显著性差异(P＞0.05)。术后2周，B组动物体重下降明显，与A组、C组相比差异均有显著性(P＜0.05)；A组与C组相比差异无显著性(P＞0.05)。
介入治疗前1d，3组动物ALT、AST水平差异无显著性；介入治疗后3、7d，3组动物ALT和AST水平相比差异有统计学意义(F=28.411，55.537 P＜0.05；F=8.565，6.401 P＜0.05)，C组与B组相比差异有统计学意义(P＜0.05)。
治疗后2周肿瘤体积、肿瘤生长率和肿瘤坏死率B组、C组与A组相比差异均具有显著性(P＜0.01)，B组与C组相比差异亦具有统计学意义(P＜0.05)。
肝和肺的转移B组与A组相比差异无显著性(P＞0.05)，C组与B组、A组相比差异均具有统计学意义(P＜0.05)；腹腔淋巴结转移3组相比差异无显著性(P＞0.05)。
3组动物MVD值和VEGF的表达强度以C组最低，B组最高，3组相比差异有统计学意义(F=5.22，P=0.011；x~2=7.247，P=0.027 P＜0.05)，C组与B组相比差异有统计学意义(P＜0.05)。
A组的肿瘤细胞凋亡指数最小(7.31±2.85％)，C组的肿瘤细胞凋亡指数最高(15.73±2.89％)；3组动物肿瘤细胞的凋亡指数相比差异有统计学意义(P＜0.05)，C组与B组(12.83±3.51％)相比差异有统计学意义(P＜0.05)。
3组动物肿瘤细胞的Bax蛋白表达阳性率A组(16.67％)最低，C组(83.33％)最高；P53和Bcl-2蛋白表达阳性率C组最低(25.00％，16.67％)，A组最高(91.67％，83.33％)；3组动物肿瘤细胞的P53、Bax和Bcl-2蛋白表达阳性率相比差异有统计学意义(P＜0.05)，C组与B组相比相比差异均有统计学意义(P＜0.05)。
结论
槐耳清膏体外能抑制Hep-G2细胞增殖，诱导Hep-G2细胞凋亡；槐耳清膏改善动物TACE术后的生存质量；槐耳清膏改善动物TACE术后的肝功能；槐耳清膏抑制肿瘤生长，促进肿瘤坏死；槐耳清膏抑制肿瘤转移；槐耳清膏通过减少VEGF表达，抑制肿瘤血管生成；槐耳清膏通过促进肝癌细胞Bax蛋白表达增高，使P53和Bcl-2蛋白表达下降，导致肝癌细胞的凋亡增加；槐耳清膏对HCC有治疗作用。
Objective
To explore the mechanism that extract of fungi of huaier inhibits the growth of thehuman hepatocellular carcinoma cells (Hep-G2) in vitro.
To investigate the therapeutic effect of transcatheter arterial chemoembolizationcombined with extract of fungi of huaier on the expression of VEGF、MVD and hepaticfunction, the growth and metastasis of hepatic tumor of rabbit VX2.
To investigate the therapeutic effect of extract of fungi of huaier combined withtranscatheter arterial chemoembolization on the apoptosis and the expression of associatedprotein P53、Bax and Bcl-2 of rabbit VX2 hepatocarcinoma.
To provide the theory of treatment Hepatocellular carcinoma by extract of fungi ofhuaier combined with transcatheter arterial chemoembolization (TACE).
Materials and Methods
Human liver cancer cell line Hep-G2 was cultured in DMEM medium containing 10%fetal bovine serum. The human hepatocellular carcinoma cells lines (Hep-G2) in log phasewere cultured in DMEM medium containing 10% fetal bovine serum and extract of fungiof huaier of different concentrations (1mg/ml, 2mg/ml, 4mg/ml, 8mg/ml) in DMEMmedium containing 10% fetal bovine serum for 24h, 48h and 72h respectively. The growthinhibitory effect of extract of fungi of huaier on Hep-G2 was observed by MTT assay andthe apoptosis rates of cells were also observed by flow cytometry.
VX2 tumor pieces were surgically implanted into the left liver lobe of New Zealandwhite rabbits, two weeks later, 36 rabbits with experimental hepatic tumors diagnosed byMRI were randomly divided into 3 groups, 12 rabbits in each group. The rabbits in Group A (control group) were infused with 0.2ml/kg physiological saline via the hepatic artery;infused with lipiodol 0.2ml/kg+MMC 0.5mg/kg via the hepatic artery in Group B (TACEgroup); given by oral administration with extract of fungi of huaier 500mg/kg.d afterTACE in Group C (TACE+administration with extract of fungi of huaier).The commonconditions of the rabbits were observed daily after operation. The level of AST and ALT inserum was respectively tested at 1 day before operation, 3 days, 7 days and 14 days afteroperation. Two weeks after treatment, all experimental rabbits were sacrificed and thetumors were taken out. The volume and necrotic area of the implanted tumor weremeasured. The metastases in the liver, lungs and celiac lymph node were recorded.Immunohistochemical staining was performed to detect the expression and localization offactorⅧ、VEGF、P53、Bax and Bcl-2 proteins. The MVD was calculated by counting thefactorⅧpositive endothelial cells. The apoptosis index of tumor cells was assessed withthe method of TUNEL.
Results
The growth inhibitory rates of liver cancer Hep-G2 cells were observed by MTT assay:The growth of Hep-G2 cells were significantly inhibited by extract of fungi huaier. Uponthe extract of fungi of huaier administration in the concentration of 1-8mg/ml and culturedfor 24h, 48h and 72h respectively, the growth inhibitory rates was an obvious difference ascompared to the control group (p＜0.05). The growth inhibitory rates raised with increasingthe concentration of extract of fungi huaier and there is evident dose-dependent manner.
The apoptosis rates of liver cancer Hep-G2 cells were observed by flow cytometry:Extract of fungi of huaier significantly induced apoptosis of liver cancer Hep-G2 cells.Upon the extract of fungi of huaier administration in the concentration of 1-8mg/ml andcultured for 24h, 48h and 72h respectively, there was an obvious difference as compared tothe control group (p＜0.05). With the increased concentration and the prolonged time ofextract of fungi of huaier with Hep-G2 cells, the apoptosis rates induced by extract of fungiof huaier gradually elevated.
There were no significant difference in rabbits' weight and tumors' volume among 3groups at 1 day before operation (P＞0.05). The average rabbits' weight in group B wassignificant lower than those in group A and Group C at 14 days after operation (P＜0.05),but there was no significant difference between group A and group C (P＞0.05).
There was no significant difference of rabbits' AST and ALT in serum among 3 groups at1 day before operation; There was significant difference of rabbits' AST and ALT in serumamong 3 groups at 3 and 7 days after operation (F=28.411, 55.537 P＜0.05; F=8.565,6.401 P＜0.05), and there was significant difference between group B and group C(P＜0.05).
Two weeks after treatment, the average volume, the necrotic rate and growth rate of theimplanted tumor was significantly different between group B, group C versus group A(P＜0.01); Meanwhile there was statistical different between group B and group C(P＜0.05).
The metastases rate in the livers and lungs was not significantly different between groupA and group B (P＞0.05), while was significantly different in group A and group C(P＜0.05), group B and group C(P＜0.05). The incidence of metastases in celiac lymphnode was of no statistical difference among group A, B and C(P＞0.05).
The MVD and the positive express of VEGF is the lowest in group C, the highest ingroup B, there was significant difference among 3 groups (F=5.22, P=0.011;χ~2=7.247,P=0.027 P＜0.05), and there was significant difference between group B and group C(P＜0.05).
The apoptosis index of tumor cells was the minimum in Group A(7.31±2.85%) and themaximum in Group C(15.73±2.89%); there was significant difference of the apoptosisindex of tumor cells among 3 groups (P＜0.05), and there was significant differencebetween group B(12.83±3.51%) and group C (P＜0.05).
The positive expression rate of Bax proteins was the minimum in Group A(16.67%) andthe maximum in Group C(83.33%); the positive expression rate of P53 and Bcl-2 proteins was the minimum in Group C(25.00%, 16.67%) and the maximum in Group A (91.67%,83.33%); there was significant difference of the positive expression rate of P53、Bax andBcl-2 proteins among 3 groups (P＜0.05), and there was significant difference betweengroup B and group C(P＜0.05).
Conclusion
The extract of fungi of huaier can induce apoptosis and inhibit the proliferation ofHep-G2 cells.
The extract of fungi of huaier can improve the rabbits' quality of life after TACE. It cannot only inhibit the growth and promote the necrosis of tumor, but also prevent the tumorfrom developing metastasis to the liver and lungs.
The extract of fungi of huaier can improve the hepatic function of rabbit VX2hepatocarcinoma after TACE; it can treat hepatocarcinoma through diminishing theexpression of VEGF and inhibiting the angiogenesis of tumor.
The extract of fungi of huaier may increase the expression of Bax proteins and decreasethe expression of P53 and Bcl-2 proteins; it may treat hepatocarcinoma through increasingthe apoptosis of hepatoma carcinoma cell.
The extract of fungi of huaier is an ideal drug for clinical treatment of hepatic cancer.
观察槐耳清膏体外抑制肝癌细胞生长的影响；观察槐耳清膏联合经肝动脉化疗栓塞对兔VX2肝癌肝功能、VEGF、MVD、肿瘤生长及转移的影响。观察槐耳清膏联合经肝动脉化疗栓塞对兔VX2肝癌细胞凋亡及其相关蛋白P53、Bax和Bcl-2表达的影响。为槐耳清膏联合TACE治疗肝癌提供理论依据。
材料与方法
人肝癌细胞株Hep-G2采用DMEM培养基(含10％胎牛血清)培养。取对数生长期的肝癌Hep-G2细胞，分别与DMEM(含10％胎牛血清)和含槐耳清膏不同浓度(1mg／ml，2mg／ml，4mg／ml，8mg／ml)的DMEM培养液(含10％胎牛血清)作用24小时、48小时、72小时后，应用MTT比色法检测肝癌细胞的增殖情况，用流式细胞仪检测肝癌细胞的凋亡情况。
将VX2瘤粒直接种植于新西兰大白兔左肝内2周，将36只MRI证实已成功接种VX2的荷瘤兔随机分为3组，每组12只，开腹经肝动脉穿刺分别给予不同处理：A组为生理盐水对照组，经肝动脉注入0.2ml／kg体重生理盐水；B组为TACE组，经肝动脉注入超液态碘化油0.2ml／kg+丝裂霉素0.5mg／kg乳剂；C组为TACE+槐耳清膏灌服组，TACE方法同B组，同时术后每天灌服槐耳清膏500mg／kg。介入治疗前1天及治疗后3天、7天、14天抽血检测血清的ALT和AST。每天观察动物生长情况，术后二周处死动物，测量动物体重、肿瘤体积、肿瘤坏死区面积，计算肿瘤生长率、坏死率；观察肝、肺及腹腔淋巴结转移情况；用免疫组织化学方法检测各组肿瘤细胞Ⅷ因子、VEGF、P53、Bax和Bcl-2蛋白的表达情况，对Ⅷ因子染色阳性血管内皮细胞进行MVD计数；用TUNEL法检测各组肿瘤细胞的凋亡指数。
结果
MTT结果显示：在槐耳清膏浓度为1-8mg／ml，分别处理细胞24小时、48小时、72小时后，与对各自对照组相比(p＜0.05)，其能明显抑制Hep-G2细胞的增殖，随着槐耳清膏药物浓度的增加，细胞存活率降低，A值变小，IR值增大，呈量效关系。
流式细胞仪检测结果显示：槐耳清膏能明显诱导肝癌细胞凋亡，在槐耳清膏浓度为1-8mg／ml时，分别处理细胞24小时、48小时、72小时后，其诱导Hep-G2细胞的凋亡率与对照组相比均有显著性差异(p＜0.05)，随着槐耳清膏药物浓度的增加，时间延长，其诱导Hep-G2细胞的凋亡率逐步增加。
术前1天3组动物体重、肿瘤体积无显著性差异(P＞0.05)。术后2周，B组动物体重下降明显，与A组、C组相比差异均有显著性(P＜0.05)；A组与C组相比差异无显著性(P＞0.05)。
介入治疗前1d，3组动物ALT、AST水平差异无显著性；介入治疗后3、7d，3组动物ALT和AST水平相比差异有统计学意义(F=28.411，55.537 P＜0.05；F=8.565，6.401 P＜0.05)，C组与B组相比差异有统计学意义(P＜0.05)。
治疗后2周肿瘤体积、肿瘤生长率和肿瘤坏死率B组、C组与A组相比差异均具有显著性(P＜0.01)，B组与C组相比差异亦具有统计学意义(P＜0.05)。
肝和肺的转移B组与A组相比差异无显著性(P＞0.05)，C组与B组、A组相比差异均具有统计学意义(P＜0.05)；腹腔淋巴结转移3组相比差异无显著性(P＞0.05)。
3组动物MVD值和VEGF的表达强度以C组最低，B组最高，3组相比差异有统计学意义(F=5.22，P=0.011；x~2=7.247，P=0.027 P＜0.05)，C组与B组相比差异有统计学意义(P＜0.05)。
A组的肿瘤细胞凋亡指数最小(7.31±2.85％)，C组的肿瘤细胞凋亡指数最高(15.73±2.89％)；3组动物肿瘤细胞的凋亡指数相比差异有统计学意义(P＜0.05)，C组与B组(12.83±3.51％)相比差异有统计学意义(P＜0.05)。
3组动物肿瘤细胞的Bax蛋白表达阳性率A组(16.67％)最低，C组(83.33％)最高；P53和Bcl-2蛋白表达阳性率C组最低(25.00％，16.67％)，A组最高(91.67％，83.33％)；3组动物肿瘤细胞的P53、Bax和Bcl-2蛋白表达阳性率相比差异有统计学意义(P＜0.05)，C组与B组相比相比差异均有统计学意义(P＜0.05)。
结论
槐耳清膏体外能抑制Hep-G2细胞增殖，诱导Hep-G2细胞凋亡；槐耳清膏改善动物TACE术后的生存质量；槐耳清膏改善动物TACE术后的肝功能；槐耳清膏抑制肿瘤生长，促进肿瘤坏死；槐耳清膏抑制肿瘤转移；槐耳清膏通过减少VEGF表达，抑制肿瘤血管生成；槐耳清膏通过促进肝癌细胞Bax蛋白表达增高，使P53和Bcl-2蛋白表达下降，导致肝癌细胞的凋亡增加；槐耳清膏对HCC有治疗作用。
Objective
To explore the mechanism that extract of fungi of huaier inhibits the growth of thehuman hepatocellular carcinoma cells (Hep-G2) in vitro.
To investigate the therapeutic effect of transcatheter arterial chemoembolizationcombined with extract of fungi of huaier on the expression of VEGF、MVD and hepaticfunction, the growth and metastasis of hepatic tumor of rabbit VX2.
To investigate the therapeutic effect of extract of fungi of huaier combined withtranscatheter arterial chemoembolization on the apoptosis and the expression of associatedprotein P53、Bax and Bcl-2 of rabbit VX2 hepatocarcinoma.
To provide the theory of treatment Hepatocellular carcinoma by extract of fungi ofhuaier combined with transcatheter arterial chemoembolization (TACE).
Materials and Methods
Human liver cancer cell line Hep-G2 was cultured in DMEM medium containing 10%fetal bovine serum. The human hepatocellular carcinoma cells lines (Hep-G2) in log phasewere cultured in DMEM medium containing 10% fetal bovine serum and extract of fungiof huaier of different concentrations (1mg/ml, 2mg/ml, 4mg/ml, 8mg/ml) in DMEMmedium containing 10% fetal bovine serum for 24h, 48h and 72h respectively. The growthinhibitory effect of extract of fungi of huaier on Hep-G2 was observed by MTT assay andthe apoptosis rates of cells were also observed by flow cytometry.
VX2 tumor pieces were surgically implanted into the left liver lobe of New Zealandwhite rabbits, two weeks later, 36 rabbits with experimental hepatic tumors diagnosed byMRI were randomly divided into 3 groups, 12 rabbits in each group. The rabbits in Group A (control group) were infused with 0.2ml/kg physiological saline via the hepatic artery;infused with lipiodol 0.2ml/kg+MMC 0.5mg/kg via the hepatic artery in Group B (TACEgroup); given by oral administration with extract of fungi of huaier 500mg/kg.d afterTACE in Group C (TACE+administration with extract of fungi of huaier).The commonconditions of the rabbits were observed daily after operation. The level of AST and ALT inserum was respectively tested at 1 day before operation, 3 days, 7 days and 14 days afteroperation. Two weeks after treatment, all experimental rabbits were sacrificed and thetumors were taken out. The volume and necrotic area of the implanted tumor weremeasured. The metastases in the liver, lungs and celiac lymph node were recorded.Immunohistochemical staining was performed to detect the expression and localization offactorⅧ、VEGF、P53、Bax and Bcl-2 proteins. The MVD was calculated by counting thefactorⅧpositive endothelial cells. The apoptosis index of tumor cells was assessed withthe method of TUNEL.
Results
The growth inhibitory rates of liver cancer Hep-G2 cells were observed by MTT assay:The growth of Hep-G2 cells were significantly inhibited by extract of fungi huaier. Uponthe extract of fungi of huaier administration in the concentration of 1-8mg/ml and culturedfor 24h, 48h and 72h respectively, the growth inhibitory rates was an obvious difference ascompared to the control group (p＜0.05). The growth inhibitory rates raised with increasingthe concentration of extract of fungi huaier and there is evident dose-dependent manner.
The apoptosis rates of liver cancer Hep-G2 cells were observed by flow cytometry:Extract of fungi of huaier significantly induced apoptosis of liver cancer Hep-G2 cells.Upon the extract of fungi of huaier administration in the concentration of 1-8mg/ml andcultured for 24h, 48h and 72h respectively, there was an obvious difference as compared tothe control group (p＜0.05). With the increased concentration and the prolonged time ofextract of fungi of huaier with Hep-G2 cells, the apoptosis rates induced by extract of fungiof huaier gradually elevated.
There were no significant difference in rabbits' weight and tumors' volume among 3groups at 1 day before operation (P＞0.05). The average rabbits' weight in group B wassignificant lower than those in group A and Group C at 14 days after operation (P＜0.05),but there was no significant difference between group A and group C (P＞0.05).
There was no significant difference of rabbits' AST and ALT in serum among 3 groups at1 day before operation; There was significant difference of rabbits' AST and ALT in serumamong 3 groups at 3 and 7 days after operation (F=28.411, 55.537 P＜0.05; F=8.565,6.401 P＜0.05), and there was significant difference between group B and group C(P＜0.05).
Two weeks after treatment, the average volume, the necrotic rate and growth rate of theimplanted tumor was significantly different between group B, group C versus group A(P＜0.01); Meanwhile there was statistical different between group B and group C(P＜0.05).
The metastases rate in the livers and lungs was not significantly different between groupA and group B (P＞0.05), while was significantly different in group A and group C(P＜0.05), group B and group C(P＜0.05). The incidence of metastases in celiac lymphnode was of no statistical difference among group A, B and C(P＞0.05).
The MVD and the positive express of VEGF is the lowest in group C, the highest ingroup B, there was significant difference among 3 groups (F=5.22, P=0.011;χ~2=7.247,P=0.027 P＜0.05), and there was significant difference between group B and group C(P＜0.05).
The apoptosis index of tumor cells was the minimum in Group A(7.31±2.85%) and themaximum in Group C(15.73±2.89%); there was significant difference of the apoptosisindex of tumor cells among 3 groups (P＜0.05), and there was significant differencebetween group B(12.83±3.51%) and group C (P＜0.05).
The positive expression rate of Bax proteins was the minimum in Group A(16.67%) andthe maximum in Group C(83.33%); the positive expression rate of P53 and Bcl-2 proteins was the minimum in Group C(25.00%, 16.67%) and the maximum in Group A (91.67%,83.33%); there was significant difference of the positive expression rate of P53、Bax andBcl-2 proteins among 3 groups (P＜0.05), and there was significant difference betweengroup B and group C(P＜0.05).
Conclusion
The extract of fungi of huaier can induce apoptosis and inhibit the proliferation ofHep-G2 cells.
The extract of fungi of huaier can improve the rabbits' quality of life after TACE. It cannot only inhibit the growth and promote the necrosis of tumor, but also prevent the tumorfrom developing metastasis to the liver and lungs.
The extract of fungi of huaier can improve the hepatic function of rabbit VX2hepatocarcinoma after TACE; it can treat hepatocarcinoma through diminishing theexpression of VEGF and inhibiting the angiogenesis of tumor.
The extract of fungi of huaier may increase the expression of Bax proteins and decreasethe expression of P53 and Bcl-2 proteins; it may treat hepatocarcinoma through increasingthe apoptosis of hepatoma carcinoma cell.
The extract of fungi of huaier is an ideal drug for clinical treatment of hepatic cancer.
引文
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