热休克蛋白90抑制剂Ganetespib预防肝癌微波消融术后局部复发的实验研究
发布时间：2018-08-07 17:40
【摘要】：目的1.通过细胞实验研究,探讨应用第二代热休克蛋白90 (Heat Shock Protein 90, HSP90)抑制剂Ganetespib不同浓度作用于肝癌HepG2,对细胞增殖的影响及诱导细胞凋亡的作用,并验证不同温度对HSP90表达水平的影响。2.分析微波消融联合Ganetespib台疗对裸鼠肝癌皮下移植瘤消融后坏死范围的调控作用,并比较两者共同作用后,消融区周边HSP90及caspase-3表达水平的差异,以及对Ganetespib应用的生物安全性进行评价。3.分析微波消融联合Ganetespib治疗对消融治疗后裸鼠的肿瘤体积倍增时间及终点生存时间的差异。方法1.用CCK8比色法检测不同浓度的Ganetespib抑制肝癌HepG2细胞生长的效果;药物作用后HepG2细胞凋亡率的变化应用Annexin V-FITC/PI双染法检测；用酶联免疫吸附剂实验(enzymelinkedimmunosorbentassay, ELISA)检测不同温度作用后HSP90表达水平的变化。2.给予裸鼠最高药物耐受剂量(150mg/kg),5h后取出动物内脏进行病理检查,并同时送检无药物处理的裸鼠动物内脏,对比药物的生物安全性。建立肝癌HepG2细胞裸鼠皮下移植瘤模型。将40只裸鼠随机分配到如下4个处理组中：(1)单独微波消融组;(2)单独静脉注射Ganetespib组;(3)微波消融前2h静脉注射Ganetespib组;(4)无处理阴性对照组。分别于治疗后24h将动物脱颈处死,剥离出肿瘤,经2%2,3,5-三苯基氯化四氮唑溶液染色后观察肿瘤坏死范围,免疫组化检查HSP90及caspase-3的表达。3.建立肝癌HepG2细胞裸鼠皮下移植瘤模型。待肿瘤直径达到1.0-1.2cm时,将20只裸鼠随机分配到如下4个处理组中：(1)单独微波消融组;(2)单独静脉注射Ganetespib组;(3)微波消融前2h静脉注射Ganetespib组：(4)无处理阴性对照组。治疗后每隔2-3天观察肿瘤大小,肿瘤直径长到2.5cm或生存期达到40天设为生存终点,使用Kaplan-Meier生存分析方法比较各处理组达到研究终点的时间差异。结果1. CCK8实验结果显示Ganetespib呈时间-剂量依赖性抑制肝癌HepG2细胞增殖。Annexin-FITC/PI双染法检测结果显示,Ganetespib药物作用于细胞48h后,细胞凋亡率随药物浓度升高而增大,浓度设定为30、100、250、500及1000nmol/l,30nmol/L Ganetespib干预48h后细胞晚期凋亡率为(16.3±1.22)%(与阴性对照组比较,P0.05),1000nmol/l Ganetespib干预48h后细胞晚期凋亡率为(56.6±1.83)%(与其他浓度组比较,P0.05)。ELISA法检测结果显示,45℃组的HSP90浓度最高,50℃组次之,60℃组最低。 (45℃组与其他各组比较,P0.05)2.应用Ganetespib后没有影响肝脏、脾脏、肾脏及肺的大体形态及微观病理表现。单独静脉注射Ganetespib组给药后24h没有观察到明显的坏死区域。单独微波消融组凝固坏死范围7.5±0.3mm。微波消融联合静脉注射Ganetespib组观察到较大的凝固范围9.4±0.5mm(P0.05,与单独微波组比较)。治疗后24h,与单独微波消融组比较,联合应用Ganetespib组可以在消融区周边观察到更加微弱的Hsp90表达带(表达带厚度0.19±0.07mm,染色细胞21.4±11.2% vs.0.25±0.13mm,30.3±10.7%,P0.05)。消融联合Ganetespib组显示出更多caspase-3的表达。(表达带厚度0.37±0.12mm,染色细胞数37.3±15.1% vs.0.25±0.18mm,27.6±11.9%,P0.05)。3.无处理组的平均终点生存时间(从开始治疗的到肿瘤直径达25mm)为19.1±2.0天。单独应用微波消融组的平均终点生存时间比单独静脉注射Ganetespib组长,前者平均时间为26.3±2.2天,后者平均时间为23.2±2.0天(与对照组比较P0.05)。微波消融联合静脉注射Ganetespib组表现出明显延长的生存时间,平均为33.2±1.9天(与其他组比较P0.05)。方差分析结果显示,微波消融联合药物治疗组肿瘤体积倍增时间(25.1±1.8天)明显长于单独微波消融组(17.5±1.6天)及单独药物处理组(15.4±2.3天)(P0.05)。结论1.第二代HSP90抑制剂Ganetespib能够抑制肝癌HepG2细胞增殖,随着药物浓度的增加,作用时间的延长,细胞活性逐渐下降,并能够促进肝癌HepG2细胞的凋亡。2. Ganetespib对裸鼠应用的生物安全性好。应用Ganetespib联合热消融治疗,可以抑制热休克蛋白的表达,增加微波消融后肿瘤的坏死范围,促进消融区周边亚致死温度带上肿瘤细胞的凋亡。3.联合应用微波消融及Ganetespib能够降低消融后亚致死温度带肿瘤生长速率,延长动物终点生存时间。
[Abstract]:Objective 1. to investigate the effects of different concentrations of second generation heat shock protein 90 (Heat Shock Protein 90, HSP90) inhibitor Ganetespib on hepatocellular carcinoma HepG2, the effect on cell proliferation and the induction of cell apoptosis, and to verify the effect of different temperatures on the level of HSP90 surface, and to verify the effect of.2. analysis microwave ablation combined with Ganetespib table. The effect of treatment on the extent of necrosis after ablation of subcutaneous transplanted tumor in nude mice, and compare the difference of the expression level of HSP90 and caspase-3 around the ablation area, and the evaluation of the biological safety of Ganetespib application..3. analysis of microwave ablation combined with Ganetespib on the tumor volume multiplied by the nude mice after ablation treatment. Difference of time and end-point survival time. Method 1. CCK8 colorimetry was used to detect the effect of different concentrations of Ganetespib on the growth of HepG2 cells in liver cancer; the changes of apoptosis rate of HepG2 cells after drug action were detected by Annexin V-FITC/PI double staining; enzyme linked immunosorbent assay (enzymelinkedimmunosorbentassay, ELISA) was used to detect the difference. The change of HSP90 expression level after temperature action.2. gave the highest drug tolerance dose (150mg/kg) in nude mice. After 5h, the animal viscera was taken out for pathological examination, and the non treated nude mouse viscera was examined at the same time. The biological safety of the drug was compared. The model of subcutaneous xenograft in nude mice of HepG2 cells of liver cancer was established. 40 nude mice were randomly assigned to the model of the nude mice. The next 4 treatment groups: (1) separate microwave ablation group; (2) group Ganetespib alone; (3) Ganetespib group before microwave ablation; (4) non treatment negative control group. After the treatment, the animals were removed from the neck and stripped out of the tumor. After the 2%2,3,5- three phenyl chlorinated tetrazolium solution staining, the tumor necrosis area was observed and the immunological group was observed. The expression of HSP90 and Caspase-3 expression.3. was used to establish the subcutaneous tumor model of nude mice of liver cancer HepG2 cells. When the diameter of the tumor reached 1.0-1.2cm, 20 nude mice were randomly assigned to the following 4 treatment groups: (1) the single microwave ablation group; (2) the individual intravenous injection of Ganetespib group; (3) the 2H intravenous injection of Ganetespib before microwave ablation: (4) no treatment. The size of the tumor was observed every 2-3 days after treatment, the tumor diameter was long to 2.5cm or the survival period was 40 days. The time difference between the treatment group and the end point was compared with the Kaplan-Meier survival analysis. Results the results of 1. CCK8 showed that Ganetespib was time dose dependent inhibition of liver cancer HepG2 cells. The results of proliferation.Annexin-FITC/PI double staining showed that the apoptosis rate of cell 48h increased with the increase of drug concentration, and the concentration was set to 30100250500 and 1000nmol/l. After 30nmol/L Ganetespib intervention 48h, the apoptosis rate was (16.3 + 1.22)% (P0.05) and 1000nmol/l Ganetespib compared with the negative control group. The apoptosis rate of advanced cells after 48h intervention was (56.6 + 1.83)% (compared with other concentration groups, P0.05).ELISA assay results showed that the HSP90 concentration in 45 C group was the highest, 50 degrees centigrade, and the group was the lowest. (45 centigrade and other groups, P0.05) 2. application Ganetespib did not affect the liver, spleen, kidney and lung There was no obvious necrotic area in 24h after intravenous injection of Ganetespib alone. The coagulation necrosis area of 7.5 + 0.3mm. microwave ablation group was 9.4 + 0.5mm (P0.05, P0.05, compared with the single microwave group) in the single microwave ablation group. After treatment, 24h was compared with the single microwave ablation group. In the Ganetespib group, a more weak Hsp90 expression zone could be observed around the ablation zone (the thickness of the expression band was 0.19 + 0.07mm, the staining cells were 21.4 + 11.2% vs.0.25 + 0.13MM, 30.3 + 10.7%, P0.05). The ablation combined with Ganetespib group showed more caspase-3 expression. (the thickness of the expression band was 0.37 + 0.12mm, the number of dyed cells was 37.3 + 15.1% vs.0.25 + 0.1. The average end point survival time of the 8mm, 27.6 + 11.9%, P0.05).3. non treatment group (from the beginning of treatment to the tumor diameter of 25mm) was 19.1 + 2 days. The average end point survival time of the microwave ablation group was longer than the Ganetespib group alone, the average time of the former was 26.3 + 2.2 days and the latter was 23.2 + 2 days (compared with the control group). Compared with P0.05), the survival time of microwave ablation combined with intravenous injection of Ganetespib was obviously prolonged, averaging 33.2 + 1.9 days (compared with other groups P0.05). The results of variance analysis showed that the tumor volume doubling time (25.1 + 1.8 days) of the combined microwave ablation group (25.1 + 1.8 days) was significantly longer than that of the single microwave ablation group (17.5 + 1.6 days) and the individual drug place. (15.4 + 2.3 days) (15.4 + 2.3 days) (P0.05). Conclusion the 1. second generation of HSP90 inhibitor Ganetespib can inhibit the proliferation of hepatocellular carcinoma HepG2 cells. With the increase of the drug concentration, the action time is prolonged, the cell activity gradually decreases, and it can promote the biological safety of the apoptosis.2. Ganetespib for the HepG2 cells of the liver cancer. The combined heat of Ganetespib combined heat can be used. Ablation therapy can inhibit the expression of heat shock protein, increase the extent of tumor necrosis after microwave ablation, promote the apoptosis of tumor cells in the sublethal temperature zone around the ablation zone, combined with microwave ablation and Ganetespib can reduce the growth rate of sublethal temperature after ablation and prolong the end-point survival time of the animals.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
[Abstract]:Objective 1. to investigate the effects of different concentrations of second generation heat shock protein 90 (Heat Shock Protein 90, HSP90) inhibitor Ganetespib on hepatocellular carcinoma HepG2, the effect on cell proliferation and the induction of cell apoptosis, and to verify the effect of different temperatures on the level of HSP90 surface, and to verify the effect of.2. analysis microwave ablation combined with Ganetespib table. The effect of treatment on the extent of necrosis after ablation of subcutaneous transplanted tumor in nude mice, and compare the difference of the expression level of HSP90 and caspase-3 around the ablation area, and the evaluation of the biological safety of Ganetespib application..3. analysis of microwave ablation combined with Ganetespib on the tumor volume multiplied by the nude mice after ablation treatment. Difference of time and end-point survival time. Method 1. CCK8 colorimetry was used to detect the effect of different concentrations of Ganetespib on the growth of HepG2 cells in liver cancer; the changes of apoptosis rate of HepG2 cells after drug action were detected by Annexin V-FITC/PI double staining; enzyme linked immunosorbent assay (enzymelinkedimmunosorbentassay, ELISA) was used to detect the difference. The change of HSP90 expression level after temperature action.2. gave the highest drug tolerance dose (150mg/kg) in nude mice. After 5h, the animal viscera was taken out for pathological examination, and the non treated nude mouse viscera was examined at the same time. The biological safety of the drug was compared. The model of subcutaneous xenograft in nude mice of HepG2 cells of liver cancer was established. 40 nude mice were randomly assigned to the model of the nude mice. The next 4 treatment groups: (1) separate microwave ablation group; (2) group Ganetespib alone; (3) Ganetespib group before microwave ablation; (4) non treatment negative control group. After the treatment, the animals were removed from the neck and stripped out of the tumor. After the 2%2,3,5- three phenyl chlorinated tetrazolium solution staining, the tumor necrosis area was observed and the immunological group was observed. The expression of HSP90 and Caspase-3 expression.3. was used to establish the subcutaneous tumor model of nude mice of liver cancer HepG2 cells. When the diameter of the tumor reached 1.0-1.2cm, 20 nude mice were randomly assigned to the following 4 treatment groups: (1) the single microwave ablation group; (2) the individual intravenous injection of Ganetespib group; (3) the 2H intravenous injection of Ganetespib before microwave ablation: (4) no treatment. The size of the tumor was observed every 2-3 days after treatment, the tumor diameter was long to 2.5cm or the survival period was 40 days. The time difference between the treatment group and the end point was compared with the Kaplan-Meier survival analysis. Results the results of 1. CCK8 showed that Ganetespib was time dose dependent inhibition of liver cancer HepG2 cells. The results of proliferation.Annexin-FITC/PI double staining showed that the apoptosis rate of cell 48h increased with the increase of drug concentration, and the concentration was set to 30100250500 and 1000nmol/l. After 30nmol/L Ganetespib intervention 48h, the apoptosis rate was (16.3 + 1.22)% (P0.05) and 1000nmol/l Ganetespib compared with the negative control group. The apoptosis rate of advanced cells after 48h intervention was (56.6 + 1.83)% (compared with other concentration groups, P0.05).ELISA assay results showed that the HSP90 concentration in 45 C group was the highest, 50 degrees centigrade, and the group was the lowest. (45 centigrade and other groups, P0.05) 2. application Ganetespib did not affect the liver, spleen, kidney and lung There was no obvious necrotic area in 24h after intravenous injection of Ganetespib alone. The coagulation necrosis area of 7.5 + 0.3mm. microwave ablation group was 9.4 + 0.5mm (P0.05, P0.05, compared with the single microwave group) in the single microwave ablation group. After treatment, 24h was compared with the single microwave ablation group. In the Ganetespib group, a more weak Hsp90 expression zone could be observed around the ablation zone (the thickness of the expression band was 0.19 + 0.07mm, the staining cells were 21.4 + 11.2% vs.0.25 + 0.13MM, 30.3 + 10.7%, P0.05). The ablation combined with Ganetespib group showed more caspase-3 expression. (the thickness of the expression band was 0.37 + 0.12mm, the number of dyed cells was 37.3 + 15.1% vs.0.25 + 0.1. The average end point survival time of the 8mm, 27.6 + 11.9%, P0.05).3. non treatment group (from the beginning of treatment to the tumor diameter of 25mm) was 19.1 + 2 days. The average end point survival time of the microwave ablation group was longer than the Ganetespib group alone, the average time of the former was 26.3 + 2.2 days and the latter was 23.2 + 2 days (compared with the control group). Compared with P0.05), the survival time of microwave ablation combined with intravenous injection of Ganetespib was obviously prolonged, averaging 33.2 + 1.9 days (compared with other groups P0.05). The results of variance analysis showed that the tumor volume doubling time (25.1 + 1.8 days) of the combined microwave ablation group (25.1 + 1.8 days) was significantly longer than that of the single microwave ablation group (17.5 + 1.6 days) and the individual drug place. (15.4 + 2.3 days) (15.4 + 2.3 days) (P0.05). Conclusion the 1. second generation of HSP90 inhibitor Ganetespib can inhibit the proliferation of hepatocellular carcinoma HepG2 cells. With the increase of the drug concentration, the action time is prolonged, the cell activity gradually decreases, and it can promote the biological safety of the apoptosis.2. Ganetespib for the HepG2 cells of the liver cancer. The combined heat of Ganetespib combined heat can be used. Ablation therapy can inhibit the expression of heat shock protein, increase the extent of tumor necrosis after microwave ablation, promote the apoptosis of tumor cells in the sublethal temperature zone around the ablation zone, combined with microwave ablation and Ganetespib can reduce the growth rate of sublethal temperature after ablation and prolong the end-point survival time of the animals.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
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