熱休克蛋白90抑制劑Ganetespib預防肝癌微波消融術后局部復發(fā)的實驗研究
發(fā)布時間：2018-08-07 17:40
【摘要】：目的1.通過細胞實驗研究,探討應用第二代熱休克蛋白90 (Heat Shock Protein 90, HSP90)抑制劑Ganetespib不同濃度作用于肝癌HepG2,對細胞增殖的影響及誘導細胞凋亡的作用,并驗證不同溫度對HSP90表達水平的影響。2.分析微波消融聯(lián)合Ganetespib臺療對裸鼠肝癌皮下移植瘤消融后壞死范圍的調控作用,并比較兩者共同作用后,消融區(qū)周邊HSP90及caspase-3表達水平的差異,以及對Ganetespib應用的生物安全性進行評價。3.分析微波消融聯(lián)合Ganetespib治療對消融治療后裸鼠的腫瘤體積倍增時間及終點生存時間的差異。方法1.用CCK8比色法檢測不同濃度的Ganetespib抑制肝癌HepG2細胞生長的效果;藥物作用后HepG2細胞凋亡率的變化應用Annexin V-FITC/PI雙染法檢測；用酶聯(lián)免疫吸附劑實驗(enzymelinkedimmunosorbentassay, ELISA)檢測不同溫度作用后HSP90表達水平的變化。2.給予裸鼠最高藥物耐受劑量(150mg/kg),5h后取出動物內臟進行病理檢查,并同時送檢無藥物處理的裸鼠動物內臟,對比藥物的生物安全性。建立肝癌HepG2細胞裸鼠皮下移植瘤模型。將40只裸鼠隨機分配到如下4個處理組中：(1)單獨微波消融組;(2)單獨靜脈注射Ganetespib組;(3)微波消融前2h靜脈注射Ganetespib組;(4)無處理陰性對照組。分別于治療后24h將動物脫頸處死,剝離出腫瘤,經2%2,3,5-三苯基氯化四氮唑溶液染色后觀察腫瘤壞死范圍,免疫組化檢查HSP90及caspase-3的表達。3.建立肝癌HepG2細胞裸鼠皮下移植瘤模型。待腫瘤直徑達到1.0-1.2cm時,將20只裸鼠隨機分配到如下4個處理組中：(1)單獨微波消融組;(2)單獨靜脈注射Ganetespib組;(3)微波消融前2h靜脈注射Ganetespib組：(4)無處理陰性對照組。治療后每隔2-3天觀察腫瘤大小,腫瘤直徑長到2.5cm或生存期達到40天設為生存終點,使用Kaplan-Meier生存分析方法比較各處理組達到研究終點的時間差異。結果1. CCK8實驗結果顯示Ganetespib呈時間-劑量依賴性抑制肝癌HepG2細胞增殖。Annexin-FITC/PI雙染法檢測結果顯示,Ganetespib藥物作用于細胞48h后,細胞凋亡率隨藥物濃度升高而增大,濃度設定為30、100、250、500及1000nmol/l,30nmol/L Ganetespib干預48h后細胞晚期凋亡率為(16.3±1.22)%(與陰性對照組比較,P0.05),1000nmol/l Ganetespib干預48h后細胞晚期凋亡率為(56.6±1.83)%(與其他濃度組比較,P0.05)。ELISA法檢測結果顯示,45℃組的HSP90濃度最高,50℃組次之,60℃組最低。 (45℃組與其他各組比較,P0.05)2.應用Ganetespib后沒有影響肝臟、脾臟、腎臟及肺的大體形態(tài)及微觀病理表現(xiàn)。單獨靜脈注射Ganetespib組給藥后24h沒有觀察到明顯的壞死區(qū)域。單獨微波消融組凝固壞死范圍7.5±0.3mm。微波消融聯(lián)合靜脈注射Ganetespib組觀察到較大的凝固范圍9.4±0.5mm(P0.05,與單獨微波組比較)。治療后24h,與單獨微波消融組比較,聯(lián)合應用Ganetespib組可以在消融區(qū)周邊觀察到更加微弱的Hsp90表達帶(表達帶厚度0.19±0.07mm,染色細胞21.4±11.2% vs.0.25±0.13mm,30.3±10.7%,P0.05)。消融聯(lián)合Ganetespib組顯示出更多caspase-3的表達。(表達帶厚度0.37±0.12mm,染色細胞數(shù)37.3±15.1% vs.0.25±0.18mm,27.6±11.9%,P0.05)。3.無處理組的平均終點生存時間(從開始治療的到腫瘤直徑達25mm)為19.1±2.0天。單獨應用微波消融組的平均終點生存時間比單獨靜脈注射Ganetespib組長,前者平均時間為26.3±2.2天,后者平均時間為23.2±2.0天(與對照組比較P0.05)。微波消融聯(lián)合靜脈注射Ganetespib組表現(xiàn)出明顯延長的生存時間,平均為33.2±1.9天(與其他組比較P0.05)。方差分析結果顯示,微波消融聯(lián)合藥物治療組腫瘤體積倍增時間(25.1±1.8天)明顯長于單獨微波消融組(17.5±1.6天)及單獨藥物處理組(15.4±2.3天)(P0.05)。結論1.第二代HSP90抑制劑Ganetespib能夠抑制肝癌HepG2細胞增殖,隨著藥物濃度的增加,作用時間的延長,細胞活性逐漸下降,并能夠促進肝癌HepG2細胞的凋亡。2. Ganetespib對裸鼠應用的生物安全性好。應用Ganetespib聯(lián)合熱消融治療,可以抑制熱休克蛋白的表達,增加微波消融后腫瘤的壞死范圍,促進消融區(qū)周邊亞致死溫度帶上腫瘤細胞的凋亡。3.聯(lián)合應用微波消融及Ganetespib能夠降低消融后亞致死溫度帶腫瘤生長速率,延長動物終點生存時間。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7
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