JAK/STAT信号通路阻断剂AG490联合DDP对子宫颈癌裸鼠移植瘤生长的影响
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摘要
目的:通过体内试验观察JAK/STAT通路阻断剂A6490联合化疗药物DDP对子宫颈癌裸鼠移植瘤生长的影响,探讨AG490通过阻断STAT3通路对人子宫颈癌裸鼠移植瘤生长抑制作用及与Caspase-3蛋白表达的关系。以期对临床上药物治疗与预防子寓颈癌寻找更加完善的途径。
方法:建立人子宫颈癌(Hela)细胞裸鼠异种移植瘤模型并分组。A6490单用或联合DDP处理各组裸鼠。用药期间测量裸鼠体重和肿瘤体积,检测瘤重计算抑瘤率。流式细胞分析术测肿瘤组织中细胞凋亡情况,免疫组化检测移植瘤组织中P-stat3和Caspase-3蛋白表达。所有数据均采用SPSS10.0统计软件进行处理。
结果:
1、药物对裸鼠移植瘤体积生长的影响:分别在用药后第1天、第4天、第7天及第10天测量肿瘤长径(a)、短径(h),计算体积,描绘移植瘤生长曲线图。结果可见从用药第7天开始DDP组、AG490组以及联合用药组(DDP+AG490组)肿瘤体积均小于对照组,有显著性差异(P＜0.001)。联合用药组与DDP组、AG490组比抑瘤作用显著增强(P＜0.05、P＜0.001)。DDP组对肿瘤的生长抑制与A6490组相比显著增强(P＜0.05)。
2、药物对移植瘤裸鼠体重及副作用的影响:药物治疗子宫颈癌裸鼠移植瘤生长期间,从用药第7天开始,顺铂组和联合用药组与对照组比差异有统计学意义(P＜0.001)。A6490组与对照组比差异无统计学意义(P＞0.05)且裸鼠一般情况良好,进食和体重无明显变化。DDP组于治疗开始后第7天裸鼠逐渐出现严重消瘦,营养不良,并且出现明显食欲减退,活动迟缓,皮肤无光泽等毒副反应,体重变化与联合用药组间存在明显差异(P＜0.05),该组有1只裸鼠死亡。
3、各组裸鼠移植瘤瘤重的变化:处死裸鼠,剥出瘤块用电子天平称量瘤重(W),计算抑瘤率。抑瘤率分别为对照组0%、顺铂组51.2%、AG490组33.6%、联合用药组59.2%与对照组相比,各药物作用组均对裸鼠移植瘤有抑制作用,差异有显著性意义(P＜0.05)。A6490与DDP联合应用比两者单独应用抑瘤作用更明显,差异有显著性意义(P＜0.05)。DDP组比A6490组抑瘤作用强,差异有统计学意义(P＜0.05)。
4、流式细胞仪分析凋亡率:DOP组、AG490组和联合用药组有明显G_1期形成的凋亡峰,三个用药组的凋亡率分别是52.1±1.3%、39.5±0.4%、66.7±1.8%。与对照组10.2±2.4%相比均有显著性差异(P＜0.05)。流式细胞分析的各组DNA组方图,可见各用药组有明显的G_1期形成的凋亡峰。
5、光学显微镜下观察Hela细胞裸鼠移植瘤组织形态学的改变:肉眼观移植瘤呈结节状,肿瘤组织血供丰富,无明显周围组织和皮肤浸润,界限清楚,易分离,切而呈鱼肉样。光镜下观察肿瘤细胞呈巢、片、条索状排列,瘤细胞体大、核大深染,核仁明显,病理性核分裂相易见,瘤周组织内血管扩张充血,并有少量淋巴细胞、中性粒细胞浸润,瘤巢之间有少量的纤维及帆管。瘤组织内见小灶性凝固性坏死。用药后,肿瘤组织出现不同程度的坏死。肿瘤细胞平均坏死率为:对照组21.8±9.0%、AG490组39.4±11.3%、DDP组40.8±7.3%、联合用药组61.9±9.6%,各组与对照组差异有统计学显著意义(P＜0.05),联合用药组与AG490组、DDP组差异有统计学意义(P＜0.05)。
6、免疫组化结果:P-stat3的阳性表达主要位于宫颈癌细胞的胞浆内,染色较深,散在分布,Caspase-3的阳性表达亦位于胞浆内,散在或灶性分布。各组阳性率依次为:P-STAT3对照组92.5%;DDP组64%;AG490组55%;联合用药组40.9%;Caspase-3对照组30.4%;DDP组60%;AG490组55.6%;联合用药组92.3%;在AG490组中,P-stat3蛋白与Caspase-3蛋白的表达无负相关性(r=-0.074,P＞0.05)
结论:
1、AG490能抑制人宫颈癌Hela细胞裸鼠移植瘤的生长,还可以增加顺铂对寓颈癌的药物敏感性,降低顺铂化疗的毒副作用,与顺铂联合应用抑制作用明显增强。
2、AG490能诱导移植瘤细胞凋亡,与顺铂联合应用后其凋亡效应增强。
3、AG490与顺铀联合应用能降低移植瘤中P-stat3的表达,间接上调Caspase-3表达。
Objective: In this study, preliminary studies on the basis of the test through in vivo observation of the effects of AG490 combined DDP chemotherapy drugs on tumor growth in nude mice. Discussing AG490 by blocking STAT3 pathway on the effects of tumor and the relationship between the Caspase-3 expression and STAT3 pathway. With a view to looking for better ways of treatment.
Methods: We established the cervical cancer model in nude mice with Hela cell and gave the control group, DDP, AG490, AG490 +DDP by intraperitoneal injection. The weight of nude mice and the tumor size was measured. The tumor weight and inhibition rate were evaluated. When the nude mice were sacrificed after 2 weeks treatment. Cell apoptosis was detected by flow cytometry (FCM). The necrotic degree and range were observed by the microscope. Statistical software SPSS10.0 deal with all data.
Results:
1. The effect of the drug to nude mice tumor volume : Tumor volume in DDP group, AG490 group and combination group was less than the control group, there are significant differences(P<0.001). In the treatment of the first 7 days. Compared with the combination group, there are significant differences(P<0.001) in DDP group and AG490 group(P<0.05,P<0.001). There are significant differences between DDP group and AG490 group at inhibiting tumor growth(P<0.05).
2、The effect of the drug to nude mice body weight: During the drug treatment of cervical cancer nude mice growth , there have a change in body weight compared with the control group, there are significant differences in DDP group and combination group (P<0.001). There are no significant differences between AG490 group and the control group (P>0.05). Nude mice are generally good to eat and no significant changes in body weight in AG490 group. However, in DDP group, at the beginning of the treatment on the 7th day the nude mice has become more and more severe weight loss, malnutrition, there are significant differences compared with combination group in weight change (P<0.05). One nude mice death.
3. Each group compared with inhibition rate DDP group, AG490 group, DDP group + AG490 group of nude tumor weight inhibition rate was respectively 51.2%, 33.6% and 59.2%,which have a significant reduction compare with control group(P<0.05).Combination group have a significant reduction in tumor weight compared with the DDP group, AG490 group(P<0.05). There are significant differences between DDP group and the AG490 group (P<0.05)
4. Flow cytometry: DDP group, AG490 group and the combination group marked the peak of apoptosis in G1 phase. The apoptosis rate of the three groups were 52.1±1.3%, 39.5±0.4% and 66.7±1.8%, there are significant differences compared with the control group 10.2±2.4% (P<0.05).
5. Pathological observation: Seeing with naked-eye that tumor was nodular, tumor tissue rich in blood supply, no surrounding tissue and skin infiltration, the boundaries are clear and easy separation, the cut surface like fish. Tumor cells were arranged in nests, films, cord-like and were large, obviously nucleolus, deep-dyeing in light microscope to observe. Pathological mitotic phase are easy to see. In the tumor tissue, blood vessels are expansion congestive and a small amount of lymphocytes, neutrophil infiltration. There have a small number of fibers and blood vessels in between the tumor nest. Small focal necrosis and coagulation necrosis can be seen in the tumor tissue. After the treatment, tumor necrosis occurred in varying degree, the results showed that the degrees of necrosis were varied in each group. The average rate of tumor necrosis: control group: 21.8±9.0%; DDP group: 40.8±7.3%; AG490 group: 39.4±11.3%; the combination group: 61.9±9.6%. There are significant differences compared with the control group (P<0.05). There are significant differences between the combination group and AG490 group、DDP group(P<0.05).
6. The results of immunohistochemistry: P-stat3 positive expression of the cancer cells are mainly located in the cytoplasm, deep-dyeing and scattered. Caspase-3 positive expression of the cancer cells are also located in the cytoplasm, scattered or focal distribution. P-stat3 positive rate was 92.5% in control group、64% in DDP group、55% in AG490 group and 40.9% in combination group . Caspase-3 positive rate was 30.4% in control group、60% in DDP group、55.6% in AG490 group and 92.3% in combination group. There are no significant differences between P-stat3 protein and caspase-3 protein in AG490 group. (r=-0.074,P>0.05).
Conclusion:
1. There is inhibition of AG490 on human cervical cancer Hela cells growth of nude mice , it can increase the DDP sensitivity to cervical cancer drug and reduce the DDP toxicity. AG490 therapy combined with DDP therapy significantly enhance the effect of inhibition.
2.AG490 can induce apoptosis of transplanted tumor, combined application of cisplatin enhanced its effect.
3.AG490 therapy combined with DDP therapy can reduce the expression of P-stat3 at nude mice, indirectly increase the expression of caspase-3, however, no direct effects.
方法:建立人子宫颈癌(Hela)细胞裸鼠异种移植瘤模型并分组。A6490单用或联合DDP处理各组裸鼠。用药期间测量裸鼠体重和肿瘤体积,检测瘤重计算抑瘤率。流式细胞分析术测肿瘤组织中细胞凋亡情况,免疫组化检测移植瘤组织中P-stat3和Caspase-3蛋白表达。所有数据均采用SPSS10.0统计软件进行处理。
结果:
1、药物对裸鼠移植瘤体积生长的影响:分别在用药后第1天、第4天、第7天及第10天测量肿瘤长径(a)、短径(h),计算体积,描绘移植瘤生长曲线图。结果可见从用药第7天开始DDP组、AG490组以及联合用药组(DDP+AG490组)肿瘤体积均小于对照组,有显著性差异(P＜0.001)。联合用药组与DDP组、AG490组比抑瘤作用显著增强(P＜0.05、P＜0.001)。DDP组对肿瘤的生长抑制与A6490组相比显著增强(P＜0.05)。
2、药物对移植瘤裸鼠体重及副作用的影响:药物治疗子宫颈癌裸鼠移植瘤生长期间,从用药第7天开始,顺铂组和联合用药组与对照组比差异有统计学意义(P＜0.001)。A6490组与对照组比差异无统计学意义(P＞0.05)且裸鼠一般情况良好,进食和体重无明显变化。DDP组于治疗开始后第7天裸鼠逐渐出现严重消瘦,营养不良,并且出现明显食欲减退,活动迟缓,皮肤无光泽等毒副反应,体重变化与联合用药组间存在明显差异(P＜0.05),该组有1只裸鼠死亡。
3、各组裸鼠移植瘤瘤重的变化:处死裸鼠,剥出瘤块用电子天平称量瘤重(W),计算抑瘤率。抑瘤率分别为对照组0%、顺铂组51.2%、AG490组33.6%、联合用药组59.2%与对照组相比,各药物作用组均对裸鼠移植瘤有抑制作用,差异有显著性意义(P＜0.05)。A6490与DDP联合应用比两者单独应用抑瘤作用更明显,差异有显著性意义(P＜0.05)。DDP组比A6490组抑瘤作用强,差异有统计学意义(P＜0.05)。
4、流式细胞仪分析凋亡率:DOP组、AG490组和联合用药组有明显G_1期形成的凋亡峰,三个用药组的凋亡率分别是52.1±1.3%、39.5±0.4%、66.7±1.8%。与对照组10.2±2.4%相比均有显著性差异(P＜0.05)。流式细胞分析的各组DNA组方图,可见各用药组有明显的G_1期形成的凋亡峰。
5、光学显微镜下观察Hela细胞裸鼠移植瘤组织形态学的改变:肉眼观移植瘤呈结节状,肿瘤组织血供丰富,无明显周围组织和皮肤浸润,界限清楚,易分离,切而呈鱼肉样。光镜下观察肿瘤细胞呈巢、片、条索状排列,瘤细胞体大、核大深染,核仁明显,病理性核分裂相易见,瘤周组织内血管扩张充血,并有少量淋巴细胞、中性粒细胞浸润,瘤巢之间有少量的纤维及帆管。瘤组织内见小灶性凝固性坏死。用药后,肿瘤组织出现不同程度的坏死。肿瘤细胞平均坏死率为:对照组21.8±9.0%、AG490组39.4±11.3%、DDP组40.8±7.3%、联合用药组61.9±9.6%,各组与对照组差异有统计学显著意义(P＜0.05),联合用药组与AG490组、DDP组差异有统计学意义(P＜0.05)。
6、免疫组化结果:P-stat3的阳性表达主要位于宫颈癌细胞的胞浆内,染色较深,散在分布,Caspase-3的阳性表达亦位于胞浆内,散在或灶性分布。各组阳性率依次为:P-STAT3对照组92.5%;DDP组64%;AG490组55%;联合用药组40.9%;Caspase-3对照组30.4%;DDP组60%;AG490组55.6%;联合用药组92.3%;在AG490组中,P-stat3蛋白与Caspase-3蛋白的表达无负相关性(r=-0.074,P＞0.05)
结论:
1、AG490能抑制人宫颈癌Hela细胞裸鼠移植瘤的生长,还可以增加顺铂对寓颈癌的药物敏感性,降低顺铂化疗的毒副作用,与顺铂联合应用抑制作用明显增强。
2、AG490能诱导移植瘤细胞凋亡,与顺铂联合应用后其凋亡效应增强。
3、AG490与顺铀联合应用能降低移植瘤中P-stat3的表达,间接上调Caspase-3表达。
Objective: In this study, preliminary studies on the basis of the test through in vivo observation of the effects of AG490 combined DDP chemotherapy drugs on tumor growth in nude mice. Discussing AG490 by blocking STAT3 pathway on the effects of tumor and the relationship between the Caspase-3 expression and STAT3 pathway. With a view to looking for better ways of treatment.
Methods: We established the cervical cancer model in nude mice with Hela cell and gave the control group, DDP, AG490, AG490 +DDP by intraperitoneal injection. The weight of nude mice and the tumor size was measured. The tumor weight and inhibition rate were evaluated. When the nude mice were sacrificed after 2 weeks treatment. Cell apoptosis was detected by flow cytometry (FCM). The necrotic degree and range were observed by the microscope. Statistical software SPSS10.0 deal with all data.
Results:
1. The effect of the drug to nude mice tumor volume : Tumor volume in DDP group, AG490 group and combination group was less than the control group, there are significant differences(P<0.001). In the treatment of the first 7 days. Compared with the combination group, there are significant differences(P<0.001) in DDP group and AG490 group(P<0.05,P<0.001). There are significant differences between DDP group and AG490 group at inhibiting tumor growth(P<0.05).
2、The effect of the drug to nude mice body weight: During the drug treatment of cervical cancer nude mice growth , there have a change in body weight compared with the control group, there are significant differences in DDP group and combination group (P<0.001). There are no significant differences between AG490 group and the control group (P>0.05). Nude mice are generally good to eat and no significant changes in body weight in AG490 group. However, in DDP group, at the beginning of the treatment on the 7th day the nude mice has become more and more severe weight loss, malnutrition, there are significant differences compared with combination group in weight change (P<0.05). One nude mice death.
3. Each group compared with inhibition rate DDP group, AG490 group, DDP group + AG490 group of nude tumor weight inhibition rate was respectively 51.2%, 33.6% and 59.2%,which have a significant reduction compare with control group(P<0.05).Combination group have a significant reduction in tumor weight compared with the DDP group, AG490 group(P<0.05). There are significant differences between DDP group and the AG490 group (P<0.05)
4. Flow cytometry: DDP group, AG490 group and the combination group marked the peak of apoptosis in G1 phase. The apoptosis rate of the three groups were 52.1±1.3%, 39.5±0.4% and 66.7±1.8%, there are significant differences compared with the control group 10.2±2.4% (P<0.05).
5. Pathological observation: Seeing with naked-eye that tumor was nodular, tumor tissue rich in blood supply, no surrounding tissue and skin infiltration, the boundaries are clear and easy separation, the cut surface like fish. Tumor cells were arranged in nests, films, cord-like and were large, obviously nucleolus, deep-dyeing in light microscope to observe. Pathological mitotic phase are easy to see. In the tumor tissue, blood vessels are expansion congestive and a small amount of lymphocytes, neutrophil infiltration. There have a small number of fibers and blood vessels in between the tumor nest. Small focal necrosis and coagulation necrosis can be seen in the tumor tissue. After the treatment, tumor necrosis occurred in varying degree, the results showed that the degrees of necrosis were varied in each group. The average rate of tumor necrosis: control group: 21.8±9.0%; DDP group: 40.8±7.3%; AG490 group: 39.4±11.3%; the combination group: 61.9±9.6%. There are significant differences compared with the control group (P<0.05). There are significant differences between the combination group and AG490 group、DDP group(P<0.05).
6. The results of immunohistochemistry: P-stat3 positive expression of the cancer cells are mainly located in the cytoplasm, deep-dyeing and scattered. Caspase-3 positive expression of the cancer cells are also located in the cytoplasm, scattered or focal distribution. P-stat3 positive rate was 92.5% in control group、64% in DDP group、55% in AG490 group and 40.9% in combination group . Caspase-3 positive rate was 30.4% in control group、60% in DDP group、55.6% in AG490 group and 92.3% in combination group. There are no significant differences between P-stat3 protein and caspase-3 protein in AG490 group. (r=-0.074,P>0.05).
Conclusion:
1. There is inhibition of AG490 on human cervical cancer Hela cells growth of nude mice , it can increase the DDP sensitivity to cervical cancer drug and reduce the DDP toxicity. AG490 therapy combined with DDP therapy significantly enhance the effect of inhibition.
2.AG490 can induce apoptosis of transplanted tumor, combined application of cisplatin enhanced its effect.
3.AG490 therapy combined with DDP therapy can reduce the expression of P-stat3 at nude mice, indirectly increase the expression of caspase-3, however, no direct effects.
引文
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