南美洲响尾蛇神经毒素（Crotoxin）对人食管鳞状细胞癌作用的实验研究
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摘要
目的:观察南美洲响尾蛇神经毒素(Crotoxin)对体外培养的人食管癌Eca-109细胞株生长的抑制作用,体内局部用药对荷瘤裸鼠人食管癌Eca-109细胞移植瘤的抑制作用,探讨南美洲响尾蛇神经毒素(Crotoxin)的抗肿瘤作用及其可能的机制。
方法:以人食管癌Eca-109细胞为实验对象,体外培养人食管癌Eca-109细胞株,分别以不同浓度的Crotoxin及顺铂作用于Eca-109细胞,并设阴性对照组,通过四氮唑盐还原法(MTT)观察Crotoxin对Eca-109细胞的生长抑制作用;倒置相差显微镜观察细胞的形态学改变,Hoechst33342染色后荧光显微镜观察细胞形态及细胞核的变化,应用流式细胞术检测Crotoxin对Eca-109细胞凋亡及细胞周期的影响;实时荧光定量逆转录聚合酶链式反应(RT-PCR)测定Crotoxin作用后Eca-109细胞内BCL-2、P15及Caspase3-P17基因扩增水平。裸小鼠肩背部皮下注射人食管癌Eca-109细胞悬液,建立皮下移植瘤动物模型,分对照组和Crotoxin组(Crotoxin组为以生理盐水稀释至浓度为25-100μg/kg的Crotoxin注射液,对照组为相同剂量的生理盐水),均为瘤周皮下注射,隔日1次,共给药10次,停药后次日杀死所有动物,比较移植瘤瘤重,计算抑瘤率;蛋白免疫印迹法(Western blot)测定移植瘤内BCL-2、P15及Caspase3-P17蛋白表达水平。
结果:Crotoxin对人食管癌Eca-109细胞生长有抑制作用,与顺铂比较,有相似甚至更强的抑制肿瘤细胞效果(P<0.05),Crotoxin对人食管癌Eca-109细胞有诱导凋亡的作用,倒置相差显微镜和荧光显微镜下均能观察到凋亡细胞的核固缩、染色质边集、核碎裂、凋亡小体形成等形态学特征。Crotoxin可引起Eca-109细胞内P15、Caspase3-P17基因扩增增加,BCL-2基因扩增减少,流式细胞仪可检测到Eca-109细胞的凋亡峰,Crotoxin药物浓度越大,检测到凋亡峰的时间越早,并有G1期细胞增多。Crotoxin对人食管癌Eca-109细胞裸小鼠移植瘤生长有抑制作用,抑瘤率为35.7%(Crotoxin 50ug/ml组);Western blot检测到Crotoxin作用后瘤体标本P15、Caspase3-P17蛋白表达升高,BCL-2蛋白表达降低
结论:南美洲响尾蛇神经毒素(Crotoxin)对人食管癌Eca-109细胞体外生长有抑制作用,其作用与诱导凋亡和G1期阻滞有关,其中诱导凋亡作用可能和活化Caspase3有关。Crotoxin局部用药能抑制裸小鼠皮下人食管癌Eca-109细胞移植瘤的生长,且抑制作用与用药剂量相关。
Objective: To observe the suppression effects of south america rattlesnake neurotoxin ( Crotoxin) on Eca-109 cells line of human carcinoma of esophagus , and observe the inhibitory efects of Crotoxin on Eca-109 cells xenografts in nude mice. To investigate the anti-tumor effect of the neurotoxin of south america rattlesnake ( Crotoxin) and explore its related mechanisms.
Method: Eca-109 cells,a cell line of human carcinoma of esophagus,was used as the experimental materials. Eca-109 cells were cultured in vitro , use different saturation,s Crotoxin to Eca-109 cells, to set up negative group. MTT colorimetry was used to test the growth inhibition ratio of cells.The light microscope to be used to observe the morphology alter of Eca-109 cells,the fluorescence microscope to the alter of cell nucleus. The flow cytometry was used to detect the apoptosis rate and the cell cycle of Eca-109 cells treated with Cortoxin. Additionally, RT-PCR also be used to detect the gene amplification of BCL-2.P15 and P17. Eca-109cell suspension were implanted into the subcutaneous tissue of back,and establish nude mice ethotopic transplantation tumor model with human carcinoma of esophagus. Fractionate them to Crotoxin group and control group(Crotoxin group to be dilute to 25-100μg/kg by solution of 09%NcaI, nad control group were treated by the same way with equal volum solution of 09% NacI). The nude mices of both group were injected subcutancously to the perimeter of tumor once every other days,All animals were sacrificed at the next day after the 10th treatment. The inhibitory rate was calculated according to the weights of xenografts. Western blot to detect the expression of some proteins in the transplantation tumor,such asBcl-2.P15 and p17.
Result: Crotoxin can inhibit the growth of Eca-109 cells,it is more effection than Cisplatin. Cortoxin can induce Eca-109 cells to apoptosis, The morphological chnages of apoptosis such as
Cell shrinkage,chromatin condensation,nuclear fragmentation and apoptotic body were obeserved by both light and fluorescence microscope.Crotoxin can increases the gene amplification of P15 and P17, and grow downwards the Bcl-2. The flow cytometry can detect the apoptotic peak of Eca-109 cells ,and the higher concentration,the earer appearance of apoptotic peak,and the G1 stage cells were also grow in number. Crotoxin inhibited the xenografts growth significantly and the inhibitory rate was 35.7%.Western blot to be used to detect the expression of sproteins,it shows that P15 and P17 were increased,and Bcl-2 was decreased.
Conclusion: Crotoxin can inhibit the growth of Eca-109 cells and the anti-tumor effect was closely related to the induction of apoptosis and cell cycle blockage. The mechanism of Crotoxin to induce apoptosis was contribute to the activation of Caspase-3. Crotoxin can inhibit the growth of Eca-109 cells xenografts in nude mice,and it,s inhibition relate to the dosage.
方法:以人食管癌Eca-109细胞为实验对象,体外培养人食管癌Eca-109细胞株,分别以不同浓度的Crotoxin及顺铂作用于Eca-109细胞,并设阴性对照组,通过四氮唑盐还原法(MTT)观察Crotoxin对Eca-109细胞的生长抑制作用;倒置相差显微镜观察细胞的形态学改变,Hoechst33342染色后荧光显微镜观察细胞形态及细胞核的变化,应用流式细胞术检测Crotoxin对Eca-109细胞凋亡及细胞周期的影响;实时荧光定量逆转录聚合酶链式反应(RT-PCR)测定Crotoxin作用后Eca-109细胞内BCL-2、P15及Caspase3-P17基因扩增水平。裸小鼠肩背部皮下注射人食管癌Eca-109细胞悬液,建立皮下移植瘤动物模型,分对照组和Crotoxin组(Crotoxin组为以生理盐水稀释至浓度为25-100μg/kg的Crotoxin注射液,对照组为相同剂量的生理盐水),均为瘤周皮下注射,隔日1次,共给药10次,停药后次日杀死所有动物,比较移植瘤瘤重,计算抑瘤率;蛋白免疫印迹法(Western blot)测定移植瘤内BCL-2、P15及Caspase3-P17蛋白表达水平。
结果:Crotoxin对人食管癌Eca-109细胞生长有抑制作用,与顺铂比较,有相似甚至更强的抑制肿瘤细胞效果(P<0.05),Crotoxin对人食管癌Eca-109细胞有诱导凋亡的作用,倒置相差显微镜和荧光显微镜下均能观察到凋亡细胞的核固缩、染色质边集、核碎裂、凋亡小体形成等形态学特征。Crotoxin可引起Eca-109细胞内P15、Caspase3-P17基因扩增增加,BCL-2基因扩增减少,流式细胞仪可检测到Eca-109细胞的凋亡峰,Crotoxin药物浓度越大,检测到凋亡峰的时间越早,并有G1期细胞增多。Crotoxin对人食管癌Eca-109细胞裸小鼠移植瘤生长有抑制作用,抑瘤率为35.7%(Crotoxin 50ug/ml组);Western blot检测到Crotoxin作用后瘤体标本P15、Caspase3-P17蛋白表达升高,BCL-2蛋白表达降低
结论:南美洲响尾蛇神经毒素(Crotoxin)对人食管癌Eca-109细胞体外生长有抑制作用,其作用与诱导凋亡和G1期阻滞有关,其中诱导凋亡作用可能和活化Caspase3有关。Crotoxin局部用药能抑制裸小鼠皮下人食管癌Eca-109细胞移植瘤的生长,且抑制作用与用药剂量相关。
Objective: To observe the suppression effects of south america rattlesnake neurotoxin ( Crotoxin) on Eca-109 cells line of human carcinoma of esophagus , and observe the inhibitory efects of Crotoxin on Eca-109 cells xenografts in nude mice. To investigate the anti-tumor effect of the neurotoxin of south america rattlesnake ( Crotoxin) and explore its related mechanisms.
Method: Eca-109 cells,a cell line of human carcinoma of esophagus,was used as the experimental materials. Eca-109 cells were cultured in vitro , use different saturation,s Crotoxin to Eca-109 cells, to set up negative group. MTT colorimetry was used to test the growth inhibition ratio of cells.The light microscope to be used to observe the morphology alter of Eca-109 cells,the fluorescence microscope to the alter of cell nucleus. The flow cytometry was used to detect the apoptosis rate and the cell cycle of Eca-109 cells treated with Cortoxin. Additionally, RT-PCR also be used to detect the gene amplification of BCL-2.P15 and P17. Eca-109cell suspension were implanted into the subcutaneous tissue of back,and establish nude mice ethotopic transplantation tumor model with human carcinoma of esophagus. Fractionate them to Crotoxin group and control group(Crotoxin group to be dilute to 25-100μg/kg by solution of 09%NcaI, nad control group were treated by the same way with equal volum solution of 09% NacI). The nude mices of both group were injected subcutancously to the perimeter of tumor once every other days,All animals were sacrificed at the next day after the 10th treatment. The inhibitory rate was calculated according to the weights of xenografts. Western blot to detect the expression of some proteins in the transplantation tumor,such asBcl-2.P15 and p17.
Result: Crotoxin can inhibit the growth of Eca-109 cells,it is more effection than Cisplatin. Cortoxin can induce Eca-109 cells to apoptosis, The morphological chnages of apoptosis such as
Cell shrinkage,chromatin condensation,nuclear fragmentation and apoptotic body were obeserved by both light and fluorescence microscope.Crotoxin can increases the gene amplification of P15 and P17, and grow downwards the Bcl-2. The flow cytometry can detect the apoptotic peak of Eca-109 cells ,and the higher concentration,the earer appearance of apoptotic peak,and the G1 stage cells were also grow in number. Crotoxin inhibited the xenografts growth significantly and the inhibitory rate was 35.7%.Western blot to be used to detect the expression of sproteins,it shows that P15 and P17 were increased,and Bcl-2 was decreased.
Conclusion: Crotoxin can inhibit the growth of Eca-109 cells and the anti-tumor effect was closely related to the induction of apoptosis and cell cycle blockage. The mechanism of Crotoxin to induce apoptosis was contribute to the activation of Caspase-3. Crotoxin can inhibit the growth of Eca-109 cells xenografts in nude mice,and it,s inhibition relate to the dosage.
引文
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