南美响尾蛇神经毒素对人肺腺癌A549细胞生长抑制作用研究
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摘要
第一部分南美响尾蛇神经毒素对人肺腺癌A549细胞体外生长抑制作用及其相关机制
目的观察南美响尾蛇神经毒素(Crotoxin)体外抗肿瘤作用,并探讨其相关机制。
方法以人肺腺癌A549细胞(野生型P53,wild-typeP53,wtP53)为实验对象。通过四氮唑盐还原法(MTT)、细胞集落平板克隆实验观察Crotoxin及其与吉非替尼(Gefitinib,Iressa)联合应用对人肺腺癌A549细胞的生长抑制作用;应用流式细胞术检测Crotoxin对人肺腺癌A549细胞凋亡率、细胞周期的影响;蛋白免疫印迹法(Western blot)测定Crotoxin作用后人肺腺癌A549细胞内wtP53、caspase3活性亚单位P17、phospho-P38MAPK蛋白表达水平,并以SB203580(吡啶咪唑化合物,P38信号转导通路特异性抑制剂)抑制P38MAPK活性后观察Crotoxin对人肺腺癌A549细胞的影响。
结果Crotoxin对人肺腺癌A549细胞有生长抑制作用,与Iressa联用可增强Iressa抗肿瘤效果(P<0.05);Crotoxin对人肺腺癌A549细胞有诱导凋亡和细胞周期G1期阻滞作用,可引起A549细胞胞内wtP53、caspase3活性亚单位P17、phospho-P38MAPK蛋白表达上调,以SB203580抑制P38MAPK活性后,Crotoxin对A549细胞诱导凋亡作用基本消失,无caspase3活性亚单位P17表达上调,但G1期阻滞作用无明显变化,仍有wtP53表达上调。
结论Crotoxin对人肺腺癌A549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激活P38MAPK信号转导通路进而活化Caspase3有关,G1期阻滞可能由胞内wtP53蛋白表达上调所致。
第二部分南美响尾蛇神经毒素对人肺腺癌A549细胞裸鼠移植瘤生长抑制作用
目的观察南美响尾蛇神经毒素(Crotoxin)对人肺腺癌A549细胞(wtP53)裸鼠移植瘤生长抑制作用,及Crotoxin对移植瘤组织微血管生成的影响。
方法接种人肺腺癌A549细胞建立裸鼠移植瘤模型,分对照组和Crotoxin组,给药四周后(Crotoxin10μg/kg生理盐水稀释后腹腔注射2次/周)比较移植瘤瘤重,计算抑瘤率;免疫组化法测定移植瘤微血管密度(microvessd density,MVD);电镜观察移植瘤微血管内皮改变。
结果Crotoxin对人肺腺癌A549细胞裸鼠移植瘤生长有抑制作用,抑瘤率为36.3%;免疫组化MVD计数Crotoxin组(13.56±3.27)较对照组(34.29±8.64)明显减少,差异有统计学意义(P<0.05);电镜观察Crotoxin组移植瘤微血管数量较对照组明显减少,微血管内皮细胞结构有不同程度损害。
结论Crotoxin对人肺腺癌A549细胞裸鼠移植瘤有生长抑制作用,可减少移植瘤组织微血管生成。
Part1 In vitro inhibition of Crotoxin on human lung adenocarcinoma cell line A549 and the related mechanisms
Objective To observe the anti-tumor effect of Crotoxin in vitro and explore its related mechanisms.
Methods A549 cells, a cell line of human lung adenicarinoma (wild-type p53), was used as the experimental materials. MTT colorimetry was used to test the growth inhibition ratio, and the flat colony experiment reflected the colony formation of cells. Both of the experiments were used to estimate the effects of Crotoxin and its combination with Iressa. The flow cytometry was used to detect the apoptosis rate and the cell cycle of A549 cells treated with Cortoxin . Additionally, we also used Western Blot to detect the expression of some proteins, such as wt p53, p17, the active subunit of caspase-3, phospho-P38MAPK. Futhermore, we used SB203580 to inhibit the activity of P38MAPK to observe whether A549 cells have some changes after treated by Crotoxin.
Results Crotoxin can not only inhibit the growth of A549 but also has synergetic or additive effect when it is combined with Iressa.Cortoxin can induce A549 cells to apoptosis and block them in G1 period, and it can also up-regulate the expression of wt p53, p17, phospho-P38MAPK. When using SB203580 to inhibit the activity of P38MAPK, apoptosis was almost disappeared and there was no expression of p17, but the blockage of G1 period remained unchanged, wt p53 was still upregulated.
Conclusion Crotoxin can inhibit the growth of A549 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 P38MAPK, which further activate Caspase-3.The G1 blockage possibly due to up-regulation of wt p53.
Part2 Growth--inhibiting efects of Crotoxin on A549 cells xenografts in nude mice
Objective To observe the inhibitory efects of Crotoxin on A549 cells xenografts in nude mice and the influence of angiogenesis.
Methods A549 cells were incubated and were inoculated subcutaneous1y in athymic nude mice to establish xenograft models.The mice were divided into control group and Crotoxin group. The inhibitory rate was calculated according to the weights of xenografts.The intratumoral microvessel density(MVD) was evaluated by immunohistochemical staining. The electron microscop was used to observe the morphological change of microvess endothelium.
Results Inhibited the xenografts growth significantly and the inhibitory rate was 36.3%. Immunohistochemical results revealed that the MVD in Crotoxin group(13.56±3.27)was signifieanfly lower than those in control group(34.29±8.64),(P<0.05). Decrease of microvessels in the xenograft tumor and morphological change of endothelium were observed under electron microscop.
Conclusion Crotoxin can inhibit the growth of A549 cells xenografts in nude mice, which may be due to the inhibit tumor angiogenesis.
目的观察南美响尾蛇神经毒素(Crotoxin)体外抗肿瘤作用,并探讨其相关机制。
方法以人肺腺癌A549细胞(野生型P53,wild-typeP53,wtP53)为实验对象。通过四氮唑盐还原法(MTT)、细胞集落平板克隆实验观察Crotoxin及其与吉非替尼(Gefitinib,Iressa)联合应用对人肺腺癌A549细胞的生长抑制作用;应用流式细胞术检测Crotoxin对人肺腺癌A549细胞凋亡率、细胞周期的影响;蛋白免疫印迹法(Western blot)测定Crotoxin作用后人肺腺癌A549细胞内wtP53、caspase3活性亚单位P17、phospho-P38MAPK蛋白表达水平,并以SB203580(吡啶咪唑化合物,P38信号转导通路特异性抑制剂)抑制P38MAPK活性后观察Crotoxin对人肺腺癌A549细胞的影响。
结果Crotoxin对人肺腺癌A549细胞有生长抑制作用,与Iressa联用可增强Iressa抗肿瘤效果(P<0.05);Crotoxin对人肺腺癌A549细胞有诱导凋亡和细胞周期G1期阻滞作用,可引起A549细胞胞内wtP53、caspase3活性亚单位P17、phospho-P38MAPK蛋白表达上调,以SB203580抑制P38MAPK活性后,Crotoxin对A549细胞诱导凋亡作用基本消失,无caspase3活性亚单位P17表达上调,但G1期阻滞作用无明显变化,仍有wtP53表达上调。
结论Crotoxin对人肺腺癌A549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激活P38MAPK信号转导通路进而活化Caspase3有关,G1期阻滞可能由胞内wtP53蛋白表达上调所致。
第二部分南美响尾蛇神经毒素对人肺腺癌A549细胞裸鼠移植瘤生长抑制作用
目的观察南美响尾蛇神经毒素(Crotoxin)对人肺腺癌A549细胞(wtP53)裸鼠移植瘤生长抑制作用,及Crotoxin对移植瘤组织微血管生成的影响。
方法接种人肺腺癌A549细胞建立裸鼠移植瘤模型,分对照组和Crotoxin组,给药四周后(Crotoxin10μg/kg生理盐水稀释后腹腔注射2次/周)比较移植瘤瘤重,计算抑瘤率;免疫组化法测定移植瘤微血管密度(microvessd density,MVD);电镜观察移植瘤微血管内皮改变。
结果Crotoxin对人肺腺癌A549细胞裸鼠移植瘤生长有抑制作用,抑瘤率为36.3%;免疫组化MVD计数Crotoxin组(13.56±3.27)较对照组(34.29±8.64)明显减少,差异有统计学意义(P<0.05);电镜观察Crotoxin组移植瘤微血管数量较对照组明显减少,微血管内皮细胞结构有不同程度损害。
结论Crotoxin对人肺腺癌A549细胞裸鼠移植瘤有生长抑制作用,可减少移植瘤组织微血管生成。
Part1 In vitro inhibition of Crotoxin on human lung adenocarcinoma cell line A549 and the related mechanisms
Objective To observe the anti-tumor effect of Crotoxin in vitro and explore its related mechanisms.
Methods A549 cells, a cell line of human lung adenicarinoma (wild-type p53), was used as the experimental materials. MTT colorimetry was used to test the growth inhibition ratio, and the flat colony experiment reflected the colony formation of cells. Both of the experiments were used to estimate the effects of Crotoxin and its combination with Iressa. The flow cytometry was used to detect the apoptosis rate and the cell cycle of A549 cells treated with Cortoxin . Additionally, we also used Western Blot to detect the expression of some proteins, such as wt p53, p17, the active subunit of caspase-3, phospho-P38MAPK. Futhermore, we used SB203580 to inhibit the activity of P38MAPK to observe whether A549 cells have some changes after treated by Crotoxin.
Results Crotoxin can not only inhibit the growth of A549 but also has synergetic or additive effect when it is combined with Iressa.Cortoxin can induce A549 cells to apoptosis and block them in G1 period, and it can also up-regulate the expression of wt p53, p17, phospho-P38MAPK. When using SB203580 to inhibit the activity of P38MAPK, apoptosis was almost disappeared and there was no expression of p17, but the blockage of G1 period remained unchanged, wt p53 was still upregulated.
Conclusion Crotoxin can inhibit the growth of A549 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 P38MAPK, which further activate Caspase-3.The G1 blockage possibly due to up-regulation of wt p53.
Part2 Growth--inhibiting efects of Crotoxin on A549 cells xenografts in nude mice
Objective To observe the inhibitory efects of Crotoxin on A549 cells xenografts in nude mice and the influence of angiogenesis.
Methods A549 cells were incubated and were inoculated subcutaneous1y in athymic nude mice to establish xenograft models.The mice were divided into control group and Crotoxin group. The inhibitory rate was calculated according to the weights of xenografts.The intratumoral microvessel density(MVD) was evaluated by immunohistochemical staining. The electron microscop was used to observe the morphological change of microvess endothelium.
Results Inhibited the xenografts growth significantly and the inhibitory rate was 36.3%. Immunohistochemical results revealed that the MVD in Crotoxin group(13.56±3.27)was signifieanfly lower than those in control group(34.29±8.64),(P<0.05). Decrease of microvessels in the xenograft tumor and morphological change of endothelium were observed under electron microscop.
Conclusion Crotoxin can inhibit the growth of A549 cells xenografts in nude mice, which may be due to the inhibit tumor angiogenesis.
引文
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