摘要
目的：食管癌系指由食管鳞状上皮或腺上皮的异常增生所形成的恶性病变，约占所有恶性肿瘤的2%。我国是食管癌的高发国家,目前治疗食管癌的方法主要是手术治疗辅助放化疗。虽然对食管癌采取多种治疗方法，但其死亡率依然没有明显下降。大多数患者就诊时已出现转移症状，食管癌的转移对其生存率的影响至关重要。
肿瘤的发生是一个多基因参与的长期连续的过程，其中涉及了许多信号传导通路。细胞外信号调节激酶（ERK）在30%的人类肿瘤中均存在异常表达，其基本的信号传递途径为Ras→Raf→MEK→ERK，通过此途径将细胞外刺激转导至细胞内，从而调节细胞的增殖分化和侵袭。该通路中任何一个因子异常激活，都会导致ERK通路的表达异常。Raf-1作为ERK通路中的重要一员，可被Ras或一些蛋白因子激活，活化的Raf-1可以进一步激活其下游的MEK、ERK。有研究发现食管癌的发生、发展和ERK通路有着密切的关系。
蟾蜍灵是从蟾蜍耳后提取的毒素，具防癌、抗癌,促进白细胞生长,提高机体免疫功能等作用。有研究表明蟾蜍灵具有抗食管癌作用，但是关于蟾毒灵抑制食管癌转移的作用和机制研究并不是很清楚。本实验通过培养食管细胞癌株TE13，用不同浓度的蟾蜍灵处理该细胞株，观察蟾蜍灵对食管癌细胞株TE13的迁移和趋化作用的影响。从蛋白水平观察蟾蜍灵作用下的食管癌细胞株TE13中Raf-1活化及Raf-1的表达水平的变化，进一步探讨蟾毒灵抑制食管癌转移的机制。
方法：本实验在食管癌细胞株TE13培养的基础上进行研究，通过MTT方法检测细胞药物毒性，通过细胞划痕试验检测不同浓度的蟾蜍灵对该细胞株的增殖和迁移能力的抑制作用。通过Boyden小室实验检测不同浓度的蟾蜍灵处理后的食管癌细胞株TE13的趋化运动情况。采用免疫组化方法和Western blot方法检测经不同浓度的蟾蜍灵处理后细胞株中Raf-1、磷酸化Raf-1蛋白的表达情况。
结果：
1MTT检测结果
不同浓度的蟾蜍素（0nM/L，10nM/L，25nM/L，50nM/L，100nM/L，200nM/L，400nM/L，800nM/L）干预24h后平均OD值（0.991、0.869、0.735、0.561、0.499、0.371、0.265、0.258），蟾蜍素在100nM/L及其以下的药物浓度作用下，细胞存活率大于50%，即TD0为100nM/L。
2不同浓度的蟾蜍素对食管癌细胞株TE13的迁移和趋化运动的影响
细胞划痕实验显示,不同溶度的蟾蜍素处理TE13细胞后划痕24h后，蟾蜍素浓度的增加，可降低TE13细胞的迁移能力。Boyden小室实验显示，不同浓度的蟾蜍素（0nM/L，10nM/L，25nM/L，50nM/L，100nM/L）处理后的食管癌细胞穿过滤膜的细胞数量分别为164±3,160±5,131±4,83±2,54±3个处理组与对照组相比有统计学意义（P＜0.05），（25nM/L，50nM/L，100nM/L）的蟾蜍素处理组间两两比较具有统计学意义（P＜0.05）。
3不同浓度的蟾蜍素对食管癌细胞株TE13中Raf-1、P-Raf-1蛋白表达的影响
3.1Western结果显示Raf-1蛋白在不同溶度（0nM/L，10nM/L，25nM/L，50nM/L，100nM/L）的蟾蜍素处理后的TE13细胞中表达的相对灰度值（IDV）分别为0.742±0.056、0.754±0.040、0.752±0.040、0.748±0.040、0.753±0.061，处理组与对照组相比较没有明显差别（P＞0.05）.磷酸化Raf-1在不同溶度（0nM/L，10nM/L，25nM/L，50nM/L，100nM/L）的蟾蜍素处理后的TE13细胞中表达的相对灰度值（IDV）分别为0.748±0.011、0.732±0.056、0.685±0.160、0.455±0.075、0.365±0.019，蟾蜍素处理组（25nM/L，50nM/L，100nM/L）两两比较具有统计学意义（P＜0.05）。
3.2镜下观察免疫组化，Raf-1、P-Raf-1蛋白阳性表达于TE13细胞的胞浆，呈棕黄色颗粒。不同浓度的蟾蜍素处理细胞后，Raf-1实验组和对照组的阳性细胞数量及棕黄色颗粒数均未见明显减少，没有统计学意义。结果表明蟾蜍素对Raf-1蛋白的表达影响，无显著性差异，无统计学意义(P>0.05）。而经不同药物浓度处理后的细胞中P-Raf-1的阳性率为（80.23，78.67，52.22，49.28，47.59）%,蟾蜍素处理组间（25nM/L，50nM/L，100nM/L）两两比较差异均具有统计学意义（P＜0.05）。
结论：
1蟾蜍素对食管鳞状细胞癌TE13细胞株的迁移能力具有明显的抑制作用。
2蟾蜍素对食管鳞状细胞癌TE13细胞株的趋化运动能力具有明显的抑制作用，并且在一定药物浓度内存在剂量依赖关系。
3蟾蜍灵不抑制Raf-1蛋白表达、但可以降低磷酸化Raf-1在食管鳞状细胞癌TE13细胞株的表达，提示可能通过抑制Raf-1的激活抑制了ERK通路从而抑制了食管鳞状细胞癌TE13细胞株的转移。
Objective: Esophageal cancer is one kind of malignant tumor in theesophageal epithelium. It accounts for2%of all malignant tumors in theworldwide. Some evidences show that there are high rate incidence ofesophageal cancer in our country. Until now, the treatment of esophagealcancer is surgical with or without adjuvant radiotherapy or chemotherapy.Although there are several methods to treat esophageal cancer, the mortalityrate is still high. Most of the patients already have transfer symptoms whenthey go to outpatient. The transfer of esophageal cancer has an importanteffect on survival rate.
The occurrence of the tumor is more than one gene involved in long-termcontinuous process, which involves a number of signaling transductionpathways. Extracellular signal-regulated kinase (ERKs) have abnormalexpressed in30%of human tumors，the basic signal transduction isRas→Raf→MEK→ERK. ERKs transducts signal to the nucleus, involves inregulating cell proliferation, differentiation, apoptosis and invasion. If onefactor in this pathway be activated abnormally, the whole pathway willexpress disorder. Raf-1as one of important factor in the ERK pathway, and itcan be activated by Ras and variety of protein factors products. Raf-1kinasephosphorylates and activates MEK (MEK1and MEK2). MEK phosphorylatesand activates ERK. Research has show that Extracellular signal-regulatedkinase (ERKs) play an important role in esophageal cancer.
Bufalin is extracted from toxin of Bufo bufo gargarizans Cantor. Therehave been reported that it has inhibitory effects on esophageal cancer.However，the role of anti-metastasis and the underlying mechanism remainelusive. Our experiment performs different concentration Bufalin to cultureesophageal cancer cell strain (TE13). In order to investigate that the effect and mechanism of Bufalin in TE13cell strain.
Methods: The experiment based on esophageal carcinoma cell strainTE13culture. Wound Healing assay was perform to found effect of Bufalin oncell migration and cell chemotaxis; chemotaxis of TE13with differentconcentration Bufalin was investigated by Boyden. Western blot andimmunocytochemistry were performed to detect the expression of Raf-1andP-Raf-1in TE13with different concentration Bufalin.
Results:
1MTT method showed that Bufalin inhibited the growth of esophagealcancer cell lines TE13significantly. Bufalin in the100nM/L and below,under the action of the drug concentration, cell viability greater than50%
2The influence of different Bufalin on migration of TE13cell lines
Wound Healing assay showed that the inhibition of cell migrationincreased with the Bufalin concentration increasing after24hours; Boydencabin showed that the migration of cells through the transwell significantlyreduced with the Bufalin concentration increasing (10nM/L，25nM/L，50nM/L，100nM/L)(P＜0.05); no significant differences between in thecontrol and treatment group(10nM/L)(P＞0.05)。
3The influence of different Bufalin on expression of Raf-1
3.1The expression of Raf-1in TE13cell with different concentrationBufalin was detected by western blot. The result shows that there are nosignificant differences between control group and test group. The expressionof P-Raf-1in TE13cell with different concentration Bufalin was also detectedby western blot. The result shows that there are decreased between controlgroup and test group(25nM/L，50nM/L，100nM/L).
3.2The expression of Raf-1in TE13cell with different concentrationBufalin was detected by Immunocytochemistry. The result shows that thereare no significant differences between control group and test group. Theexpression of P-Raf-1in TE13cell with different concentration Bufalin wasalso detected by Immunocytochemistry. The result shows that there aresignificant differences between control group and test group(25nM/L， 50nM/L，100nM/L). This resule are consistent with the above conclusions.
Conclusion:
1. Bufalin has vital effect on inhibiting the migration of TE13cell strain.The inhibition effect is different with different concentration of Bufalin.
2. Bufalin has vital effect on inhibiting the chemotaxis of TE13cell strain.The inhibition effect is different with different concentration of Bufalin.
3. Bufalin has no effect on inhibiting the expression of Raf-1, but it haseffect on inhibiting the expression of p-Raf-1in TE13cell strain. Theresult shows that ERK pathway was blocked when Raf-1was inhibited,and then it can inhibit the TE13cell strain metastasis.
引文
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