目的：观察蟾毒灵对大肠癌HCT-116细胞上皮间质转化作用的影响，初步探讨其可能的机制。方法：使用TGF-β1诱导大肠癌HCT-116细胞发生上皮间质转化（epithelial-mesenchymal transition，EMT）现象，制备EMT模型。实验分为空白对照组、模型组、蟾毒灵组。分别干预72h后，观察各组细胞形态学差异；应用transwell-invasion与transwell-migration模型评价蟾毒灵（Bufalin）对HCT-116细胞侵袭与迁移能力的影响；应用Western blot检测EMT相关蛋白E-cadherin，Vimentin及β-catenin的表达情况；应用细胞免疫荧光法观察β-catenin蛋白分布情况。结果：1、细胞形态学变化：模型组细胞较空白组明显伸长变窄，细胞间连接相对疏松。Bufalin组与模型组相比，部分细胞呈不典型的鹅卵石样上皮细胞形态，部分细胞拉长变窄，细胞间连接较紧密。2、Transwell-invasion实验提示：空白对照组、模型组，Bufalin组的72h穿膜个数（200倍，3个视野）分别为：（86.67±11.72）、（413.33±41.63）、（110.00±26.46），三组间有显著性差异（P＜0.05）。Transwell-migration实验提示：空白对照组、模型组，Bufalin组的72h穿膜个数（200倍，3个视野）分别为：（475±69.41）、（898±49.49）、（507±38.16），三组间有显著性差异（P＜0.05）。3、Western blot检测EMT相关蛋白E-cadherin/ Vimentin/β-catenin的表达：模型组与空白对照组相比，上皮标志物E-cadhrin表达下降，间质标志物Vmentin表达上升；Bufalin组与模型组相比，上皮标志物E-cadhrin的表达明显上调，间质标志物Vimentin的表达明显下调。β-catenin的表达无明显差异。4、细胞免疫荧光法观察β-catenin的分布情况：实验提示，模型组较空白对照组，β-catenin的表达从细胞膜，细胞质移位至细胞核内；Bufalin组较模型组，β-catenin蛋白主要集中于细胞质，细胞核的表达较少。结论：1、TGF-β1可以诱导结肠癌HCT-116细胞发生上皮间质转化现象。2、Bufalin抑制HCT-116细胞的侵袭、迁移能力可能与细胞发生上皮间质转化有关。3、Bufalin可能通过抑制β-catenin的核移位从而抑制HCT-116细胞EMT现象的发生，最终抑制细胞的侵袭、转移。
Objective In order to explore the possible mechanism between bufalin and colorectal cancer, we have observed the effects that bufalin inhibits the epithelial mesenchymal transformation on colorectal cell HCT-116. Methods We induce the colorectal cell HCT-116 to take effect of epithelial-mesenchymal transition (EMT) by TGF-β1 to prepare the EMT models. The experiment is divided into control groups, model groups and bufalin groups. After 72h of intervention, observe the morphological difference of each groups; evaluate the effect of the invasion and migration capabilities of bufalin on HCT-116 by using transwell-invasion and transwell-migration models; To detect the expressions of EMT-related protein including E-cadherin，Vimentin and β-catenin by using Western blot; To observe the distribution of β-catenin protein by using immunofluorescence method. Results 1. Changes of cellular morphology: Compared with the control groups, the cells of the model groups became elongated and spindle, the connections between cells are relatively lax. 2. Transwell-invasion experiment shows: After 72h , the cell numbers of permeation(under ×200, 3 view) of control groups, model groups and bufalin groups are: (86.67±11.72), (413.33±41.63) and (110.00±26.46), which show significant difference(P<0.05). Transwell-migration experiment shows: After 72h , the cell numbers of permeation(under ×200, 3 view) of control groups, model groups and bufalin groups are: (475±69.41), (898±49.49) and (507±38.16), which show significant difference(P<0.05). 3. The expressions of EMT-related protein E-cadherin/Vimentin/β-catenin by Western blot: Compared with the control groups, the model groups have lower expressions of E-cadherin and higher expressions of Vimentin; compared with model groups, the Bufalin groups have higher expressions of E-cadherin while the expressions of Vimentin are significantly lower. There is no significant difference between the expressions of β-catenin. 4. The distribution of β-catenin under immunofluorescence method: The experiment shows, compared with the control groups, the model groups have β-catenin expressions moving from the cell membrane and the cytoplasm to nucleus; compared with the model groups, the Bufalin groups have more expressions in cytoplasm than in nucleus. Conclusion 1. TGF-β1 can induce the epithelial mesenchymal transformation of colorectal cell HCT-116. 2. The inhibiting ability of Bufalin to the invasion and migration of HCT-116 is related to the epithelial mesenchymal transformation. 3. Bufalin probably inhibits the EMT of HCT-116 by inhibiting the nuclear translocation of β-catenin, and thus inhibit the invasion and migration of cells.
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