EGF诱导结肠腺癌HCT-116细胞神经内分泌分化实验

Experimental Study on Neuroendocrine Differentiation of Colon Cancer Cell Line HCT-116 Induced by Epidermal Growth Factor

目的利用表皮生长因子(EGF)及表皮生长因子受体(EGFR)抑制剂西妥昔单抗(Cetuximab)诱导和阻断诱导结肠腺癌HCT-116细胞神经内分泌分化(NED),探讨诱导和阻断诱导后肿瘤细胞生物学变化特点及相关因素.方法通过HE和免疫组化染色,观察肿瘤细胞裸鼠种植成瘤后及NED诱导前后肿瘤细胞形态变化特点和免疫组化表达情况;低密度平板克隆实验检测肿瘤细胞克隆形成率;流式细胞术检测肿瘤细胞生长周期;MTT法绘制肿瘤细胞生长曲线.结果结肠腺癌HCT-116裸鼠种植后成肿瘤细胞明显异型;经EGF及EGFR作用前后HCT-11肿瘤细胞形态无明显变化;EGF组克隆形成率增加(P>0.05),肿瘤细胞Cg A、Ki-67、Bcl-2表达增强,增殖活性增高,EGF作用后肿瘤细胞与其它各组相比差异有显著统计学意义(P<0.01).而CEA、CA19-9、CDX2、COX2、Syn、NSE在各组表达差异无统计学意义(P>0.05).结论 EGF可诱导结肠腺癌细胞HCT-116 NED;肿瘤细胞增殖活性增加、抗凋亡能力增强;EGFR可阻断EGF诱导HCT-116 NED及相应生物学特性变化;而CEA、CA19-9、CDX2、COX2对肿瘤细胞NED和增殖凋亡无明显调节作用.

Objective To induce and block to induce neuroendocrine differentiation of colon adenocarcinoma cell HCT-116by using the epidermal growth factor(EGF)and epidermal growth factor receptor(EGFR)inhibitor Cetuximab,and further explore the intrinsical changes and association with the characteristics of colonic cancer cells HCT-116in proliferation,apoptosis,possible mechanism,and the adjustment factors of neuroendocrine differentiation.Methods HE and immunohistochemical stains were conducted to observe morphological characters of colonic cancer cultured cells HCT-116and tumor in the nude mouse by expression of CgA,Ki-67,Bcl-2,Syn,CEA,CA19-9,COX2,CDX2.Cell growth curve of HCT-116was drawn by MTT,flow cytometry technique was used to determine the cell cycle of HCT-116,and Low Density Plate Clone Test was applied to measure the cloned forming rate of HCT-116.Results Morphology of HCT-116in the induced courses was similar.EGF acted on cultured HCT-116cells to strengthen the expression of CgA,Ki-67and Bcl-2,to increase cell proliferation rate and proliferation activity in cell cycle of S and G2phase.The colony formation rate was also increased significantly compared with the control group.Difference with cetuximab group was statistically significant(P<0.001).Changes of proliferation index of Ki-67in cultured HCT-116cells was statistically significant in EGF group compared with the other groups(P<0.01).The differential expression of NSE,CEA,CA19-9,CDX2,COX2,and Syn in each group was no statistically significant(P>0.05).Conclusion EGF can induce neuroendocrine differentiation in cultured HCT-116cells NED,increase cell proliferation activity,strengthen antiapoptotic ability of colonic cancer cells.EGFR can block EGF to induce HCT-116NED,and inhibit changes of relevant biological features.However,CEA,CA19-9,COX2,and CDX2do not regulate NED,proliferation,and apoptosis.