β-DG过表达对结肠癌细胞系SW620细胞凋亡的影响  

Effect of β-DG overexpression on apoptosis of colon cancer cell line SW620

作　　者：钱成[1] 徐涛[2] 潘林鑫 Qian Cheng;Xu Tao;Pan Linxin(Center for Scientific Research,Anhui Medical University,Hefei 230032;School of Pharmacy,Anhui Medical University,Hefei 230032;School of Life Sciences,Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学校科研实验中心,合肥230032 [2]安徽医科大学药学院,合肥230032 [3]安徽医科大学生命科学学院,合肥230032

出　　处：《安徽医科大学学报》2020年第10期1491-1496,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81700522);安徽医科大学校科研基金(编号:2018xkj021)。

摘　　要：目的研究β-肌营养不良蛋白聚糖(β-DG)在HT29、HCT116、SW480、SW620等4种常见的结肠癌细胞系中的表达情况,及其过表达后对SW620结肠癌细胞凋亡及相关蛋白表达变化的影响。方法培养结肠癌细胞系,提取总蛋白进行免疫印迹,检测各细胞中内源性β-DG蛋白的表达情况;构建pcDGFP-β-DG真核表达质粒,瞬时转染至SW620细胞中,提取总蛋白进行免疫印迹或直接进行免疫荧光制片,检测β-DG的过表达和细胞定位情况;过表达β-DG后,利用流式细胞术检测SW620细胞凋亡的变化情况,并进一步检测细胞凋亡标志蛋白聚腺苷酸二磷酸核糖转移酶(PARP)的表达变化情况。结果与人胚肾上皮细胞(HEK 293T)相比,结肠癌细胞系中内源性β-DG(43 ku)的表达量均有所降低,以SW620细胞下降更为明显,且都检测到了不同表达程度的β-DG水解片段(31 ku)。成功构建pcDGFP-β-DG真核表达质粒,在SW620细胞中能够有效表达,且主要定位在细胞质中靠近核膜的位置。过表达β-DG后,SW620细胞的凋亡率为0.167±0.014,高于空白组(0.075±0.027)和对照组(0.084±0.023),差异有统计学意义(P<0.05),且过表达β-DG后,完整型PARP(113 ku)的蛋白含量有所降低,而裂解型PARP(89 ku)的蛋白含量有所增高。结论β-DG在结肠癌细胞系中异常表达,主要表现在其异常的水解断裂。过表达β-DG能够促进SW620细胞凋亡及相关凋亡蛋白的表达,为进一步了解β-DG功能,及其与结肠癌之间的关系提供了细胞学基础。Objective To research the expression of β-dystrophin(β-DG) in colon cancer cell lines(HT29,HCT116,SW480,SW620), and the effect of its overexpression on apoptosis and the expression of apoptosis related proteins in SW620. Methods Colon cancer cell lines were cultured, total protein was extracted for Western blot to detect the expression of endogenous β-DG protein in each cell. The eukaryotic expression plasmid of pcDGFP-β-DG was constructed and transfected into SW620 cells, total protein was extracted for Western blot or immunofluorescence directly to detect the overexpression and localization of β-DG. After overexpression of β-DG, the apoptosis of SW620 cells was measured by flow cytometry, and the expression of apoptotic marker protein PARP was further detected. Results Compared with normal epithelial cells HEK 293 T, the expression of endogenous β-DG(43 ku) was decreased in colon cancer cell lines, especially in SW620 cells, and different levels of β-DG hydrolysis fragments(31 ku) were detected. The eukaryotic expression plasmid pcDGFP-β-DG was constructed successfully, which could be effectively expressed in SW620 cells and mainly located in the cytoplasm near the nuclear membrane. After overexpression of β-DG, the apoptotic rate of SW620 cells was(0.167±0.014), significantly higher than that of blank group(0.075±0.027) and control group(0.084±0.023)(P<0.05). After overexpression of β-DG, the protein content of complete PARP(113 ku) decreased, and the fragment of PARP(89 ku) increased. Conclusion The abnormal expression of β-DG in colon cancer cell lines is mainly manifested in its abnormal hydrolytic breakdown. Overexpression of β-DG can significantly promote apoptosis of SW620 cells and expression of apoptotic protein, which provides a cytological basis for further understanding the function of β-DG and its relationship with colon cancer.

关 键 词：结肠癌 β-肌营养不良蛋白聚糖 过表达 细胞凋亡 

分 类 号：R34[医药卫生—基础医学] Q28[生物学—细胞生物学]