摘要
研究了艰难梭菌A毒素(Clostridium difficile toxin A,TcdA)诱导人黏液表皮样癌细胞(MEC-1)DNA双链损伤的机制。采用MTT法、中性彗星电泳法与免疫荧光法三联实验,分别检测TcdA对MEC-1细胞增殖抑制的影响、TcdA对MEC-1细胞造成DNA双链损伤程度以及TcdA引起MEC-1细胞DNA双链断裂相关蛋白γH_(2)AX、pATM表达的影响。结果表明:TcdA(50~800μmol·L^(-1))抑制了MEC-1细胞的生长且存在时间和浓度依赖特点;TcdA作用MEC-1细胞24 h、100~400μmol·L^(-1)浓度曲线组和TcdA浓度为800μmol·L^(-1)、作用MEC-1细胞3~24 h时间曲线组,MEC-1彗星细胞的尾长、尾部DNA、尾矩和Olive尾矩呈增加趋势,头部DNA百分比减少,800μmol·L^(-1) TcdA作用MEC-1细胞24 h,上述参数均有统计学意义;100~800μmol·L^(-1) TcdA作用MEC-1细胞24 h浓度曲线组和TcdA浓度为800μmol·L^(-1)、作用MEC-1细胞3~48 h时间曲线组、γH_(2)AX、pATM阳性细胞率基本呈增加趋势。艰难梭菌A毒素杀伤肿瘤细胞的机制是诱导肿瘤细胞的DNA双链断裂损伤,其可能与抑制MEC-1细胞增殖,影响细胞γH_(2)AX、pATM的表达水平有关。
The mechanism of DNA double strand damage induced by Clostridium difficile toxin A(TcdA)in human mucoepidermoid carcinoma MEC-1 cells was studied.MTT assay,neutral comet electrophoresis and immunofluorescence were used to detect the inhibitory effect of TcdA on the proliferation of MEC-1 cells,the degree of DNA double strand damage caused by TcdA on MEC-1 cells and the related proteinsγH_(2)AX and pATM expression of DNA double strand break caused by TcdA.The results show that TcdA(50~800μmol·L^(-1))inhibited the growth of MEC-1 cells with time and concentration dependence.The results of single cell gel electrophoresis were analyzed by CASP software:TcdA 24 h,or 100~400μmol·L^(-1) TcdA and TcdA3~24 h,or 800μmol·L^(-1) TcdA.The parameters tail length,tail DNA,tail moment and olive tail moment of MEC-1 comet cells increased,while the percentage of head DNA decreased.100~800μmol·L^(-1) TcdA for 24 h,or 800μmol·L^(-1) for 3~48 h,MEC-1 cells were treated with TcdAγH_(2)AX and pATM roughly increase.The mechanism of Clostridium difficile toxin A killing tumor cells is to induce DNA double-strand damage in tumor cells,which may be related to inhibiting the proliferation of MEC-1 cells and affecting cell expression levels ofγH_(2)AX and pATM.
作者
尤培蒙
王丽丽
杨宇琴
王晨曦
赵晋
郭忠
YOU Peimeng;WANG Lili;YANG Yuqin;WANG Chenxi;ZHAO Jin;GUO Zhong(Medical College, Northwest Minzu University, Lanzhou 730030, China)
出处
《沈阳大学学报(自然科学版)》
CAS
2021年第6期462-469,共8页
Journal of Shenyang University:Natural Science
基金
国家自然科学基金资助项目(31560254)
2017年中央高校基本科研业务费专项资金项目(31920170164)
2019年度中央高校基本科研业务费项目(31920190210)
国家级大学生创新创业训练计划项目(201910742103)。