艰难梭菌A毒素诱导人黏液表皮样癌MEC-1细胞DNA双链损伤研究

Clostridium difficile Toxin A Inducing DNA Double-Strand Breaks in Human Mucoepidermoid Carcinoma Cell Line MEC-1

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.