艰难梭菌A毒素诱导SMMC7721细胞和Vero细胞凋亡的比较研究(英文)

Comparative Study on Apoptosis Indu ction of SMMC7721 and Vero Cells by Clostridium difficile Toxin A

背景与目的:艰难梭菌是假膜性大肠炎及抗生素诱发性腹泻的主要致病菌之一,它产生的毒素具有不同的生物学活性。本实验拟研究艰难梭菌A毒素诱导人肝癌细胞株(SMMC7721)和非洲绿猴肾细胞(Vero细胞)凋亡的作用。方法:由脑心浸液对艰难梭菌VPI10463菌株培养产毒,经甲状腺球蛋白亲合层析和QSephrose层析提纯得到精制A毒素。对SMMC7721细胞的研究以Vero细胞为对照。抑制细胞增殖的检测采用MTT法;用荧光显微镜、电子显微镜、流式细胞仪观察细胞形态和细胞周期的改变;采用琼脂糖凝胶电泳法观测DNA碎片。结果:不同浓度A毒素(0.293～4.690mg·L-1)明显抑制了SMMC7721及Vero细胞的增殖,并且呈时间和浓度依赖性;两种细胞与A毒素共同培养48h,荧光显微镜和透射电镜都观察到典型的凋亡形态变化;SMMC7721细胞与A毒素共同培养48h后,琼脂糖凝胶电泳显示梯形条带。结论:A毒素明显诱导了两种细胞的凋亡;A毒素对SMMC7721细胞具有更强的诱导凋亡作用。

BACKGROUND &OBJECTIVE:Clostridium difficile is recog nized as a frequent cause of antibiotic-in duced diarrhea.This study was desig ned to investig ate whether Clostridium difficile toxin A mig ht induce apoptosis on hum an hepatoma cell line SMMC7721and African g reen monkey kidney Vero cells.METHODS :Hig hly purified toxin A was obtained by bovine thyrog lobulin affinity purification followed by ion exchan g e chromatog raphy on Q sepharose.Th e apoptosis induction of toxin A was examined on SMMC7721cells with Vero cells as a control.Inhibition of prolifer ation was measured by MTT assay.Morpholog ical assessment of apopto sis was performed with fluorescence and electronic microscopy.DNA frag men tation was observed by ag arose g el electrophoresis.Cell cycle distribution was analyzed by flow cytometr y.RESULTS :Toxin A (0.293-4.690mg ·L -1 )inhibited proliferation of SMMC7721and Vero cells in a time-and concentration-dependent manner.Morpholog ical chang es of typical ap optosis showed that SMMC7721had a hig her percentag e of apoptosis than Vero cells.Ag arose g el electrophor esis of DNA from SMMC7721treated with toxin A for 48hours revealed a 'ladder 'pattern.CONCLUSION:Clostridium difficile toxin A induced apoptosis of SMMC7721and Vero cells.The apoptos is induction on SMMC7721cells was more effective than that on Vero cell s.