Inhibition of Fas-mediated apoptosis in Yac-1 cell via Anti-Fas ribozyme.
To detect a new and more effective way against apoptosis mouse lymphomatic cell line Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.
Zhang
Min
M
Institute of Hematology Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Liu
Fang
F
He
Wei
W
You
Yong
Y
Zou
Ping
P
Liu
Ling-Bo
LB
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003001:Cloning, Molecular / Q000379:methods; D008223:Lymphoma / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016337:RNA, Catalytic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*
D016337:RNA, Catalytic; D011994:Recombinant Proteins; D019014:fas Receptor; C427204:hammerhead ribozyme; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases