(03404)Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

Min ZHANG, Fang LIU,
Wei HE, Yong YOU, Ping ZOU*, and Ling-Bo LIU
(Institute of Hematology Disease, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430022, China)

Abstract 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.

Key words transplantation
immunology; ribozyme; Fas; Yac-1; apoptosis

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Received: October 24, 2003 Accepted:
December 15, 2003
This work was supported by a grant from the National Natural Science Foundation
of China (No. 30240022).
*Corresponding author: Tel, 86-27-85726880; Fax, 86-27-85776343