[Construction of rat constitutively active recombinant caspase-3 and investigation of its pro-apoptotic effect in vitro].
OBJECTIVE
To investigate the apoptosis induction effect by the rat recombinant caspase-3.


METHODS
Reversed rat caspase-3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector to transfect human 293T cells and rat immortalized neural progenitor cells. The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTT assay.


RESULTS
The transfected cells presented obvious apoptosis as detected by immunofluorescence. MTT assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited [(48.35 +/- 0.16)%, (44.61 +/- 0.15)%] (P < 0.05). Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase-3 gene was [(30.7 +/- 1.5)%, (16.0 +/- 1.0)%] (P < 0.05), which was much higher than that of control cells.


CONCLUSIONS
Rat recombinant caspase-3 can be expressed and induce apoptosis effectively, and used safely both in vitro and in vivo. It can also cause neural cells to death.
Department of Anesthesiology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China.
Sha-sha
SS
Chen
Fei
F
Cao
Feng
F
Gao
Ai-jun
AJ
Xu
Xue-bi
XB
Tian
Xing-peng
XP
Xiao
Shao-bing
SB
Yang
Yu-ke
YK
Tian
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics*; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000196:embryology; D051381:Rats; D011995:Recombination, Genetic; D014162:Transfection
C505410:Casp3 protein, rat; D053148:Caspase 3