[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.
Chen
Sha-sha
SS
Department of Anesthesiology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China.
Cao
Fei
F
Gao
Feng
F
Xu
Ai-jun
AJ
Tian
Xue-bi
XB
Xiao
Xing-peng
XP
Yang
Shao-bing
SB
Tian
Yu-ke
YK
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