Experiment On The Mechanism Of Leukemia Cell's Apoptosis Induced By Proapoptotic Gene Smac/DIABLO

Leukemia is a common and recurrent disease,which may do great harm to human's health.The treatment of leukemia still rely on high dose of chemotherapy and/or bone marrow transplant by now.However,the curative effect isnot ideal and leukemia has a tendency to recur again.Therefore the moclular mechanism of leukemia and genetherapy become a research hot point. Since Smac/DIABLO, a new proapoptotic gene, was discovered by foreign researchers in 2000,there are more and more successful reports on the treatment of malignancies and a few leukemia in vitro delivered.These provide new thinking and basis for both the treatment of malignancies and some leukemia and exploitation of gene engineering medicine. Objective:This study set human leukemia cells Jurkat, K562 and U937 as models and studied the apoptosis of leukemia cell induced by proapoptotic gene Smac/DIABLO and the possible mechanism at the cell and molecular level. These may provide new selection for the treatment of leukemia and malignancies by research on the Smac/DIABLO. Methods:1.The construction of recombinant vector containing Smac/ DIABLO gene.(1).Acquiring the Smac mRNA. Smac total RNA was extracted from Jurkat cells,then the Smac mRNA was harvested by chromatography.(2).Obtaining Smac/DIABLO full length cDNA. According to the reference sequences in the GeneBankTM and the polyclonal site in pcDNA3.1/His A, primers containing the digestion site of XhoI/BamHI were designed.Two-step RT-PCR was performed to get Smac full length cDNA.(3).Constraction of recombinant vector.After the double digestion to Smac full length cDNA and the mammalian expression vector pcDNA3.1/His A with XhoI/BamHI,two purified target fragments were ligated by T4 DNA ligase.Then the ligation products were transformed into DH5αcompetent cells.After exam ination the size of inserted gene by PCR, recombinant vector pcDNA3.1 /His A-Smac/DIABLO was amplified and examined by sequencing. 2.Transcient transfect leukemia cells with the recombinant vector.K562 and U937 cells was trasfected with cationic liposome reagents Geneporter.After culturing for about 24 hours ,cells were harvested and smeared.3. Observation f apoptosis.The effects of transfection on the leukemia cells were examined by Annexin V-PI-FCM. Meanwhile, overexpression of Smac/DIABLO and caspase-3 was analized by immunocytochemistry. Result: 1. Successfully construction of recombinant vector pcDNA 3.1/His A-Smac/DIABLO.The inserted cDNA in recombinant vector and the target fragment of double digestion to Smac/DIABLO full length cDNA are in the same size.The result of sequencing proved that the sequence of inserted DNA is the same as the reference sequence in the GeneBankTM.These proved that the recombinant mammalian expression vector was successfully constructed. 2. The result of immunocytochemistry showed that the expression of Smac/DIABLO protein in the cells transfected with recombinant vector were higher than havingnot transfected cells.48-72 hrs after transfection,expression of caspase-3 still increased sharply.3. 48-72 hours after transfection, overexpresed Smac/ DIABLO induced leukemia cells to apoptosis.Apoptosis can be detected in K562 cells by light microscope. Conclusion:This study successfully constructed recombi- nant vector pcDNA3.1/His A-Smac/DIABLO.Apoptosis and expres- sion of Smac/DIABLO were detected after the leukemia cells was trensfected with the constructed vector.Its possible mechanism is performed by activation of caspase-3. This provides experi- ment basis for putting forward new method on the chemistherapy of leukemia and exploitation new gene engineering medicine,it still needs more experiments in vivo and further research to support.