Recombinant Human Decorin Leukemia K562 Cells In Vitro Effects Of Experimental Research

Objective:To investigate the anti-tumorigenesis function of rhDCN on the leukemia K562 cells in vitro and analyze the possible mechanism.Methods:1. pcDNA3.1(+)-DCN eukaryotic expression vector was verified by EcoRI, NotI double digestion and sequencing.2.Verified recombinant plasmids were amplified. After 48h, total RNA was extracted from liposome-mediated transfection K562 cells and carried out RT-PCR identification.3.They were divided into physiological saline group,pcDNA3.1(+) vector group,pcDNA3.1(+)-DCN group and liposome group. Morphology change of K562 cells was detected by Wright stain.4.Cell proliferation activity was detected by MTT. Cell cycle and apoptosis of K.562 cells was assessed by propidium iodide (PI) staining FCM.5. The expression of apoptosis-related protein, BCL-XL, Mcl-1 and Bax was detected by Western blot.Results:1. The size of gene fragment obtained by double digestion was the same to expected gene fragment size. Sequencing results completely consist with the DCN in Genebank sequences.2. pcDNA3.1 (+)-DCN recombinant plasmids were transformed into K562 cells by liposome, RT-PCR results confirmed the successful transfection.3.Wright stain showed that typical apoptotic morphological change of K562 cells in transfected group. There were no morphological changes of apoptosis in other groups.4.MTT method results showed that the transfected cell proliferation inhibition rate(24h,16±1.08%; 48h,14±1.01%; 72h,20±1.19%)was higher than that of the other corresponding groups (P<0.05). FCM results showed that the apoptosis index (20.15±1.31%)of the transfected group was higher than that of the other groups (P<0.05), cells were arrested in the G0/G1 phase (51.15±0.57%) (P<0.05).5. Western blot showed the expression levels of Bax were increased and that of BCL-XL and Mcl-1 was decreased in pcDNA3.1(+)-DCN/K562 group.Conclusion:rhDCN can inhibit the growth of K562 cells and induce the apoptosis, the effect of cell cycle and apoptosis-related protein BCL-XL, Mcl-1, Bax may play a role in its mechanism. rhDCN has provided a new way for the biological treatment of leukemia.Objective:To investigate the effect of rhDCN and ADM on suppression, apoptosis and TGF-β1 mRNA expression of leukemic K562 cell line. Methods:K562 cells were cultured by containing 10% fetal calf serum RPMI1640 culture fluid.K562 cells in Logarithmic growth phase were divided into Saline group, pcDNA3.1(+)-DCN group, ADM group, pcDNA3.1(+)-DCN-ADM group. Morphology change of cell was detected by Wright stain, cell proliferation activity was assessed by MTT. The apoptosis index of K562 cells was assessed by FCM. TGF-β1 mRNA of cell was assessed by RT-PCR.Results:Wright stain showed that typical apoptotic morphological change of K562 cells in combined group. MTT method results showed that the proliferation inhibition rate of the combined group was (61±1.32%) higher than that of individual intervention group (DCN group,20±1.9%;ADM group,47±1.04%)(P<0.05). FCM results showed that the apoptosis index of the combined group was (61.30±0.9%) higher than that of individual intervention group(DCN group,28.25±1.3%;ADM group,31.85±1.5%) (P＜0.05). TGF-β1 mRNA synthesis of combined group cell was significantly decreased.Conclusions:rhDCN can markedly enhance cytotoxicity of ADM on K562 cells, and the mechanisms of apoptosis may be down-regulation of TGF-β1 mRNA. Specific mechanisms will be further studied.