Résumé
Objective To observe expressions of mRNA for Par-4 and WT1 in bone marrow cells from acute leukemia patients and non-leukemia patients, and to approach the correlation between CR rate and Par-4, WT1 expression level. Methods To detect Par-4 and WT1 mRNA expression level in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients by means of Real-time Fluorescent Quantitation RT-PCR. Results FQ-RT-PCR result showed that Par-4 mRNA was expressed in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients. Compared with control groups, the expression levels of Par-4 mRNA were significantly suppressed (9.35×10-4±8.4×10-5, P ＜0.05). Compared with initial treatment groups and relapse groups, the expression levels of Par-4 mRNA in remission groups were significantly up-regulated (1.26×10-3±1.1×10-4) but were still significantly lower than that in control groups (3.25×10-3±2.9×10-4). There was no significance difference between initial treatment groups and relapse groups. No apparent association was found between Par-4 expression level and CR rate (P ＞0.05). WT1 gene was overexpressed in bone marrow cells from acute leukemia patients(2.98× 10-3±2.1×10-4), but the expression levels of WT1 mRNA were significantly lower in bone marrow cells from control groups (7.25×10-5±6.7×10-6,P ＜0.05). Compared with initial treatment groups and relapse groups, the expression levels of WT1 mRNA in remission groups were significantly down-regulated (6.86×10-4±5.2× 10-5) but were still significantly higher than that in control groups. There was no significant difference between initial treatment groups and relapse groups.There was significant difference between different WT1 expression levels and CR rates (P ＜0.05). Conclusion The result of FQ-RT-PCR testing confirmed that Par-4 mRNA expression is lower, while WT1 is higher in acute leukemia. Par-4 and WT1 gene present mutually exclusive expression patterns. There was no apparent association between Par-4 expression level and CR rate.
Résumé
Objective To construct an eukaryotic expression vector of human Par-4 gene with green fluorescent protein gene which iS named pIRES2-EGFP/Par-4 and transfect jt into K562 cell line. Methods Using pDNR/Par-4 plasmid as a template.the full length Par-4 cDNA was amplified by PCR and subsequently cloned into T-A vector.Then subcloned into pIRES2-EGFP vector.After identified by digestion of restriefive endonucleases.pIRES2-EGFP/Par-4 was further confirmed by sequencing.Then it was transfected into K562 cells with Superfect reagents.The total proteins were isolated and Par-4 was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/Par-4 vector were confirmed by digestion of restrictive endonucleases and sequencing.After transfection,the expressions of green fluorescent protein were present.The protein expression of Par-4 has been detected in transfected ceils hv Western blotting.Conclusion The vector pIRES2-EGFP/Par-4 has been constructed and could Successfully express Par-4 gene in K562 cells.