The Expression Of Gfi1 In Chronic Myelognous Leukemia And The Effect On The Proliferation Of 32D Cells

Objective:To establish SYBR Green I real-time fluorescence quantitative polymerase chain reaction method for detection of human Gfi1 mRNA expression and studying function of new gene.Method:We designed Gfi1 specific primers and constructed plasmid pLOX-Gfi1 carring GfilcDNA,then purified,quantified spectrophotometrically,calculated copies and used a 10-fold serial dilutions of quantified plasmids.At last,we established standard curve and melting curve to measure Gfil using ABI stepone PCR instrument with real-time fluorescent polymerase chain reaction.Result:A good linearity was found from 6.36×102 to 6.36×108 copies/μl,and the correlation coefficient was 0.996.Melting curve analysis showed a single peak,and Tm was (89.98±0.22)℃.The intraassay and interassay variation of cycle threshold was 1.35%-3.61% and 1.49%-3.42% respectively.conclusion:SYBR Green I real-time fluorescent quantitative polymerase chain reaction of Gfi1 mRNA expression is a rapid,sensitive,specific,repeatable and stable method.It can be used to further research on human Gfi1. Objective:To study Gfi1 expression profile in patients of chronic myelocytic leukemia (CML) before and after bone marrow transplantation or imatinib treatment. To study the relationship of Gfi1 expression and BCR/ABL, to probe the Gfi1 function in the pathogenesis of CML.Methods:1.Quantitative real time PCR was performed to quantify the Gfil mRNA in CML patients, which included 62 cases of CML-CP without any treatment,9 cases of CML-AP/ BC without treatment,21 cases of CML-CP after bone marrow transplantation,7 cases after imatinib treatment and 11 healthy volunteers; 2.CD34+ cells were separated from monocytes by flowcytometry from 6 patients of CML and 9 healthy donors. Sorted cells were analyzed by real time PCR to quantify mRNA of Gfi1 and Western-blot was performed to analyze the Gfi1 protein expression; 3.Quantitative real time PCR was used to quantify mRNA of BCR/ABL fusion gene in monocytes of CML patients; 4.Statistics was performed by software Prism3.Result:1.The mRNA expression of Gfil in mononuclear cells was significantly higher in comparison to healthy control group as determined by qPCR (2.041 vs.0.342, p= 0.005).Although patients in accelerated phase or blast crisis phase also exhibited increased expression of Gfi1, the progression of the disease from chronic phase to more advanced stage was not accompanied by more profound change of Gfi1 expression level (p=0.012 for CML-AP/BC vs. control and p=0.189 for CML-CP vs. CML-AP/BC); 2.The qPCR results showed that the mean Gfil mRNA level in 21 patients decreased from 2.242 before transplant to 0.368 after transplant (p<0.0001).Severn patients receiving imatinib and more than 10-fold reduction in Gfil mRNA expression after imatinib treatment.(2.891 vs.0.208, p=0.014); 3.Compared to healthy donors, Gfil mRNA expression is higher in CD34+BMMC of CML patients; 4.BCR/ABL expression is highly related to Gfi1mRNA expression (r2=0.65,p<0.0001); 5.Compared to healthy donors, Gfi1 protein expression is higher in CML patients BMMC and CD34+ cell by flowcytometry; 6.Gfi1 expression is higher in CD34+ cells of CML than healthy volunteers by western-blot.Conclusion:Gfi1 mRNA and proteins expression increased dramatically in CML patients. Gfil expression decreased after treatment and was related to expression of BCR/ABL. Those results suggested that Gfi1 is involved in the pathogenesis of CML, whose expression profile is affected by the activation of ABL tyrosine kinase caused by the expression of BCR/ABL. Objective:To establish an efficient human Gfi1 expressing 32D cell line by lentiviral mediated delivery system, study expression profile and effect of proliferation in 32D cells.Methods:293T cells were tranfected with recombinant lentiviral system plasmids, which included Gfi1 encoding plamid pLOX-Gfi1/pLOX, packaging plasmid pCMV△R8.2 and envelope encoding plasmid pMD.G.Recombinant virus was harvested and titrated.Gfi1 expressing 32D cells were selected FACS.Viable cell counting and colony forming units were performed to analyze the effect of Gfi1 on the proliferation of 32D cells.Results:(1)High titer (3.5×105TU/ml-4.3×105TU/ml) of recombinant Gfi1 virus was obtained by cotransfection of lentiviral plamids to 293T cells.(2)Expression of Gfi1 was detected by Westerblot after recombinant virus infection of 32D cells.(3)Low concentration of IL-3 favored the proliferation of 32D/Gfi1cells;(4)Viability of 32D/Gfi1 cells was enhanced even after IL-3 retraction;(5)More colonies forming units were detected in 32D/Gfi1.Conclusion:Gfi1 expressed efficiently in 32D cells when delivered by lentiviral system. Gfi1 played an important role in the proliferation of 32D cells.