【作者】 黄敏；

【导师】 张义成；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的：建立测定人类Gfi1mRNA表达量的SYBR GreenⅠ实时荧光定量PCR方法,为进一步研究新基因的功能奠定基础。方法：以Gfi1基因为靶基因设计引物。构建携带Gfi1cDNA的质粒pLOX-Gfi1,经过纯化,质粒浓度测定,拷贝数的计算及10倍的梯度稀释制备标准品。应用ABI steponePCR仪对Gfi1进行实时荧光定量PCR检测,建立标准曲线和熔解曲线。结果：建立了Gfi1mRNA表达的实时荧光定量PCR方法,本法线性范围为6.36x102-6.36×108copies/μl,线性相关系数γ2为0.996,熔解曲线为单峰,Tm值为(89.98±0.22)℃,标准品的循环阈值批内变异系数和批间变异系数分别为1.35%-3.61%和1.49%-3.42%。结论：本研究建立的Gfi1mRNA表达实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性及稳定性好,可用于Gfi1基因的定量检测。目的：研究Gfil在慢性粒细胞白血病患者中的表达情况,了解慢性粒细胞白血病经骨髓移植或格列卫治疗后Gfi1表达的变化,以及Gfi1与BCR/ABL表达的关系,探讨Gfi1在CML发生和发展中的作用。方法：1.实时荧光定量PCR法检测99例慢性粒细胞白血病患者单个核细胞中Gfi1 mRNA的表达情况,包括CML-CP62例,CML-AP/BC9例,有21例CML-CP的患者进行骨髓移植,7例给予格列卫治疗,11例正常健康供者作对照；2.流式细胞仪分选骨髓单个核细胞中CD34+细胞,包括6例CML,9例正常对照,采用实时荧光定量PCR法及western-blot法检测Gfi1 mRNA及蛋白的表达；3.实时荧光定量PCR法检测CML患者骨髓单个核细胞BCR/ABL融合基因的表达情况；4.采用Prism3软件对数据进行统计学处理。结果：1.CML患者慢性期骨髓单个核细胞Gfi1 mRNA的表达为2.042,而正常对照为0.342,其表达明显增高p=0.005；尽管加速期和急变期Gfi1的表达也有所增加,但是增加的程度与疾病的进展没有很大的关系,CML-AP/BC:正常的P值为0.012,CML-CP:CML-AP/BC的P值为0.189；2.对21例进行异基因造血干细胞移植的患者移植前Gfi1 mRNA的表达为2.242,移植后为0.368,P<0.0001；7例格列卫治疗前Gfi1 mRNA的表达为2.891,治疗后为0.208下降约10倍,P=0.014；3.CML患者骨髓单个核CD34+的细胞中Gfi1 mRNA的表达比正常对照显著升高p=0.026；4.BCR/ABL融合基因与Gfi1 mRNA的表达水平具有显著的相关性(r2=0.65,p<0.0001)；5.通过流式细胞分析,我们发现CML患者慢性期骨髓单个核细胞及CD34+细胞Gfi1的蛋白表达水平比正常对照组明显升高；6.western blot检测显示CML患者慢性期CD34+细胞Gfi1的蛋白表达水平比正常对照明显升高。结论：慢性粒细胞白血病患者Gfi1mRNA和蛋白水平的表达明显上调,治疗后Gfi1转录本会明显下降,并且与BCR/ABL的表达具有显著的相关性。Gfi1可能是慢性粒细胞白血病的致病因子,慢性粒细胞白血病患者BCR/ABL融合基因形成导致ABL酪氨酸蛋白激酶活化可能引起Gfi1表达的改变。目的：研究慢病毒介导人Gfi1在32D细胞的表达,建立稳定高表达Gfi1的32D细胞株,观察其对32D细胞增殖的影响。方法：制备完整的重组慢病毒载体三质粒系统：转移质粒(pLOX-Gfi1/pLOX),包装质粒(pCMVAR8.2)及包膜蛋白质粒(pMD.G),共转染293T细胞,获得慢病毒颗粒,病毒滴度测定,并转染32D细胞,无菌流式分选获取稳定高表达Gfi1的32D细胞,采用细胞计数法,集落形成实验检测Gfi1对32D细胞增殖的影响。结果：(1)慢病毒的三种质粒可以高效转染293T细胞,并成功包装出高滴度慢病毒,病毒滴度在3.5×105TU/ml-4.3×105TU/ml之间,可满足后续实验需要；(2)Western-blot能检测到Gfi1蛋白在及32D细胞中的表达,获得稳定表达Gfi1的32D细胞克隆；(3)低浓度IL-3细胞增殖实验显示32D/Gfi1的增殖能力增强；(4)IL-3撤退实验显示32D/Gfi1的存活能力增强；(5)软琼脂集落形成实验显示32D/Gfi1克隆形成能力增强。结论：慢病毒介导的Gfi1基因可以高效稳定转染32D细胞,并持续表达,可以作为研究Gfi1基因功能的重要技术手段；Gfi1对32D细胞的增殖有重要影响。更多还原

【Abstract】 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.更多还原