Par-4基因在急性白血病的表达及其促凋亡作用研究

【作者】 秦洁；

【导师】 王宏伟；

【作者基本信息】 山西医科大学 ， 内科学， 2009， 博士

【摘要】 目的前列腺凋亡反应因子4（Prostate apoptosis response-4, Par-4）是从凋亡的前列腺肿瘤细胞中分离出来的一种促凋亡基因,是第一个被证实的WT1（Wilms’tumor suppressor-1）的分子伴侣和抑制因子。WT1基因在各类白血病中异常高表达,而且过度表达的WT1基因与正常细胞恶性转化有关。本实验拟采用荧光定量RT-PCR及Western blotting的方法,首先观察Par-4基因、WT1基因在急性白血病及非白血病患者骨髓细胞中的表达情况,并进一步探讨其在白血病细胞凋亡过程中的表达变化。然后构建人Par-4基因真核表达载体,转染人白血病细胞K562,观察Par-4基因表达对WT1基因表达的影响,进一步研究其对白血病细胞增殖、凋亡、细胞周期改变等生物学行为的影响。本课题将为白血病的发病机理提供进一步的理论依据,为白血病的基因治疗提供更好的治疗靶点。方法1.荧光定量RT-PCR检测人Par-4及WT1基因在急性白血病患者骨髓细胞中的表达制备Par-4基因、WT1基因及β-actin阳性标准品,建立荧光定量RT-PCR扩增体系。采用荧光定量RT-PCR方法检测78例急性白血病患者及23例非白血病患者骨髓细胞中Par-4、WT1mRNA的表达。同时探讨Par-4、WT1基因表达水平与完全缓解率的关系。2.检测As₂O₃诱导K562细胞凋亡过程中Par-4及WT1基因表达的变化用不同终浓度三氧化二砷（2-10umol/L）处理K562细胞24-72h,细胞涂片,Wright-Giemsa染色,观察细胞形态学改变,用MTT法测定细胞增殖活力,流式细胞术检测细胞凋亡。采用荧光定量RT-PCR检测Par-4、WT1基因mRNA表达变化,Western blotting检测Par-4和WT1蛋白表达的变化。3.构建人Par-4基因真核表达载体以pDNR/Par-4质粒为模板,PCR扩增Par-4基因, T-A克隆,亚克隆至pIRES2-EGFP上,并对重组表达载体进行酶切、测序鉴定。构建的重组质粒命名为pIRES2-EGFP/Par-4。4.转染Par-4基因对K562细胞WT1基因表达及生物行为的影响用最佳配比的质粒pIRES2-EGFP/Par-4（或pIRES2-EGFP）和转染试剂Superfect的复合物转染K562细胞,分别于转染前及转染后48h,荧光显微镜下观察绿色荧光蛋白（EGFP）的表达,流式细胞术（FCM）检测转染效率。经流式细胞仪分选EGFP阳性细胞,荧光定量RT-PCR检测Par-4、WT1mRNA表达水平,Western blotting检测Par-4、WT1蛋白表达水平。pIRES2-EGFP/Par-4转染K562细胞后,MTT法检测细胞增殖抑制率,流式细胞仪Annexin-V/PI法检测细胞凋亡。同时设立未转染组、空质粒（pCon）组作为对照。5.人Par-4基因对K562细胞的促凋亡作用研究pIRES2-EGFP/Par-4质粒转染K562细胞48h,加用小剂量（2umol/L）As₂O₃,MTT法检测细胞增殖抑制率,流式细胞术检测细胞周期变化及细胞凋亡。同时设立未转染组、未转染加As₂O₃组、Par-4质粒转染未加As₂O₃组作为对照。结果1.急性白血病患者骨髓细胞中Par-4及WT1基因的表达情况荧光定量RT-PCR检测结果示:Par-4基因在78例急性白血病患者与23例非白血病患者骨髓细胞中都有表达,在急性白血病患者表达量明显下降,具有统计学差异（p<0.05 ）。在缓解组Par-4基因表达增强,与初治组和复发组之间有统计学差异（p<0.05 ）,但仍低于正常对照组。初治组和复发组之间表达量无统计学差异。Par-4基因表达水平与CR率无明显关系（p>0.05 ）。WT1基因在急性白血病患者骨髓细胞中高表达,正常对照组WT1基因低表达（p<0.05 ）。在缓解组WT1基因表达下降,与初治组和复发组之间有统计学差异（p<0.05 ）,但仍高于正常对照组。初治组和复发组之间表达量无统计学差异。不同WT1基因表达水平与CR率之间有统计学差异（p<0.05 ）。2. As₂O₃诱导K562细胞凋亡过程中Par-4及WT1基因表达的变化不同浓度As₂O₃作用于K562细胞,随浓度增加能明显抑制细胞生长,诱导细胞发生凋亡。同时Par-4基因mRNA表达和Par-4蛋白表达水平逐渐增加;而WT1基因mRNA及蛋白表达逐渐下降。3.构建人Par-4基因真核表达载体经酶切、测序鉴定,pIRES2-EGFP/Par-4真核表达载体构建成功。4.转染Par-4基因对K562细胞WT1基因表达及生物行为的影响4.1 Par-4质粒转染采用1.5μg pIRES2-EGFP/Par-4质粒DNA,DNA与转染试剂SuperFect为1:4的比例转染生长状态良好的K562细胞（2×106/ml）。随着时间的延长,转染效率逐渐增高,在48h达到峰值（75.34±5.84）%,与对照组相比差异具有显著性（p<0.05）。4.2转染Par-4基因对K562细胞WT1基因表达的影响转染48h流式细胞仪分选EGFP阳性的细胞,纯度可达98.0%±1.2%,荧光定量RT-PCR及Western-blotting检测结果显示:与未转染组及pCon组相比, Par-4mRNA及蛋白表达明显增强,而WT1mRNA及蛋白表达水平无明显变化（p>0.05）。4.3 Par-4基因表达对K562细胞增殖、凋亡的影响Par-4基因真核表达载体转染K562细胞后,MTT法检测细胞增殖抑制率,结果显示:转染Par-4质粒及空质粒组K562细胞增殖抑制率与对照组相比,无显著性差异（P >0.05）;经流式细胞仪对EGFP阳性的细胞采用AnnexinV/PI双参数法进行细胞凋亡分析,结果显示:与未转染组相比,转染Par-4质粒及空质粒组K562细胞凋亡率无显著性差异（P >0.05）。5.人Par-4基因对K562细胞的促凋亡作用研究5.1 MTT法检测细胞增殖抑制率结果显示:与未转染组相比,各转染组细胞增殖抑制率无统计学差异（p>0.05）;2μmol/L As₂O₃作用后,各组细胞增殖抑制率较未加药组逐渐升高,随时间延长差异出现显著性（p<0.05）;Par-4质粒+As₂O₃组细胞增殖抑制率各个时间点均高于其他各组（p<0.05）。5.2流式细胞仪检测Par-4基因表达对K562细胞周期的影响结果显示:未转染组K562细胞S期细胞比例较高。与未转染组相比,Par-4质粒未加As₂O₃组及未转染加As₂O₃组G1期细胞比例略有下降,G2期细胞比例略有上升,但S期细胞无明显变化（P>0.05）;而Par-4质粒+As₂O₃组G1期及S期的细胞比例明显降低（P<0.05）,G2期的细胞比例明显上升,出现G2/M期阻滞（P<0.05）。5.3 AnnexinV/PI双参数法分析K562细胞凋亡情况结果显示:与未转染组相比,各转染组细胞凋亡率无统计学差异（p>0.05）;2μmol/L As₂O₃作用后,各组细胞凋亡率较未加药组逐渐升高,随时间延长差异出现显著性（p<0.05）;Par-4质粒+As₂O₃组细胞凋亡率各个时间点均明显高于其他各组,差异具有显著性（P<0.05）。结论1采用荧光定量RT-PCR的方法证实Par-4基因在急性白血病低表达,而WT1基因高表达,二者成相反的表达模式。Par-4基因表达水平与CR率无明显关系。2高浓度As₂O₃能明显抑制K562细胞生长,诱导细胞发生凋亡。在诱导细胞发生凋亡过程中,Par-4基因的表达上调,而WT1基因的表达下调。3成功构建了人Par-4基因真核表达载体。4通过采用SuperFect高分子树脂转染试剂,成功介导了Par-4基因在K562细胞中的表达。Par-4基因表达对WT1基因无明显抑制作用,对K562细胞增殖、凋亡无直接作用。5 Par-4基因表达上调可以加强小剂量As₂O₃对K562细胞的增殖抑制及凋亡作用,引起K562细胞G2/M期阻滞。更多还原

【Abstract】 ObjectiveProstate apoptosis response-4 （Par-4） is a proapoptotic gene originally isolated from apoptotic prostate cancer cells. It was firstly identified as a molecular chaperones and inhibitive factor of Wilms’tumor suppressor-1（WT1）. WT1 gene is abnormally overexpressed in different kinds of leukemias and is related to malignant transformation of normal cells. In this study, we will chiefly observe expressions of mRNA and protein for Par-4 and WT1 in bone marrow cells from acute leukemia patients and non-leukemia patients by means of Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction （RQ-RT-PCR） and Western blotting, and furtherly approach their expression changes in apoptotic leukemia cells. Then we will design and construct the eukaryotic expression vector of human Par-4 gene, transfect it into human leukemia cells K562, investigate the effect of Par-4 to WT1mRNA and protein expression level in K562, and finally observe the influence of Par-4 gene up regulation on K562 biological behaviour such as cell proliferation, apoptosis and cell cycle. This study will provide further theoretical evidences to leukemia pathogenesis and therapy target to leukemia gene therapy .Methods1. Detection of Par-4 and WT1 mRNA expression level by means of Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction （FQ-RT-PCR） in bone marrow cells from acute leukemia patients.Prepared Par-4、WT1gene andβ-actin positive standard substances, constructed Real-time Fluorescent Quantitation RT-PCR amplification system, 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. Meanwhile, to approach the correlation between CR rate and Par-4、WT1 expression level.2. Exploration of changes of Par-4 and WT1 genes expression during the apoptosis of human leukemic K562 cells induced arsenic trioxide （As₂O₃）.After K562 cells had been treated with arsenic trioxide （2-10umol/L） for 24-72 hours, cell smear was stained by Wreigt-Gmisa, cell morphous observed by microscope; cell survival rate evaluated by MTT assay and cell apoptosis analyzed using flow cytometry. Real-time Fluorescent Quantitation RT-PCR was used to test the expression of Par-4 and WT1 mRNA, and Western blotting was used to test the expression of protein for Par-4 and WT1.3. Construction of human Par-4 gene eukaryotic expression vector . 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. Finally,restriction enzyme digestion and sequencing analysis was conducted to the recombinant plasmids which was named pIRES2-EGFP/Par-4.4. Influence of transfection of Par-4 gene on WT1 gene expression level and the biological behaviour of K562 cells.K562 cells were transfected with transfection complex comprising optimal proportion of pIRES2-EGFP/Par-4 plasmid and Superfect reagents, before and 48h after the transfection, obeservation for EGFP was made by fluorescent microscope, and transfection efficiency was detected by flow cytometery （FCM）. Total RNA and protein was extracted from EGFP positive K562 cells （ sorted by FCM）. Par-4、WT1mRNA and protein expression level was evaluated by Real-time Fluorescent Quantitation RT-PCR and Western-blotting. Then cytostasis rates was measured by MTT assay; cell apoptosis was detected by FCM. Total RNA and protein of pCon group （K562 cells transfected with pIRES2-EGFP） and non transfected group were also evaluated as control.5. Study on the auxo-apoptosis action of Par-4 gene in K562 cells.48h after K562 cells being transfected with pIRES2-EGFP/Par-4, 2umol/L As₂O₃ was added, then cytostasis rates was measured by MTT assay,cell apoptosis and distribution of cell cycle was detected and analysed by FCM. Meanwhile, non- transfected group、non- transfected group with As₂O₃ and Par-4 transfected non- As₂O₃ group were also evaluated as control.Results1. Expressions of Par-4 and WT1 mRNA by Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction （FQ-RT-PCR） in bone marrow cells from acute leukemia patients.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 （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 but were still significantly lower than that in control groups. 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, but the expression levels of WT1 mRNA were significantly lower in bone marrow cells from control groups（p<0.05）. Compared with initial treatment groups and relapse groups, the expression levels of WT1 mRNA in remission groups were significantly down-regulated but were still significantly higher than that in control groups. There was no significance difference between initial treatment groups and relapse groups . There was significance difference between different WT1 expression levels and CR rates （p<0.05）.2. Changes of Par-4 and WT1 genes expression lever during human leukemic K562 cells apoptosis induced by arsenic trioxide （As₂O₃）.After the K562 cells were treated with As₂O₃ of different concentrations, arsenic trioxide could efficiently inhibit the growth of K562 cells and induce apoptosis with the increase of As₂O₃ concentration. At the same time, the mRNA and protein expressions of Par-4 were up-regulated, while the mRNA and protein expressions of WT1 were down-regulated.3. Construction of human Par-4 gene eukaryotic expression vector.Restriction enzyme digestion and sequencing analysis showed that human Par-4 gene eukaryotic expression vector had been constructed successfully.4. Influence of transfection of Par-4 gene on WT1 gene expression level and the biological behaviour of K562 cells.4.1 Transfection of Par-4 plasmidAfter K562 cells in well growth conditions（2×106/ml）was transfected with the optimal transfection proportion of pIRES2-EGFP/Par-4 to Superfect reagent as 1.5μg: 6μl, transfection efficiency was evaluated by fluorescence microscope and FCM, it showed that the transfection efficiency was increased with the prolongation of time and reached its peak（75.34±5.84）% at 48h. There were significance difference compared with control groups（p<0.05）.4.2 Influence of transfection of Par-4 gene on WT1 gene expression level48h after K562 cells was transfected, EGFP positive cells were sorted by FCM, purity rates reached 98.0%±1.2%. Total RNA and protein were extracted, then Par-4、WT1mRNA and protein were evaluated by FQ-RT-PCR and Western blotting. The result showed: compared with control groups and the pCon group, the expression levels of Par-4 mRNA and protein in Par-4 plasmid group were significantly increased （p<0.05）; but there were no significance difference in expression levels of WT1 mRNA and protein（p>0.05）.4.3 Influence of the expression of Par-4 gene on the proliferation and apoptosis of K562 cellsAfter K562 cells’being transfected with pIRES2-EGFP/Par-4, cytostasis rates was measured by MTT. The result showed: compared with non-transfected group and pCon group, proliferation inhibition ratio had no significance difference （p>0.05）. Apoptosis rates of EGFP positive cells were analyzed applying AnnexinV/PI double parameter method by FCM, the result showed: compared with non-transfected group and pCon group, apoptosis rates had no significance difference （p>0.05）.5. Study on the auxo-apoptosis action of human Par-4 gene in K562 cells.5.1 Cell proliferative inhibition rates assayed by MTTThe result showed:compared with non-transfected group, cell proliferative inhibition rates of different transfection groups had no significance difference（p>0.05）; When As₂O₃ was added, cell proliferative inhibition rates of each group were increased gradually; significance difference emerged gradually with the prolongation of time （P<0.05）; cell proliferative inhibition rates of Par-4 plasmid +As₂O₃ group was higer than other groups at each time point （P<0.05）.5.2 Influence of Par-4 gene on K562 cells cycle distribution analyzed by FCMThe result showed:cells rates of S phase were higher in non-transfected group. Compared with non-transfected group, cell rates of G1 phase were a little decreased, cell rates of G2 phase were a little increased in Par-4 plasmid group and non-transfected+As₂O₃ group, while cells rates of S phase showed no obvious change （p>0.05）; But cell rates of G2 phase were increased significantly （P<0.05）, while cells rates of S and G1 phase were decreased significantly（P<0.05） in Par-4 plasmid +As₂O₃ group. Inhibition of G2/M phase emerged.5.3 K562 Cell apoptosis rate analyzed by FCM AnnexinV/PIThe result showed:compared with non-transfected group, cell apoptosis rates of different transfected groups had no significance difference（p>0.05）; when 2μmol/L As₂O₃ was added , cell apoptosis rates of each group were increased gradually; significance difference emerged gradually with the prolongation of time （P<0.05）; cell apoptosis rates of Par-4 plasmid +As₂O₃ group was higer than other groups at each time point （P<0.05）.Conclusion1. The result of FQ-RT-PCR testing confirmed that Par-4 mRNA expression is lower, while WT1 is higer in acute leukemia. Par-4 and WT1 gene presentates mutually exclusive expression patterns. There was no apparent association between Par-4 expression level and CR rate.2. Higher concentration As₂O₃ could efficiently inhibit the growth of K562 cells and induce apoptosis. The induction of K562 cells apoptosis was accompanied by up-regulation of Par-4 mRNA and protein expression but down-regulation of WT1.3. Human Par-4 gene eukryotic expression vector was constructed successfully.4. Giant molecule balsam tranfection reagent Superfect can transfect pIRES2-EGFP/Par-4 into K562 cells efficiently and mediate Par-4 gene expression in K562 cells. Par-4 gene expression in K562 cell could not efficienly inhibit the expression of WT1mRNA and protein. There was no straight inhibition action to cell proliferation and apoptosis.5. Up-regulation of Par-4 gene in K562 cells can promote inhibition action to cell proliferation and apoptosis induced by As2O3. Inhibition of G2/M phase emerged.更多还原