【作者】 宋婷婷；

【导师】 任金海；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:白血病是血液系统最常见的一种异质性造血干细胞的恶性克隆性疾病,随着分子生物学技术的发展,白血病的病因学已经从群体医学、细胞生物学进入分子生物学的研究。通过这些研究,目前较为详尽地阐述了白血病发生过程的分化基因学说日益受到关注,该学说认为分化基因突变可以阻止细胞的终末分化,使早期细胞堆积导致癌变。c-fes基因是参与并调节正常造血细胞的生成及其功能的酪氨酸激酶原癌基因,其表达与髓系分化的程度相关,是一个与分化相关的基因。在细胞的生长过程中,原癌基因通过基因易位、基因扩增、插入及点突变等途径被激活转变成癌基因,使细胞获得不死性和恶性增殖的能力。研究表明,c-fes在髓系白血病及其他肿瘤性疾病中均有表达,在白血病细胞分化过程中具有重要作用,尤其在髓系细胞分化过程中的作用更为明显,同时在凋亡途径中具有重要作用。将特异性的c-fes反义寡核苷酸和HL-60细胞共同孵育以抑制c-fes的表达,细胞并没有分化,而是呈现了早期的细胞死亡,该死亡表现出了凋亡的形态学和分子学特征。我们可以推断c-fes基因产物在粒细胞的分化过程中发挥了抗凋亡的作用。RNAi技术是针对转录后阶段的基因沉默,能特异性地抑制如癌基因、癌相关基因或突变基因的过度表达,使这类基因保持在静默或休眠状态,进而下调相应蛋白水平及功能。为此,本实验中我们采用RNAi技术将c-fes shRNA序列转染入HL-60细胞,初步探讨下调c-fes基因表达对白血病细胞增殖、分化和凋亡的影响。同时进一步观查干扰前后不同时段,不同浓度亚砷酸对HL-60细胞增殖、凋亡和分化情况的影响,探讨以c-fes基因作为急性髓系白血病治疗靶点的可行性。方法:1应用体外细胞培养技术,采用脂质体转染的方法干扰HL-60细胞的c-fes mRNA。实验分四组:空白对照组、脂质体对照组、空质粒转染对照组、c-fes shRNA重组质粒转染组。应用流式细胞仪检测c-fes蛋白,以评估转染效率。2以不同浓度亚砷酸(0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L)干预上述4组,于不同时段(0h、24h、48h、72h)采用RQ-PCR技术检测c-fes mRNA水平变化,采用瑞式染色观察细胞的生长情况,采用四甲基偶氮唑蓝(MTT)实验了解细胞增殖抑制情况,NBT还原实验检测细胞分化,应用Annexin-V/FITC检测细胞凋亡。结果:1 c-fes shRNA转染抑制了c-fes蛋白的表达:FCM结果显示c-fes shRNA可显著下调c-fes mRNA的表达,且对c-fes蛋白的下调从干扰后12h即出现,最高下调率为76.36%,出现在干扰后48h,但持续时间较短,72h后c-fes蛋白略有回升。2 c-fes shRNA组经As2O3 (0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L)诱导后,细胞更早地呈现染色质固缩,核碎裂等凋亡特征,最为明显的是在浓度为2.0μmol/L的As2O3处理后在72h即出现了凋亡特征性的高峰改变,可见细胞核染色质凝集,核边集,细胞被分裂成数个质膜包裹的小体即凋亡小体,而且随着作用时间的延长,视野中的活性细胞逐渐减少,凋亡细胞相应增多。3 CON组、LIP组及KB组随着As2O3浓度的增加和时间的延长,RQ-PCR检测显示c-fes mRNA的表达水平均呈逐渐增强趋势,但三组之间c-fes mRNA表达水平无明显差异(P>0.05)。c-fes shRNA组在与低浓度(0.1μmol/L、0.5μmol/L)As2O3处理不同时段(0h、24h、48h、72h)后,c-fes mRNA表达水平均未升高。72h后c-fes mRNA的表达水平略有回升,但仍旧比同时期的CON组、LIP组及KB组低。4 CON组、LIP组、KB组的之间的细胞抑制率、细胞分化指数、细胞凋亡指数均无显著性差异(P>0.05)。5 CON组、LIP组及KB组HL-60细胞随着As2O3浓度的增加,白血病细胞的增殖抑制率呈明显升高趋势,c-fes shRNA组HL-60细胞随着As2O3浓度的增加及作用时间的延长,白血病细胞的抑制率又显著高于同期对照组(P<0.05)。6 As2O3在0.1μmol/L、0.5μmol/L浓度时对CON组、LIP组、KB组的HL-60细胞有明显的诱导分化作用,并且呈现一定的剂量依赖性和时间依赖性的量效关系。As2O3在0.1μmol/L、0.5μmol/L时,c-fes shRNA组HL-60细胞在12h时分化指数与未经任何处理的HL-60细胞无显著性差异(P>0.05),24h、48h时分化指数降低,到48h时分化指数降到最低,明显低于未经任何处理的HL-60细胞,有统计学差异(P<0.05)。在1.0μmol/L、2.0μmol/L浓度的As2O3诱导下,分化指数一直处于较低水平,在24h时分化指数降到最低。7用流式细胞仪与Annexin-V/FITC试剂盒检测,c-fes shRNA组经As2O3 (0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L)诱导后,FITC+ /PI-和FITC+ /PI+细胞数显著增加,显著高于同期对照组(P<0.05)。其中在2.0μmol/L的浓度下,24h时FITC+/PI-和FITC+ /PI+细胞数明显增加,达到了41.36%。结论: 1 c-fes shRNA序列阻断了HL-60细胞c-fes mRNA表达,成功地使c-fes的表达水平受抑制,有效地阻滞了HL-60的分化和增殖,诱导了HL-60细胞的凋亡。2亚砷酸与c-fes shRNA联合作用于白血病细胞,能够更加有效地抑制肿瘤细胞的增殖,诱导HL-60细胞的凋亡,而且二者联合应用可以减少亚砷酸的剂量,使其在较小剂量,更早期时即发挥了强大的生物学效应。更多还原

【Abstract】 The leukemia is one of the most common heterogeneity hematopoietic malignancies, and the research has been into molecular biology from colonia medicine and cell biology as the development of the technology of molecular biology. Now cell differentiation can be seen as a much more predominant theory. It presumed that genetic mutation could prevent terminal differentiation of cells and cause cumulate of the immature cells to induce canceration.c-fes belongs to protein-tyrosine kinase family which implicated in the regulation of the generation and fuction of the common hematopoietic cells, c-fes mRNA protein levels and kinase activity increase upon induction of granulocyte cell differentiation. During the process of the cell growth, proto-oncogene is activated into oncogene through gene translocation、gene amplification、insertion and point mutation, then cell will have immortality and malignancy proliferation .Recent research has shown that c-fes expressed in many kinds of neoplastic disease, the c-fes proto-oncogene is expressed at high levels in the terminal stages of granulocytic differentiation. In addition, c-fes has been implicated in apoptosis pathways. It was observed that inhibition c-fes by incubating the HL-60 cells with a specific c-fes antisense oligodeoxynucleotide, the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.RNA interference (RNAi) is the most effective gene silencing technology, which can specifically inhibit the transcription of target genes, and in turn reduce the expression level and function of the corresponding protein. Therefore, using RNAi technology, we choose c-fes as the target gene. We transiently transfected c-fes shRNA into HL-60 cells by lipofectamine reagent. After observing the proliferation、differentiation and apoptosis, the experiment advanced to investigate the effect of combination use arsenious oxide with siRNA.Methods:1 Using in vitro cell culture technique, we transfected c-fes shRNA into HL-60 cells by lipofectamine reagent.The cells were divided into four groups:①blank control group;②transfection reagent group of lipofectamineTM2000;③plasmid KB group;③pGenesil1.1c-fes shRNA group. In order to detect the interference effect, we observe the expression of c-fes protein in HL-60 cells using FCM.2 The four groups of HL-60 cells were cocultured with different concentrations of As2O3 (0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L). By using the RQ-PCR technology,we detected the changes of c-fes mRNA level in different time interval(0h,24h,48,72h). The following detection regarding the proliferation、differentiation and apoptosis was observed as above-mentioned. Cells were stained with Wright-Giemsa for general morphology. The inhibition rate of HL-60 cells was examined by methyl-thiazolyl-tetrazolium(MTT) test . The differentiation of HL-60 cells were examined by NBT reduction reaction. Annexin-V/FITC staining was used to evaluated the apoptosis rate.Results:1 c-fes shRNA inhibited c-fes protein expression: FCM results showed that c-fes protein could be significantly reduced by c-fes shRNA. The reduction appeared from 12h after interference, and the maximum reduction rate was 76.36%, which appeared at 48h. However, transient transfection of c-fes shRNA could only present for a limited period, and the c-fes protein had gone up at 72h after transfection.2 The cell morphology appeared to apoptosis earlier after treated with low concentrations of As2O3(0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L)in c-fes shRNA group, it is peaked up 72 hours after treated with 2.0μmol/L As2O3.The nucleolus almost disappearance, karyoplasmic ratio diminished, karyomorphism irregular, more vacuole in intracytoplasm, the effect was in a time-dependent manner.3 With the growing of As2O3 concentration and time, RQ-PCR analysis of CON group, LIP group and KB Group showed that there was a gradually increased tendency of c-fes mRNA expression levels ,but no significant differences were found among the three groups (P>0.05). The c-fes mRNA expression levels of c-fes shRNA group did not elevated after the treatment with low concentration (0.1μmol/L, 0.5μmol/L) As2O3 in different time interval (0h, 24h, 48h, 72h). The expression level of c-fes mRNA increased slightly after 72h, but still lower than the CON group, LIP group and KB of the same period.4 There were no significant difference among the CON group, LIP group and KB group regarding the proliferation、differentiation and apoptosis(P>0.05). 5 Treated with the different concentrations of As2O3(0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L), the inhibition rate increased as the concentration raised in the CON group, LIP group and KB group. The inhibition rate in c-fes shRNA group was higher than the concurrent group as does and time extend(P<0.05).6 Treated with the concentrations of As2O3(0.1μmol/L、0.5μmol/L), HL-60 showed significantly differentiated and in dose-dependent and time-dependent manner in the CON group, LIP group and KB group. The differentiation rate of HL-60 cells in c-fes group had no significant difference with the untreated group 12 hours after cocultured with As2O3 by 0.1μmol/L、0.5μmol/L(P>0.05), 24 hours and 48 hours descended, and were lowest at 48 hours(P<0.05). With the concentrations of As2O3(1.0μmol/L、2.0μmol/L), the differentiation rate of HL-60 cells kept in a low level, with a maximum reduction at 24 hours.7 Flow cytometry (FCM) with the Annexin-V/FITC staining displayed a promoted apoptosis with concentrations of As2O3(0.1μmol/L、0.5μmol/L、1.0μmol/L、2.0μmol/L)after interference The apoptosis rate in c-fes shRNA group was higher than the concurrent group as does and time extend(P<0.05),with 41.36% at 24 hours by 2.0μmol/L.Conclusion:1 RNA interference targeting c-fes shRNA reduced the c-fes expression successfully. In the meantime, the growth status and differentiation of HL-60 cells were significantly inhibited, and HL-60 cells were induced apoptosis.2 Cocultured with different concentrations of As2O3 after RNA interference, the growth status and differentiation of HL-60 cells were more severe inhibited, and HL-60 cells were induced apoptosis more effective, what’s more, the combination use can decrease the dose of As2O3.更多还原