FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

Rb在细胞周期调控中的作用

Ｒｂ是细胞周期调控及细胞分化中重要的调控因子。它通过与细胞内的调控元件,细胞蛋白相互作用来控制细胞生长和分化。同时它又受到胞内、外多种因子在多个层次上的调控,使Ｒｂ功能与细胞生长、分化状态相适应。     

敲除LSD1基因对人慢性髓系白血病K562细胞周期的影响

为了探讨敲除LSD1基因后抑制人慢性髓系白血病 K562细胞增殖的原因,使用前期CRISPR/Cas9技术构建的人慢性髓系白血病 K562 LSD1基因敲除株,通过细胞凋亡Annexin V/PI（碘化丙啶）双染色、细胞PI染色以及流式细胞术技术,探究敲除LSD1基因后,K562细胞的凋亡水平是否改变,细胞周期是否受到影响。结果表明敲除LSD1基因后K562细胞被阻滞在G0/G1期,进入DNA复制期的细胞变少,因此导致细胞增殖速度减慢;通过细胞凋亡Annexin V/PI双染色并分析早期以及晚期凋亡细胞总比例,显示敲除LSD1基因后,不影响K562细胞的凋亡。研究结果表明,敲除LSD1基因后人慢性髓系白血病 K562细胞的增殖受到抑制,这是由于K562细胞增殖周期发生了改变,进入DNA复制期和分裂期的细胞减少;而与细胞凋亡水平的变化无关。     

EDAG—1，一种与造血调控密切相关新基因的分离和确认

以代表性差异显示分析技术（RDA）获得一种在人胎肝组织中高丰度表达的胚胎发育相关基因1（embry-onic develop associated gene1,EDAG-1）,经筛选cDNA文库及5′末端cDNA快速扩增技术（rapid amplification of cDNA 5′end,5′RACE）获得其全长为2166bp的cDNA。该基因含编码484个氨基酸组成蛋白质的ORF,Blast检索表明编码蛋白质不含任何已知蛋白质功能结构域,与小鼠Hemogen及大鼠RP59基因同源。其mRNA3′端非释译区序列含2拷贝AUUUA结构,表明该基因mRNA稳定性差。体外翻译证明该基因编码蛋白质分子量为55.3kD,与理论预测值一致。Northern杂交及PCR检测表明该基因主要表达在成人在及胚胎胪组织,在K562和MO7e两株人髓性白血病细胞系中也有较高丰度表达。氯高铁血红素及红细胞生成素EPO（erythropoietin）诱导K562细胞向红系分化过程中,EDAG－1的表达随细胞的分化迅速下调;这些结果提示EDAG－1可能是一种与造血细胞分化调控密切相关的新基因。     

抗菌肽CM4与绿色荧光蛋白融合基因的真核载体构建及表达

目的：构建绿色荧光蛋白（GFP）与抗菌肽CM4融合基因的真核表达载体。方法：采用递归PCR（rPCR）将GFP基因与CM4基因通过一段核苷酸片段连接成GFP-CM4融合基因,构建真核表达载体pcDNA3-GFP-CM4,用脂质体介导转染人K562白血病细胞,用MTT检测其活性。结果：融合基因可在K562细胞中表达,抗菌肽CM4的表达可抑制K562细胞的生长。结论：为全面了解抗菌肽的生物学活性及其在基因治疗中的可能作用提供了重要的理论依据。     

过表达KLF4重组质粒的构建及其在白血病K562细胞中的表达

为了构建过表达锌指转录因子Krüppel样因子4(Krüppel-like factor 4,KLF4)基因的重组质粒pEGFP-KLF4,观察其在白血病K562细胞中的表达,以RT-PCR法扩增人KLF4基因,构建pEGFP-KLF4重组质粒;经酶切及测序鉴定后,将pEGFP-KLF4质粒电穿孔转染白血病K562细胞(K562/pEGFP-KLF4组),设pEGFP-C1空质粒转染K562细胞(K562/pEGFP-C1组)及空白K562细胞作为对照,荧光显微镜观察EGFP-KLF4融合蛋白的表达情况,同时用抗生素G418筛选阳性克隆并扩大培养建立稳定过表达KLF4基因的K562细胞株,随后用RT-PCR检测3组K562细胞中KLF4的m RNA表达水平。实验结果显示,pEGFP-KLF4重组质粒构建成功。该质粒转染K562细胞24 h后能观察到较高强度的绿色荧光;经G418筛选出的阳性克隆扩大培养后建立了稳定过表达KLF4基因的K562细胞株;与对照组相比,K562/pEGFP-KLF4细胞组KLF4的m RNA表达水平明显升高,其过表达率为65.71%(P0.05)。实验中构建的过表达KLF4基因的重组质粒pEGFP-KLF4能在K562细胞中高效表达,为进一步研究其在白血病中的作用奠定了基础。     

促凋亡基因Bad在胰腺癌中的调控及靶向治疗研究进展

胰腺癌是一种预后极差的恶性消化道肿瘤,5年生存率小于5%,这与胰腺癌细胞的凋亡异常有着密切的关系。Bad基因是Bcl-2家族中的一种促凋亡基因,被认为可以通过浓度依赖性方式替换Bcl-xL/Bax、Bcl-2/Bax二聚体中的Bax,从而达到促进细胞凋亡、防止胰腺癌的发生。Bad基因在胰腺癌中的调控主要以磷酸化/去磷酸化调控最为常见,研究显示,胰腺癌中Bad表达总量变化不明显,而在Ser112位点上的磷酸化为灭活的pBad112形式表达却明显增多。胰腺癌中过表达的14-3-3sigma、Pim-3、cAMP等均可诱导Bad蛋白磷酸化,而PKC、MAP4K3等可诱导其去磷酸化。同时,针对Bad的靶向治疗在胰腺癌中的应用研究已经有所进展,其中有研究发现人参皂苷Rg3、Cantharidin、Stemonamid等药物均可通过Bad途径促胰腺癌细胞凋亡。对胰腺癌中的Bad的深入研究,有利于了解其与胰腺癌发病机制的关系,为胰腺癌的靶向治疗提供新的方向。     

促凋亡基因Bad在胰腺癌中的调控及靶向治疗研究进展

胰腺癌是一种预后极差的恶性消化道肿瘤,5年生存率小于5%,这与胰腺癌细胞的凋亡异常有着密切的关系。Bad基因是Bcl-2家族中的一种促凋亡基因,被认为可以通过浓度依赖性方式替换Bcl-xL/Bax、Bcl-2/Bax二聚体中的Bax,从而达到促进细胞凋亡、防止胰腺癌的发生。Bad基因在胰腺癌中的调控主要以磷酸化/去磷酸化调控最为常见,研究显示,胰腺癌中Bad表达总量变化不明显,而在Ser112位点上的磷酸化为灭活的pBad112形式表达却明显增多。胰腺癌中过表达的14-3-3sigma、Pim-3、cAMP等均可诱导Bad蛋白磷酸化,而PKC、MAP4K3等可诱导其去磷酸化。同时,针对Bad的靶向治疗在胰腺癌中的应用研究已经有所进展,其中有研究发现人参皂苷Rg3、Cantharidin、Stemonamid等药物均可通过Bad途径促胰腺癌细胞凋亡。对胰腺癌中的Bad的深入研究,有利于了解其与胰腺癌发病机制的关系,为胰腺癌的靶向治疗提供新的方向。     

ZnPcH1介导的光动力疗法对K562细胞的作用及其机制研究

探讨新型光敏剂ZnPcH1介导的光动力疗法对K562细胞的杀伤效应及其杀伤机制.应用MTT比色法检测光动力疗法对K562细胞增殖能力的影响;采用AO/EB荧光染色法、DNA片段化分析、TUNNEL、AnnexinV-FTTC/PI双染法等检测ZnPcH1-PDT诱导K562细胞的死亡方式;以RT-PCR法检测ZnPcH...     

Characterization of the expression of HTm4 (MS4A3), a cell cycle regulator,in human peripheral blood cells and normal and malignant tissues

HTm4 (MS4A3) is a member of a family of four‐transmembrane proteins designated MS4A. MS4A proteins fulfil diverse functions, acting as cell surface signalling molecules and intracellular adapter proteins. Early reports demonstrated that HTm4 is largely restricted to the haematopoietic lineage, and is involved in cell cycle control, via a regulatory interaction with the kinase‐associated phosphatase, cyclin A and cyclin‐dependent kinase 2 (CDK2). Here we describe the expression pattern of HTm4 in peripheral blood cells using gene expression microarray technology, and in normal foetal and adult human tissues, as well as adult human cancers, using tissue microarray technology. Using oligonucleotide microarrays to evaluate HTm4 mRNA, all peripheral blood cell types demonstrated very low levels of HTm4 expression; however, HTm4 expression was greatest in basophils compared to eosinophils, which showed lower levels of HTm4 expression. Very weak HTm4 expression is found in monocytes, granulocytes and B cells, but not in T cells, by lineage specific haematopoietic cell flow cytometry analysis. Interestingly, phytohaemagglutinin stimulation increases HTm4 protein expression in peripheral blood CD4‐T‐lymphocytes over nearly undetectable baseline levels. Western blotting and immunohistochemical studies show strong HTm4 expression in the developing haematopoietic cells of human foetal liver. Immunohistochemical studies on normal tissue microarrays confirmed HTm4 expression in a subset of leucocytes in nodal, splenic tissues and thymic tissue, and weak staining in small numbers of cell types in non‐haematopoietic tissues. Human foetal brain specimens from 19 to 31 gestational weeks showed that the strongest‐staining cells are ventricular zone cells and the earliest‐born, earliest‐differentiating ‘pioneer’ neurons in the cortical plate, Cajal‐Retzius and, to a lesser extent, subplate‐like neurons. Malignant tissue microarray analysis showed HTm4 expression in a wide variety of adenocarcinomas, including breast, prostate and ovarian. These findings warrant the further study of the role of HTm4 in the cell cycle of both haematopoietic and tumour cells.     

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm⁻¹, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G₁ cells is less affected by electroporation than that of cells in late S and G₂/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

A significant role for the heme oxygenase-1 gene in endothelial cell cycle progression

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression, using a retroviral vector, on cell cycle progression in the presence and absence of pyrrolidine dithiocarbamate (PDTC). The addition of PDTC (25 and 50 microM) to human microvessel endothelial cells over 24 h resulted in significant (P < 0.05) abnormalities in DNA distribution and cell cycle progression compared to cells overexpressing the HO-1 gene. The addition of PDTC resulted in a significantly decreased G(1) phase and an increased G(2)/M phase in the control cells, but not in cells transduced with the human HO-1 gene (P < 0.05). Further, PDTC had a potent effect on DNA distribution abnormalities in exponentially grown cells compared to subconfluent cells. Upregulation of HO activity in endothelial cells, as a result of overexpressing human HO-1, prevented PDTC-mediated abnormalities in DNA distribution. Inhibition of HO activity by tin-mesoporphyrin (SnMP) (30 microM) resulted in enhancement of PDTC-mediated abnormalities in cell cycle progression. Bilirubin or iron did not mediate DNA distribution. We conclude that an increase in endothelial cell HO-1 activity with subsequent generation of carbon monoxide, elicited by gene transfer, reversed the PDTC-mediated abnormalities in cell cycle progression and is thus a potential therapeutic means for attenuating the effects of oxidative stress-causing agents.     

氯化高铁血红素诱导K562细胞红系分化对其细胞周期的影响

细胞周期的测量是细胞增殖动力学的研究基础。通过添加30μmol·L-1氯化高铁血红素(Hemin)诱导人慢性髓系白血病K562细胞红系分化,利用5-溴脱氧尿嘧啶核苷(BrdU)与7-AAD双染的方法检测Hemin诱导的K562红系分化细胞对细胞周期各期比例的影响,未诱导的K562细胞周期各期比例作为对照,检测发现Hemin诱导的K562红系分化细胞对其细胞周期相对值无明显影响。应用BrdU间隔染色结合流式细胞术的方法,通过分析BrdU间隔染色后BrdU阳性细胞群的动态变化规律,从而推算出K562红系分化细胞的倍增时间及细胞周期各期时长。根据测量结果发现,未诱导的K562细胞总倍增时间约为20 h,与通过生长曲线公式法计算倍增时间的结果相符,Hemin诱导的K562细胞的细胞周期倍增时长约为23 h。Hemin诱导的K562红系分化细胞较未诱导的K562细胞倍增时间与各期时长无明显差异。因此,Hemin诱导K562细胞红系分化对其细胞周期绝对值及相对值均无明显影响。