肌肉特异性基因启动子的上游转录调控元件研究进展

真核生物启动子位于基因5’端上游转录起始位点附近,是包含核心启动子以及上游转录调控元件的一段DNA序列,这些转录调控元件控制着基因表达的强度和特异性。肌肉特异性启动子的上游调控元件种类、数量和排列顺序决定着基因在肌肉中的特异性表达。深入研究肌肉启动子的上游调控元件,可以进一步了解肌肉基因表达机制,从而为肌肉性状的改良、增殖分化的机理和疾病的基因治疗等研究提供重要依据。该文回顾了近年来肌肉特异性启动子研究领域中的新发现,包括肌肉特异性启动子转录调控元件的分子机制、建立人工合成肌肉启动子的方法及应用,并探讨该领域中急需解决的问题和发展前景。     

BCL9与肿瘤及肿瘤靶点治疗研究

Wnt信号传导通路与肿瘤的关系是近年来肿瘤研究的热点,该通路包含许多信号成员,其中BCL9是近年来该通路上发现的新的癌基因,其在多种Wnt信号通路失调肿瘤中的异常表达表明其与肿瘤的发生、发展有密切关系.而针对Wnt信号通路不同基因靶点的高特异性基因药物开发以及肿瘤的分子诊断也相继出现,尤其是针对BCL9与β-catenin作用界面的小分子抑制剂基因药物的开发更是引起广泛的注意.现就BCL9及其与肿瘤的关系和BCL9基因为靶点的肿瘤基因治疗等三方面做一综述.     

选择性复制型腺病毒介导的自杀基因HSV -tk 在 肿瘤基因治疗中的应用

自杀基因治疗是肿瘤基因治疗的手段之一,治疗效果与自杀基因能否被高效、选择性的导入肿瘤细胞有关。肿瘤选择性复制型腺病毒（conditionally replication adenovirus,CRADs）可以特异性的在肿瘤细胞中复制,在复制的同时所携带的治疗基因也大量表达。由CRAds介导的自杀基因,实现了对肿瘤的病毒治疗和基因治疗的结合,提高了治疗效率和使用复制型腺病毒的安全性。     

与人PC-1同源的鼠PC-1基因的启动子的分子克隆和特性分析

为了鉴定鼠mPC-1基因表达的调控元件,克隆并分析了该基因的启动子.构建了一系列mPC-1基因启动子的截短序列.通过荧光素酶报道基因,分析了它们在前列腺癌细胞和其它细胞中的表达.结果表明,在AR阳性细胞系中,mPC-1基因启动子活性远远高于SV40和p61-PSA 启动子,mPC-1基因启动子 599 bp 至449bp 可能含有一个负调控元件; mPC-1 1.1 kb 启动子控制的表达主要在前列腺癌细胞系中; 雄激素可调控mPC-1 1.1kb 启动子表达.mPC-1 1.1kb 序列是一个有前列腺癌细胞特异性和较强的启动子,经过进一步的修饰有可能作为一种有用的前列腺癌基因治疗元件.     

目的基因表达调控研究进展

载体技术的发展使基因治疗中的安全性问题及外源基因的转移效率等技术难题已部分解决,越来越多的研究显示目的基因体内表达的可控性是决定基因治疗成功的关键,组织特异性启动子,诱导性调控元件,增强子,静息子及反式作用因子作用机制的阐明为改造,增强基因治疗载体表达特性,实现人类基因治疗目的基因表达的可控性,靶向性目标,为基因治疗真正进入临床奠定了基础。     

EGR-1启动子在肿瘤基因治疗中的应用

EGR-1的启动子为早期生长反应因子-1基因上游约-550-0bp的一顺式作用元件。其活性受控于一些诱导剂,如电离辐射,联合EGR-1的启动子与治疗基因（如TNF-α、自杀基因）,用辐射能在肿瘤局部从时,空调控治疗基因的表达,使其产物局限于肿瘤局部,并发挥放疗的杀肿瘤效应,而放疗与转基因产物又有协同作用。放射治疗与基因治疗的配伍为肿瘤的治疗提供了新思路。     

组织特异性启动子介导的反义FGF8b RNA对前列腺癌细胞生长增殖能力的影响

利用启动子的组织特异性和治疗基因组织特异表达的特点 ,设计出前列腺癌靶向基因治疗的新方案 .利用DNA重组技术将前列腺组织特异性启动子 (probasin基因启动子 )和在前列腺癌细胞中高表达成纤维细胞生长因子 (FGF) 8b反义cDNA克隆到逆转录病毒载体pSIR中构建成重组体PB 反义FGF8b pSIR .经转染包装细胞PT 6 7后将产生的复制缺陷型逆转录病毒体外感染前列腺癌细胞系PC 3M ,体外检测其生长增殖和侵袭转移能力的变化 .结果表明 ,与对照组相比 ,前列腺癌细胞感染产生反义FGF8bRNA的逆转录病毒后生长速度减慢 ,集落形成能力下降 ,体外侵袭转移能力降低 (P <0 0 1) .体外试验表明 ,前列腺组织特异性启动子介导的反义FGF8bRNA可有效降低前列腺癌细胞的体外生长增殖和转移能力 ,这为体内靶向前列腺癌基因治疗奠定了可靠的基础 .     

靶向Wnt/β-catenin通路的溶瘤腺病毒携带抑癌基因TSLC1抑制肿瘤细胞增殖的研究

溶瘤病毒(oncolytic viruses)可选择性地感染肿瘤细胞并在其中复制,使宿主细胞裂解并在体内产生强烈的免疫应答反应,从而抑制或者杀死肿瘤细胞。因其特有的溶瘤性和靶向性,溶瘤病毒成为肿瘤基因治疗的热门领域之一。基于Wnt信号的肿瘤特异性,本研究使用Wnt信号启动子TCF/LEF结合位点序列,调控腺病毒早期基因E1A,并删除E1A基因序列上能与Rb蛋白结合的24 bp片段。将抑癌基因TSLC1插入腺病毒的E3区,形成重组腺病毒Ad.wnt-E1A(Δ24)-TSLC1。通过双荧光素酶报告系统、Western blot实验、MTT法、结晶紫染色法、Hoechst染色实验分别检测Wnt信号活性、肿瘤细胞的存活率和凋亡的变化情况。结果发现,肿瘤细胞较正常肝细胞具有较高的Wnt通路活性和病毒敏感性,其中重组病毒处理Hep G2后杀伤效果显著,并可通过Caspase通路诱导细胞凋亡,为肝癌的临床治疗提供参考。     

通过启动子同源重组研究人凝血因子Ⅸ的组成型表达

为了探索定点整合基因治疗血友病B的可行性,开展了在HeLa细胞中的hFIX组成型表达研究.同源重组载体p921构建后,电穿孔转入细胞,经过GCV和G418克隆选择,通过特异性的PCR证实了同源重组的发生,hCMV启动子定点替换hFIX基因在非肝细胞中可以被启动子hCMV转录表达,显示人为进行基因表达调控的可行性.     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

P13K—Akt—mTORC1信号途径在细胞生长增殖中的调控作用

P13K-AKT—mTORCl信号途径在细胞生长增殖中起重要调控作用,P13K-Akt—mTORl信号途径能够调节细胞周期相关蛋白基因的表达来调控细胞的增殖;同时,P13K—Akt-mTORl信号途径也能够调控细胞的生长和大小;P13K-Akt-mTORCl信号途径的异常活化与肿瘤发生紧密相关。就P13K—AKT-mTORCl信号途径在细胞生长增殖中的作用作一综述。     

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.     

hK-Fc融合蛋白的改良、表达及其生物活性的分析

为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5～10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。     

Analysis of CD4 gene expression in human fetal brain and neuroblasts

1. The expression of the gene codifying for CD4, the most important human immunodeficiency virus type 1 (HIV-1) receptor molecule, was analyzed in 11 fetal brains at various gestational ages and in 9 human neuroblastoma (NB) cell lines. CD4 gene expression in fetal and malignant neural cells was then compared with that observed in a hematopoietic cell line and adult hippocampus. 2. In addition, CD4 mRNA was evaluated in two NB cell lines induced to differentiate in vitro with retinoic acid (RA) or 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H7), a protein kinase C inhibitor. 3. All fetal brains and NB cell lines express a 1.8-kb signal when hybridized with pT4BcDNA probe, while a 3.0-kb signal such as observed in hematopoietic human cells was found in 1 of 11 fetal brains and in 0 of 9 NB cell lines. The 1.8-kb signal was lost in all analyzed poly(A)+ mRNA samples. 4. Moreover, CD4 gene expression was not induced in either RA- or H7-treated NB cells at any tested time and dose. The analysis of NB cells by polymerase chain reaction failed to demonstrate CD4 expression in either poly(A)+ or poly(A)- RNA. 5. In conclusion, the results show that the 1.8-kb signal observed in RNA extracted from fetal or transformed human neural cells is probably due to an aspecific hybridization. However, the gene codifying for CD4 can rarely be expressed by fetal brain cells early during gestation, in still unclear circumstances.     

A Systems Approach Uncovers Restrictions for Signal Interactions Regulating Genome-wide Responses to Nutritional Cues in Arabidopsis

As sessile organisms, plants must cope with multiple and combined variations of signals in their environment. However, very few reports have studied the genome-wide effects of systematic signal combinations on gene expression. Here, we evaluate a high level of signal integration, by modeling genome-wide expression patterns under a factorial combination of carbon (C), light (L), and nitrogen (N) as binary factors in two organs (O), roots and leaves. Signal management is different between C, N, and L and in shoots and roots. For example, L is the major factor controlling gene expression in leaves. However, in roots there is no obvious prominent signal, and signal interaction is stronger. The major signal interaction events detected genome wide in Arabidopsis roots are deciphered and summarized in a comprehensive conceptual model. Surprisingly, global analysis of gene expression in response to C, N, L, and O revealed that the number of genes controlled by a signal is proportional to the magnitude of the gene expression changes elicited by the signal. These results uncovered a strong constraining structure in plant cell signaling pathways, which prompted us to propose the existence of a “code” of signal integration.     

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12–0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair. © 1994 Wiley-Liss, Inc.     

Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture.

We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.     

融合gp67信号肽的慈菇蛋白酶抑制剂基因在蚕体内表达

用ＡｃＭＮＰＶ的ｇｐ６７信号肽与慈菇蛋白酶抑制剂基因（ＡＰＩ２）融合,并整合到昆虫病毒表达载体ＢｍＢａｃＰＡＫ６中,受多角体蛋白基因启动子控制。慈菇蛋白酶抑制剂在蚕体内成功地得到了高效表达。比较了ｇｐ６７信号肽和慈菇蛋白酶抑制剂信号肽在昆虫表达系统中对表达产物的影响,发现表达产物都能分泌到血淋巴中,表达量很相似,但这两种信号肽在表达过程中都没有被切除,且不同信号肽对表达产物的生物活性有很大影响。