人骨碱性磷酸酶基因启动子的克隆及高水平尿酸对其活性影响的实验研究

目的克隆人骨碱性磷酸酶(BALP)基因的启动子序列并构建荧光素酶报告基因重组质粒,初步探索高水平尿酸(UA)对BALP基因启动子活性是否产生影响。方法首先用聚合酶链反应扩增目的基因5′端侧翼上游627bp(距转录起始位点-473bp至154bp)的片段,纯化扩增产物后行双酶切,后亚克隆至pGL3-basic质粒,构建BALP基因启动子荧光素酶报告基因重组质粒。通过质粒转染到HEK293细胞内,最后借助双荧光素酶报告基因活性检测的方法,比较3种UA水平条件下(0.0、0.2和0.4mmol/L)对基因启动子活性的影响。结果通过双酶切电泳、基因组测序比对分析,成功构建了BALP启动子荧光素酶报告基因重组质粒。随着UA水平的升高,HEK293细胞中启动子重组质粒的相对荧光素酶活性明显降低,差异有统计学差异(P0.05)。结论高水平UA能够抑制BALP基因启动子的活性。     

急性单核细胞白血病新抗原基因MLAA-34启动子的克隆及其核心区鉴定

目的:克隆急性单核细胞白血病新抗原基因MLAA-34上游启动子序列并检测其活性、鉴定核心区域。方法:PCR扩增MLAA-34基因启动子区全长片段,将其连接至p GL3-Basic载体,克隆重组质粒;构建一系列MLAA-34基因启动子5'侧翼区截短质粒;将含MLAA-34启动子序列的重组质粒及其截短型质粒转染至U937、HEK293细胞,应用双荧光素酶报告基因检测各片段的启动子活性,确定启动子最小活性区域,并通过生物信息学方法分析转录因子结合位点。结果:构建了含有MLAA-34启动子序列的重组质粒及其截短型质粒;与空载体相比,其启动子活性明显增加(P 0. 001)。MLAA-34最小活性区域位于转录起始位点-402 bp至-200 bp之间,其中包含E2F1,MZF-1,SP1,USF2和STAT3等多个转录因子结合位点。结论:成功构建急性单核细胞白血病新抗原基因MLAA-34不同缺失片段的启动子荧光素酶报告基因重组质粒,鉴定出核心启动子区,其中含有多个潜在的转录因子结合序列。     

丁苯酞对1-甲基-4-苯基吡啶离子诱导细胞自噬的影响

目的研究丁苯酞对1-甲基-4-苯基吡啶离子(MPP+)诱导的SH-SY5Y细胞自噬相关因子的影响,探讨丁苯酞对帕金森病细胞模型的神经保护作用机制。方法将SH-SY5Y细胞分为空白对照组(A组)、MPP+组(B组)、雷帕霉素预处理+MPP+组(C组)和丁苯酞预处理+MPP+组(D组)。采用噻唑蓝(MTT)比色法测定细胞相对存活率,倒置相差显微镜观察细胞形态的变化,Western blotting检测微管相关蛋白1轻链3(LC3)-Ⅱ/Ⅰ、Beclin 1蛋白表达,RT-PCR检测LC3-Ⅱ/Ⅰ和Beclin 1 m RNA表达。结果 B组SH-SY5Y细胞存活率显著低于A组(t=20.270,P0.001),C组和D组显著高于B组(t8.770,P0.001),且C组和D组间无显著性差异(t=2.270,P=0.064)。与A组相比,B组LC3-Ⅱ/Ⅰ、Beclin 1蛋白和m RNA表达明显增加(t6.647,P0.01);C组和D组明显高于B组(t3.630,P0.01),C组与D组间无显著性差异(t2.238,P≥0.05)。结论丁苯酞可以提高MPP+诱导的SH-SY5Y损伤细胞存活率,其可能通过诱导帕金森病模型细胞自噬,发挥治疗作用。     

MeCP2、TH蛋白在正常SH-SY5Y细胞和帕金森病细胞模型中的表达及其意义研究

目的探讨MeCP2、TH蛋白在正常SH-SY5Y细胞和帕金森病细胞模型中的表达及其意义。方法实验分为3组:空白对照组,未转染;模型对照组;pEGFP-N1-MeCP2组,转染液加入重组pEGFP-N1-MeCP2质粒。除空白对照组外,其余二组均加入6-羟多巴胺(6-OHDA)50.0μmol/L处理24h。用CCK-8法、流式细胞仪检测正常SH-SY5Y细胞、上调MeCP2表达的SH-SY5Y细胞在6-OHDA诱导下细胞活性及细胞凋亡的变化;免疫荧光技术和Western blot法检测各组SH-SY5Y细胞中MeCP2蛋白、TH蛋白表达的变化。结果 CCK-8法检测及流式细胞仪6-OHDA诱导的SH-SY5Y细胞、上调MeCP2的SH-SY5Y细胞中细胞存活和凋亡情况发现,模型对照组与pEGFP-N1-MeCP2组、空白对照组相比,差异具有统计学意义(P0.05)。双重免疫荧光检测6-OHDA诱导的SH-SY5Y细胞中的MeCP2、TH表达情况发现,模型对照组,6-OHDA(50.0μmol/L)诱导24h,MeCP2和TH的蛋白表达显著下降(P0.05)。Western blot检测上调MeCP2表达后,6-OHDA诱导的SH-SY5Y细胞中的MeCP2、TH表达情况发现,pEGFP-N1-MeCP2组上调组和空白对照组中MeCP2、TH蛋白表达与模型对照组相比,差异具有统计学意义(P0.01)。结论转染pEGFP-N1-MeCP2重组质粒可抑制6-OHDA诱导的SH-SY5Y细胞凋亡,提高TH的表达,提高细胞存活率。     

AML1B和AML1/ETO对TSC基因启动子的转录调节作用

目的 观察AML1B、AML1/ETO对TSC基因、TSC1和TSC2启动子的转录调节作用,探讨其在白血病发生中的作用.方法 构建TSC基因启动子/增强子区包含AML1基因结合位点的荧光素酶报告基因质粒,与AML1B、AML1/ETO表达质粒共转染非洲绿猴肾细胞系CV-1细胞,测定荧光素酶的活性,分析其对TSC基因启动子转录活性的影响.结果 在CV-1细胞中,AML1B对TSC1基因启动子的转录没有明显的作用.而对于TSC2基因启动子的转录活性具有明显的促进作用.在pCMV5-AML1B的剂量为75 ng时,TSC2启动子的转录活性上升为对照组的8.55倍,且这种作用具有一定的剂量依赖性;相反,AML1/ETO对TSC1基冈启动子转录活性具有明显的促进作用而对于TSC2基因启动子的转录活性没有明显的作用,但是可以抑制AML1B对TSC2基因启动子的反义启动作用.结论 AML1B和AML1/ETO能够调控TSC基因的转录.     

高迁移率族蛋白B1在白细胞介素-2转录表达信号调控中的作用

目的 探讨高迁移率族蚩白B1(HMGB1)在白细胞介素-2(IL-2)转录表达信号调控机制中的可能作用.方法首先将HMGB1和NFAT2质粒共同转染Hela细胞,同时转染IL-2报告基因,逐步增加HMGB1的转染剂量,检测IL-2报告基因的表达活性.观察HMGB1质粒用量埘IL-2报告基凶活性的影响;然后应用sRNAi质粒对内源性及外源性HMGB1表达进行特异性抑制,观察对IL-2报告基因活性的影响,以期从反而论证HMGB1可促进IL-2转录表达.结果 在Hela细胞中,随着HMGB1质粒转染剂量增加,IL-2报告基因活性增加了2.12倍(P<0.01).应用sRNAi抑制293T细胞中外源性以及Hela细胞中内源性HMGB1表达水平后,IL-2报告基因活性分别下降1.7倍和4.76倍(P<0.05或P<0.01).结论 HMGB1在NFAT2介导IL-2报告基冈转录表达的信号调控过程中发挥重要作用.     

调控组织因子基因表达的药物筛选研究

本研究建立TF启动子转录活性的荧光素酶基因稳定细胞株,应用该细胞模型筛选调控TF基因表达的药物,并为深入研究其分子机制打下基础。构建TF启动子的一系列5′端截短型的荧光素酶报告基因质粒(包括-2174 bp~+128 bp,-684 bp~+128 bp,-247 bp~+128 bp,-201 bp~+128 bp),将质粒电转染至U937细胞中,建立表达荧光素酶报告基因的稳定细胞株。应用ATRA验证该细胞株的功能;应用bortezomib、尿多酸肽(CDA-II)等药物处理该细胞株24小时,分析荧光素酶基因活性,筛选出能够调控TF基因表达的药物。结果发现,5 nmol/L bortezomib能激活其转录活性,上调TF转录本表达水平;1 mg/ml CDA-Ⅱ抑制TF启动子的转录活性,下调TF转录本的表达水平。TF启动子逐步截短功能分析发现,bortezomib及CDA-ⅡII调控TF启动子转录活性的区域位于-201 bp—0 bp之间。结论:本研究建立了表达TF启动子荧光素酶活性的U937稳定细胞株,并筛选出能够调控TF基因转录的药物CDA-II及bortezomib,为将来筛选新药物及深入研究其分子机...     

多发性骨髓瘤细胞中P53介导的Ran转录调控的研究

目的:探讨P53是否在骨髓瘤细胞中对ran进行转录调控。方法:应用实时荧光定量PCR检测多发性骨髓瘤细胞系OPM-2、RPMI-8226、U-266、KAS6/1、ANML-6、H-929、MM1.S、MOLP-8中ran转录水平,使用Nutlin-3a培养骨髓瘤细胞株MM1.S 24、48和72 h后检测ran转录水平;蛋白免疫印迹方法检测ran和P53蛋白表达水平;使用不同剂量的表达P53荧光素酶报告基因质粒转染MM1.S细胞后检测ran表达水平。结果:在8株人骨髓瘤细胞株中H-929和MM1.S细胞中ran的转录活性最高,其差异具有统计学意义(P0.05)。Nutlin-3a处理MM1.S细胞后ran转录水平较空白对照组明显下降(P0.05),同时在蛋白表达水平这种变化呈现时间依赖性;P53蛋白表达增多(r=1.00,P=0.06),相应的ran蛋白表达减少(r=-1.00,P=0.04)。表达P53荧光素酶报告基因的质粒转染MM1.S细胞后发现,在质粒增加至25 ng后ran转录水平开始下降(P0.05)。结论:通过多发性骨髓瘤细胞中P53转录调控ran转录水平的实验证明了ran是受p53调控的一个靶基因。     

X-染色体相关凋亡抑制蛋白相关因子1启动子的克隆及其转录活性检测

目的:X-染色体相关凋亡抑制蛋白相关因子1能够诱导半胱氨酸天冬氨酸蛋白酶3凋亡,其具体转录调控机制尚不十分清楚.克隆X-染色体相关凋亡抑制蛋白相关因子1基因启动子,检测干扰素对其转录活性的影响.方法:实验于2006-05/2007-07在香港大学分子生物学研究所和洛阳华美生物工程公司完成.①材料:结肠癌细胞株Lovo和Sw1116来自美国 American Type Culture Col-lection.pGL3 basic载体,内参照pRL-CMV载体和Dual-luciferase reporter Kit,T4 DNA连接酶,限制性内切酶Kpn I和XhoI均由Promega公司提供.②实验方法:采用PCR法扩增获得X-染色体相关凋亡抑制蛋白相关因子1基因的启动子片段,将之定向克隆到含荧光素酶报告基因的pGL3 basic载体上,获得的报告基因质粒命名为pLUC107,瞬时转染Lovo和Sw1116细胞株,经α-干扰素刺激处理,启动子转录活性用荧光素酶相对表达活性表示.结果:①PCR扩增结果:在预期的271 bp处出现清晰DNA片段,无非特异扩增现象,证明其为X-染色体相关凋亡抑制蛋白相关因子1启动子的PCR扩增产物.②重组质粒的酶切鉴定及测序:重组质粒pLUC107双酶切后可见约为271 bp的DNA片段,与理论值相一致.DNA测序证实含X-染色体相关凋亡抑制蛋白相关因子1启动子荧光素酶报告基因质粒构建成功.③干扰素对X-染色体相关凋亡抑制蛋白相关因子1启动子转录活性的影响:与未经α-干扰素处理的含pLUC107的Lovo和Sw1116细胞的荧光素酶相对活性值比较,经α-干扰素处理后两种细胞的荧光素酶相对活性值均明显升高(P均 < 0.05),且升高幅度与α-干扰素呈剂量依赖性.结论:成功克隆出X-染色体相关凋亡抑制蛋白相关因子1基因启动子片段,并利用荧光素酶报告基因证实α-干扰素可上调其转录活性.     

上调AMPK可促进左旋布比卡因诱导SH-SY5Y细胞凋亡

目的:探讨过表达单磷酸腺苷激活的蛋白激酶(AMPK)在左旋布比卡因作用于SH-SY5Y细胞过程中是否激活了ROS爆发,进而导致细胞凋亡。方法:将空载质粒pEGFP-N1和质粒pEGFP-N1-AMPKα2分别转染SH-SY5Y细胞株,Western blot法对AMPKα2表达水平进行鉴定。重组质粒的SH-SY5Y细胞和未转染细胞分别经1mmol/L左旋布比卡因处理后,MTT法检测细胞活力,流式细胞术检测细胞凋亡率和细胞内ROS含量。结果:与空载质粒pEGFP-N1细胞和未转染质粒细胞相比,重组质粒pEGFP-N1-AMPKα2的SH-SY5Y细胞组的AMPKα2表达上调,1mmol/L左旋布比卡因处理后,与空载质粒pEGFP-N1细胞组及未转染质粒的细胞组相比,重组质粒pEGFP-N1-AMPKα2的SH-SY5Y细胞活力降低,ROS含量提高,细胞凋亡率增多(P<0.01)。空载质粒pEGFP-N1细胞组和未转染质粒细胞组细胞活力,细胞内ROS含量和细胞凋亡率均无明显差异(P>0.05)。结论:上调AMPKα2的表达可促进左旋布比卡因诱导ROS大量增多和细胞凋亡。     

Relative activation of human pregnane X receptor versus constitutive androstane receptor defines distinct classes of CYP2B6 and CYP3A4 inducers

Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR-/- mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR- and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.     

Suppression of cytochrome P450 2E1 promoter activity by interferon-gamma and loss of response due to the -71G>T nucleotide polymorphism of the CYP2E1*7B allele

The CYP2E1*7B allele is defined by two nucleotide sequence polymorphisms, -71G>T and -333T>A. The CYP2E1 promoter sequence flanking the -71G nucleotide is consistent with a gamma-interferon activated sequence. Inflammation and interferon (IFN)-gamma suppress expression of CYP2E1 in vivo; however, the exact mechanism is not known. The objectives of this study were to determine whether the CYP2E1 promoter is regulated by IFN-gamma and to examine the influence of the nucleotide substitutions on this function. Treatment of HepG2 cells with IFN-gamma, after transient transfection with a luciferase reporter gene bearing the native CYP2E1 (-71G) promoter sequence resulted, in a dose-dependent reduction of luciferase activity. In contrast, no suppression was observed in cells transfected with the *7B allele promoter (-333A and -71T) nor a CYP2E1 plasmid containing only the -71T polymorphism. These data indicate that IFN-gamma suppresses native CYP2E1 promoter activity and that the -71G is critical for this response.     

Selective induction of human hepatic cytochromes P450 2B6 and 3A4 by metamizole

The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8- and 2.8-fold, respectively), and 2.9-fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time- and concentration-dependent induction by metamizole of CYP2B6 (7.8- and 3.1-fold for mRNA and protein, respectively, at 100 muM) and CYP3A4 (2.4- and 2.9-fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital-like mechanism of induction. These data warrant further studies to elucidate the drug-interaction potential of metamizole, especially in patients with long-term treatment.     

Sequence analyses of CYP2B genes and catalytic profiles for P450s in Qdj:Sprague-dawley rats that lack response to the phenobarbital-mediated induction of CYP2B2

The Qdj:Sprague-Dawley (SD) rat is a mutant strain lacking in phenobarbital (PB)-mediated induction of CYP2B2. The presence of interindividual differences in the hepatic content of CYP2B proteins and testosterone 16beta-hydroxylase activity demonstrated that the breeding colony of Qdj:SD rats involves normal (+/+) and intermediate (+/-) phenotypes as well as mutant (-/-)-type rats. Although PB-treated Qdj:SD (-/-) rats expressed CYP2B1 normally, testosterone 16beta-hydroxylase activity in these rats was quite low. Analysis of regioselective metabolism of testosterone and 4-hydroxybiphenyl glucuronidation demonstrated normal catalytic activities associated with other forms of cytochrome P450s, including CYP2A, -2C, and -3A, as well as PB-inducible UDP-glucuronosyltransferase in Qdj:SD (-/-) rats. There were no serious mutations in the exons of the CYP2B1 gene in Qdj:SD (-/-) rats, demonstrating that this gene codes a functional CYP2B1. These observations suggest that CYP2B1 needs the interaction with CYP2B2 to exert the full function. The CYP2B2 gene in Qdj:SD (-/-) rats was the same as that in wild-type (+/+) rats in its length of the region containing all exon/introns and 5'-upstream up to -2.3 kilobase pairs. Malignant mutation such as stop codon formation was not observed in the exons, and no mutation was detected in the region containing the PB-responsive unit. These results strongly suggest that impaired induction of CYP2B2 in Qdj:SD (-/-) rats is attributable either to mutation at the region different from PB-responsive unit and exons or to absence or lowered expression of trans-acting factor(s) necessary for gene regulation.     

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.     

成年新生神经元NMDA受体NR2B亚型基因条件性敲除模型建立

目的:建立成年神经发生中新生颗粒细胞N-甲基-D-天冬氨酸(NMDA)受体NR2B亚型基因条件性敲除模型。方法:运用逆转录病毒基因标记技术,结合Cre-1oxp重组酶系统,在ROSA26-LacZ基因报告小鼠体内研究逆转录病毒RSV-pGFP:T2aCre的Cre酶重组的发生以及其转化效率。在NR2Bfl/fl转基因小鼠中建立成年新生颗粒细胞NR2B基因敲除模型。结果:在逆转录病毒注射后14 d,观察到LacZ标记的神经元及β-gal和Cre免疫荧光双标阳性细胞,在海马齿状回颗粒下层、颗粒层以及hilus区表达;同时在嗅球颗粒层也可见LacZ表达。28 dCre转化效率高达98.3%。在NR2Bfl/fl转基因小鼠,14 d时分别在嗅球和齿状回的颗粒细胞层,观察到GFP和Cre同时标记的阳性新生颗粒细胞。结论:成年神经发生中起源于海马齿状回颗粒下层和侧脑室旁脑室管膜下区的新生颗粒细胞NR2B基因敲除模型建立。