高产L-5-甲基四氢叶酸基因工程菌的构建与表达

L-5-甲基四氢叶酸是叶酸最具生物活性的形式,也是唯一可以穿透血脑屏障的叶酸类药物,有极大的应用价值。为提高菌体内L-5-甲基四氢叶酸的含量,将5,10-亚甲基四氢叶酸还原酶基因metF插入载体质粒,并将重组质粒pET-22b-metF转化至E.coliBL21(DE3),乳糖诱导蛋白表达。成功构建的重组菌经诱导后,L-5-甲基四氢叶酸量比原始菌株中提高28%,达到66μg/g菌体。本研究成功的为生物合成L-5-甲基四氢叶酸寻找到一种新的途径。     

菌体中L-5-甲基四氢叶酸的HPLC法检测

目的:建立高效液相色谱法测定菌体中L-5-甲基四氢叶酸(L-5-MTHF)的方法。方法:加热法破碎细胞,强碱性阴离子交换树脂纯化,高效液相色谱柱为C18反向柱,流动相为K2HPO4(0.02 mol.L-1,pH7.2)-甲醇(84∶16)溶液,流速为1.0 mL.min-1,柱温为40℃,检测波长为290 nm。结果:L-5-MTHF在0.5～50μg.mL-1范围内线性良好,r=0.999 5,RSD为1.11%。结论:色谱法灵敏、准确、重现性好,可用于菌体中L-5-甲基四氢叶酸的定性与定量。     

产L-5-甲基四氢叶酸工程菌自动诱导培养基的优化

考察了自动诱导培养基代替异丙基-β-D-巯基半乳糖苷(IPTG)培养基诱导四氢叶酸还原酶在重组大肠杆菌BL21(DE3)中表达的可行性,对自动诱导培养基自诱导时的乳糖浓度和葡萄糖添加量进行优化,并与IPTG诱导培养基进行比较,确定了自动诱导培养基配方:葡萄糖1.8 mmol/L、乳糖3 mmol/L、胰蛋白胨10 g/L、酵母膏5g/L、NaCl 10 g/L。HPLC结果显示,自诱导与IPTG诱导条件下的L-5-甲基四氢叶酸产量接近。     

亚甲基四氢叶酸还原酶的纯化及催化性质研究

对亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)的体外酶促反应性质进行研究。通过盐析、DEAE-Sepharose fast flow柱层析纯化猪肝中的MTHFR,采用荧光法分析测定该酶纯化结果和酶促动力学性质。结果表明,MTHFR的纯化倍数可达20倍以上;测得最适酶促反应条件为pH 6.8,37℃;NADPH浓度1.2 mmol/L;以5,10-亚甲基四氢叶酸为底物测得米氏常数km=70.3μmol/L。研究发现,别构抑制剂S-腺苷甲硫氨酸(AdoMet)可使MTHFR的体外活性降到原活性的60%以下。本文初步探讨了MTHFR的催化性质,对MTHFR催化活性的主要影响因素进行研究,为酶促法合成5-甲基四氢叶酸的进一步研究奠定了基础。     

5,10-亚甲基四氢叶酸还原酶基因的研究进展

叶酸是一种水溶性B族维生素,其主要生理功能是在生化反应中转移一碳单位即某些氨基酸在分解过程中产生的含有一个碳原子的基团,如甲基、亚甲基、次甲基等,为核苷酸单位从头合成DNA和RNA提供一碳单位.     

L-5-甲基四氢叶酸钙在SD大鼠体内的药代动力学研究

目的建立测定SD大鼠血浆中L-5-甲基四氢叶酸钙(5-MTHF)浓度的高效液相色谱-串联质谱法(HPLC-MS/MS法),初步考察5-MTHF在SD大鼠体内的药代动力学(PK)参数。方法给予6只雄性SD大鼠口服1.5×10^-2mmol·L^-1·kg^-15-MTHF,采集血样用于HPLC-MS/MS分析。用蛋白沉淀法处理血浆样本,色谱柱:Grace Altima HP C₁₈,流动相A:甲醇-乙腈-异丙醇-乙醇-水-甲酸-二甲基亚砜(25∶25∶25∶25∶0.1∶1);流动相B:甲醇-水-甲酸(10∶90∶0.1);梯度洗脱:0.40 mL·min^-1。考察该方法的线性范围、准确度和精密度、回收率、基质效应及稳定性。用WinNonlin软件计算5-MTHF在SD大鼠体内的PK参数。结果 5-MTHF在10.00~1.00×10~4ng·mL^-1内线性关系良好,回归方程为y=1.01×10^-3x+1.82×10^-3(r=0.998 3),日内和日间的准确性和精密度、回收率、基质效应及稳定性符合要求。5-MTHF在SD大鼠体内的PK参数:不扣除本底5-MTHF浓度值的t_1/2为(15.23±5.76)h,AUC_0~t为(2788.52±348.86)ng·mL^-1·h,扣除本底5-MTHF浓度值的t_1/2为(1.57±0.34)h,AUC_0~t平均值为(971.80±237.58)ng·mL^-1·h。结论本研究建立了一种灵敏的、准确的用于SD大鼠血浆中5-MTHF浓度测定的HPLC-MS/MS法。     

亚甲基四氢叶酸还原酶基因多态性与个体化叶酸补充概述

亚甲基四氢叶酸还原酶（MTHFR）是机体叶酸代谢过程中最为关键酶,参与机体许多生理生化过程。科学研究证实MTHFER基因多态性引起的酶活性降低可导致叶酸代谢障碍,该遗传特征与许多疾病易感性,临床药物治疗作用及不良反应相关,而适当的补充叶酸能够在一定程度上可规避这种遗传因素所致疾病,本文就目前MTHFR基因多态性与疾病关系作一综述,并就结合基因型制定个体化叶酸补充做一预测和评估。     

亚甲基四氢叶酸还原酶C667T基因多态性与晚期胃癌患者对5-FU化疗疗效的相关性

目的 观察亚甲基四氢叶酸还原酶(MTHFR)C667T基因多态性与5-氟尿嘧啶(5-FU)为基础的化疗方案治疗晚期胃癌的疗效间的关系.方法 收集经病理学确诊的晚期胃癌59例.所有病例化疗前抽取外周静脉血,提取DNA,用连接酶检测反应技术(LDR)检测研究对象的MTHFR基因型.所有患者经5-FU为基础的联合化疗方案化疗.结果 59例晚期胃癌患者中,21例(35.59%)为MTHFR C/C基因型,22例(37.29%)为MTHFR C/T基因型,16例(27.12%)为MTHFR T/T基因型.其中,4例CR,14例PR,19例SD,22例PD,总有效率为30.51%(18/59).MTHFRT/T基因型患者的化疗有效率(68.75%)明显高于C/T基因型患者(18.18%)(P＜0.01),也明显高于C/C基因型(14.29%)(P＜0.01).结论 MTHFR基因型对预测以5-FU为基础化疗方案治疗晚期胃癌的疗效具有较好的临床意义.     

妊娠期高血压疾病与亚甲基四氢叶酸还原酶基因的相关性研究进展

妊娠期高血压疾病是妊娠晚期出现的特发性高血压综合征,是导致孕产妇和围产儿发病率和死亡率升高的重要原因,其发病率高达7%～10%,其病因尚无定论.妊娠期高血压疾病的病因可能为内皮细胞的损伤或功能紊乱,随着后基因时代的到来,人们研究发现妊娠期高血压疾病与遗传因素也有重要的关系.近年来有研究显示亚甲基四氢叶酸还原酶活性的变化可能导致血高同型半胱氨酸,从而引起内皮细胞损伤或内皮功能的紊乱,由此推测亚甲基四氢叶酸还原酶基因可能与妊娠期高血压疾病的发生有关.本文章就近年来亚甲基四氢叶酸还原酶基因与妊娠期高血压疾病的关系的研究进展作一综述.     

长治市女性亚甲基四氢叶酸还原酶基因多态性分析

目的了解亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性在长治女性人群的分布情况。方法采用实时荧光定量聚合酶链反应(q PCR)方法,对829例女性的MTHFR C677T基因多态性进行检测。结果本地区女性人群MTHFR 677位点CC、CT、TT基因型频率分别为17.61%,45.11%,37.27%;C和T等位基因频率为40.17%和59.83%。本地区女性MTHFR的基因型和等位基因频率与郑州、廊坊地区比较差异无统计学意义(P>0.05),与珠海、云南、温州、苏州、西安、长春、齐齐哈尔女性比较差异有统计学意义(P<0.05)。结论本研究中MTHFR C677T基因多态性在女性人群中的分布符合Hardy-Weinberg平衡,为本地女性优生优育及叶酸代谢相关疾病提供流行病学资料。     

Bioavailability and Related Pharmacokinetics in Man of Orally Administered L-5-Hydroxytryptophan in Steady State

Abstract The bioavailability of orally administered L-5-hydroxytryptophan in steady state was investigated at four increasing multiple dose levels in five patients suffering from various myoclonic disorders. An L-aromatic amino acid decarboxylase inhibitor was co-administered in all the experiments. The disposition pharmacokinetics of the amino acid had been established in the same patients in preceding intravenous single dose experiments. The finding of a direct proportionality between the size of the oral dose level of L-5-hydroxytryptophan and the corresponding areas under the plasma concentration curves within a dosage interval at steady state strongly indicates dose independent, linear pharmacokinetics of the compound. The systemic availability of L-5-hydroxytryptophan exhibited an interindividual range of 47–84%, with a mean value of 69.2% ±4.7 S.E.M. The absorption took place at a rather slow rate as judged from times of 1.8 to 3.3 hours elapsing from administration of the compound until occurance of the maximum measured plasma concentrations. Transitory nausea and vomiting were only recognized in few instances during the gradual building up of increasing steady state levels of L-5-hydroxytryptophan in the patients, and the importance of a slow initiation of therapeutical treatment with the amino acid is emphasized.     

人纤溶酶原半胱氨酸卷曲区5(hPK-5)基因在4种工程菌株中表达差异性研究

目的:探索表达人纤溶酶原半胱氨酸卷曲区5(hPK-5)的最佳菌株,为研究其抑制内皮细胞增殖和迁移的活性,开发肿瘤治疗和预防肿瘤转移的新药提供前提.方法:在将人纤溶酶原半胱氨酸卷曲区5(hPK-5)基因与原核表达载体pBV220进行体外重组获得表达质粒pBV220/hPK-5,采用氯化钙转化法将重组质粒分别导入BL21(DE3)、DH5α,JM109和BL21(DE3)-pLyss 4种工程菌,在温控诱导表达条件下进行相同表达条件和优化表达条件下的hPK-5因子表达差异性研究.结果:工程微生物(本研究为工程菌株)的筛选、培养条件的建立、外源基因表达条件的建立是优化基因工程技术体系的重要技术环节.结论:本研究为hPK5因子的进一步深入研究以及产业化的发展奠定了基础.     

L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for alpha5-containing GABAA receptors

The in vitro and in vivo properties of L-655,708, a compound with higher affinity for GABA(A) receptors containing an alpha5 compared to an alpha1, alpha2 or alpha3 subunit have been examined further. This compound has weak partial inverse agonist efficacy at each of the four subtypes but, and consistent with the binding data, has higher functional affinity for the alpha5 subtype. In a mouse hippocampal slice model, L-655,708 was able to enhance the long-term potentiation produced by a theta burst stimulation, consistent with a potential role for the alpha5 subtype in processes involving synaptic plasticity, such as learning and memory. When administered in a formulation specifically designed to achieve relatively constant plasma drug concentrations, and therefore maintain selective occupancy of alpha5- compared to alpha1-, alpha2- and alpha3-containing receptors (75+/-4% versus 22+/-10%, respectively), L-655,708 did not alter the dose of pentylenetetrazole required to induce seizures, indicating that the inverse agonist effects of L-655,708 at the alpha5 subtype are not associated with a proconvulsant liability. In the Morris water maze, L-655,708 enhanced performance not only during acquisition but also in a probe trial, demonstrating that this compound has cognition enhancing effects. These data further support the potential of alpha5-containing GABA(A) receptors as a target for novel cognition enhancing drugs.     

Differential uptake, metabolism and behavioral effects of the D and L isomers of 5-hydroxytryptophan.

The relative contribution of the D and L isomers of 5-hydroxytryptophan (5-HTP) to the uptake and metabolism of 5-HTP and their associated behavioral effects were investigated. For the metabolic study, an injection of 25 mg/kg of D or L-5-HTP was administered IP and the rats killed 15, 30, 45 or 60 min later. Endogenous levels of 5-HTP, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured in the telencephalon following D- or L-5-HTP administration. Levels of 5-HTP, 5-HT and 5-HIAA were also measured in the brain stem (including diencephalon) following L-5-HTP administration. In the behavioral study, the effects of IP injections of D-5-HTP, L-5-HTP and D,L-5-HTP upon operant responding on a VI 1 schedule were investigated. Compared to vehicle controls, L-5-HTP significantly increased the levels of 5-HTP, 5-HT and 5-HIAA in the telencephalon and brain stem at all time points investigated. Behaviorally, 25 mg/kg of L-5-HTP and 50 mg/kg of D,L-5-HTP produced similar changes. Following the injection of either compound there was a large decrease in response rate with a duration of about 1 hr which paralleled the neurochemical changes. Injections of D-5-HTP produced an increase in the levels of 5-HTP and 5-HIAA in the telencephalon at 15 min but no change in the level of 5-HT was observed. In the operant situation, following D-5-HTP injections, a brief decrease in responding occurred in some animals which did not correlate with the neurochemical data. It was concluded that the L isomer is mainly responsible for the neurochemical and behavioral effects seen when D,L-5-HTP is administered.     

左旋丁苯酞对低灌注大鼠脑皮质Tau蛋白磷酸化的影响

目的通过观察左旋丁苯酞(1-NBP)对脑低灌注大鼠学习记忆的改善作用,并研究其皮质β-淀粉样肽(Aβ1-42)水平以及Tau蛋白磷酸化变化,以探讨1-NBP改善认识功能的作用机制。方法大鼠双侧颈总动脉结扎90d,制备脑低灌注模型。实验分假手术组,溶剂对照组,1-NBP 30、120mg/kg组。1-NBP治疗组连续给予45d后,用Morris水迷宫检测大鼠的空间学习和记忆能力,后取大鼠脑皮质组织,用酶联免疫吸附试验(ELISA)检测Aβ1-42含量,用蛋白印迹法检测Tau蛋白的磷酸化水平。结果在水迷宫空间探索实验中1-NBP明显增加脑低灌注大鼠在目标象限活动时间的百分比。1-NBP明显减少了大鼠皮质Aβ1-42含量,降低低灌注大鼠皮质Tau蛋白的磷酸化水平。结论 1-NBP改善了脑低灌注大鼠的学习记忆缺陷,可能是通过减少皮质Aβ1-42的水平,从而减轻Tau蛋白的磷酸化,最终保护了神经元免受损伤。     

Effects of Carbidopa on the Cerebral Accumulation of Exogenous L-5-Hydroxytryptophan in Mice

Abstract: The concentration of L-5-hydroxytryptophan (5-HTP) was measured in plasma and whole brain tissue from mice by use of high pressure liquid chromatography with fluorimetric detection after reaction with phthaldialdehyde. 5-HTP was injected intraperitoneally and the rise in plasma and brain 5-HTP concentrations was found directly related to the size of amino acid dose. Carbidopa, an extracerebral inhibitor of L-aromatic amino acid decarboxylation, increased dose dependently the brain bioavailability of systemically administered 5-HTP not only by peripheral enzyme inhibition, but also by inhibition of the decarboxylase activity within the blood-brain barrier as indicated by a rise in the calculated brain/plasma 5-HTP concentration ratio.     

基于戊肝病毒的嵌合病毒样颗粒对人乳头瘤病毒16型肿瘤免疫治疗作用的研究

目的 研究基于戊肝病毒(hepatitis E virus,HEV)的嵌合病毒样颗粒(virus-like particles,VLPs)对人乳头瘤病毒16型(human papillomavirus type 16,HPV 16)肿瘤免疫治疗作用。方法 将HPV 16 E7插入HEV的p239蛋白形成重组嵌合蛋白p239-HPV16 E7。所构建的重组蛋白经大肠杆菌表达、纯化、复性后,通过电镜和动态光散射对所得蛋白颗粒大小形态进行表征。将蛋白颗粒免疫C57B/L小鼠,通过流式细胞技术与酶联免疫斑点免疫试验检测脾淋巴细胞特异性免疫细胞分化情况;并且利用TC-1肿瘤细胞在C57B/L小鼠中构建肿瘤模型,以此评价蛋白颗粒在小鼠体内的抗肿瘤免疫效果。结果 体外复性后的嵌合蛋白在电镜下观察到颗粒结构,粒径大小为22.80 nm。所得蛋白颗粒在C57B/L小鼠体内诱导产生良好的特异性细胞免疫反应。与对照组相比,试验组脾淋巴细胞的CD3⁺/CD4⁺、CD3⁺/CD8⁺比例均有明显差异(P＜0.05),且分泌IFN-γ干扰素的效应T细胞显著增加。同时,所得蛋白颗粒能有效抑制TC-1荷瘤小鼠体内肿瘤细胞的生长,在实验周期内小鼠未出现死亡,而对照组小鼠体内肿瘤快速生长,且6周后全部死亡。结论 原核表达的嵌合蛋白p239-HPV16 E7形成病毒样颗粒并有效诱导针对HPV 16的抗肿瘤免疫。