Applications of Aqueous Metal Phthalocyanine Spectroscopic Probes in the Analysis of Biomacromolecules and Inorganic Anions

金属酞菁是一类化学性质稳定（耐热、耐酸、耐碱），较易合成的化合物。自Braun和Tehemiac获得第一个酞菁化合物以来，酞菁化合物由于其特殊的物理化学和光学性质而在化学和化工领域得到了广泛研究和应用。上世纪80年代开始，酞菁化合物在生物医学领域的应用也逐步得到开发应用。由于金属酞菁的吸收峰和荧光发射峰都在长波区段，可以避开绝大多数天然物质背景光的干扰，在生物化学分析中具有广泛的应用前景。本论文围绕水溶性金属酞菁作为分子探针在生物大分子和无机阴离子检测中的应用而展开。 第一章就长波长光学探针的应用进行了阐述。首先，简要介绍了光学探针的原理、长波长光学探针的特点以及长波长光学探针应用于化学及生...Phthalocyanine was first discovered by Braun and Tehemiac, the special optical properties of phthalocyanine compounds had being widely studied and applied in chemistry and biochemistry ever since. In general, metal phthalocyanines are stable compounds with easy way of synthesis. They have been attracting more and more attention of analysts because of their great potential to be candidates of mol...学位：医学硕士院系专业：医学院_药理学学号：2452012115322

Determination of Chondroitin Sulfate with High Sensitivity by Fluorescence Recovery through Shifting the Equilibrium between Anionic Phthalocyanine and Cationic Surfactant

目的建立高灵敏荧光增强法测定硫酸软骨素的新方法,并用于实际样品测定。方法对荧光光谱行为的考察显示,在PH 7.0的介质中,具有共轭结构的阳离子头部和长碳链尾部的阳离子表面活性剂对四磺基铝酞菁(AlS4PC)的荧光具有高效猝灭作用,二者形成无荧光离子缔合物。此离子缔合物可对带有磺酸基团的高分子黏多糖硫酸软骨素(CS)作出灵敏响应。在CS的存在下,反应体系荧光显著恢复,最大恢复倍数可达900倍。由于猝灭效率高,荧光恢复程度大,甚至可实现目视化观测。结果工作曲线响应区间0.2~6μg·Ml-1,检测限0.09μg·Ml-1,工作曲线lOgy=0.158 23ρ-0.035 97,r=0.998 7。本实验对该法用于批量样品分析的可行性进行了考察,结果表明,所建立的方法可望用于高通量分析。结论该法操作简便,灵敏度、稳定性与检测准确性好。所建立的方法成功地用于组成复杂的实际样品中CS的定量分析,解决了实际工作中的难点。OBJECTIVE To propose a novel method for quantitative determination of chondroitin sulfate in real samples using the ion-association phthalocyanine complex as a fluorescent probe emitting at red-region.METHODS The fluorescence of tetrasulpnonated aluminum phthalocyanine( AlS4Pc),an anionic metal phthalocyanine,was quenched dramatically by cationic surfactants which contain a positively-charged head with a conjugated structure and a long carbon chain as tail through the formation of an almost non-fluorescent association complex.Hexadecylpyridinium bromide( CPB) screened from a series of cationic surfactants was selected as a quencher because of its high fluorescence quenching efficiency.It was found that the ion-association complex( AlS4Pc-CPB) emitted strong fluorescence in the presence of chondroitin sulfate,due to the ability of chondroitin sulfate to shift the association equilibrium of the ionassociation complex,which led to the release of AlS4Pc,thus resulting in an increase in the fluorescence of AlS4Pc.The method based on the above-mentioned phenomenon was investigated in the aspects of spectral characteristics,effect of pH,influence of reaction time, order of addition of reagents,the usage of reagents,and interference of foreign substances.RESULTS Under optimal conditions,the linear range of the assay for chondroitin sulfate was 0.2- 6 μg·mL- 1with a detection limit of 0.09 μg·mL- 1.CONCLUSOIN The established method is not only sensitive,accurate but also simple and reliable.It has been used to the analysis of real samples with complicated composition with satisfactory results.It has also been successfully applied in batch test combined with well-reader technique,exhibiting great potential for high-throughput analysis.福建省科技重点资助项目(2012Y0081); 福建省公益类科研院所基本专项(2010R1101)资助项

皂术茵陈方治疗非酒精性脂肪性肝炎40例临床研究

目的:观察皂术茵陈方治疗非酒精性脂肪性肝炎的临床疗效。方法:将78例患者单盲法随机分为治疗组40例和对照组38例;治疗组予中药皂术茵陈方治疗,对照组采用水飞蓟宾葡甲胺片(西利宾安)治疗;两组均治疗2个月。检测治疗前后患者血清谷丙转氨酶(AlT)、谷草转氨酶(AST)活性、血清总胆固醇(TCH)、甘油三酯(Tg)含量;比较患者肝脏b超变化以及症状、体征积分等临床疗效。结果:经过2个月治疗,治疗组的临床总有效率达87.50%,较之对照组的73.68%,差异有统计学意义(P<0.05);两组治疗后症状、体征积分、肝脏b超改善情况、血清AlT、AST活性、血清TCH、Tg含量均较各自治疗前显著改善;较之对照组,治疗组上述改善更加显著(P<0.05)。结论:中药皂术茵陈方对改善非酒精性脂肪性肝炎有较好的临床疗效,可明显改善患者肝功能、血脂、b超影像指标及临床证候。国家自然科学基金(No.81274155); 福建省卫生厅中医药项目(No.wzpw201408); 厦门市重大科技计划项目(No.3502Z20100006

菖郁逍遥方治疗慢性乙型肝炎伴抑郁症的临床疗效及作用机制探讨

目的观察菖郁逍遥方治疗慢性乙型肝炎（chronic hepatitis B,CHB）伴抑郁症的临床疗效,并探讨其作用机制。方法 90例慢性CHB伴抑郁症患者,采用随机数字表法分为治疗组45例,对照组45例。在恩替卡韦治疗的基础上,对照组服用氟哌噻吨美利曲辛片;治疗组服用中药复方菖郁逍遥方,疗程12周。以汉密尔顿抑郁量表17项(Hamilton depression scale,HAMD17)评分观察治疗前、治疗中（治疗4周）、治疗后（治疗12周）两组患者的抑郁状态变化;以副反应量表（treatment emergent symptom scale,TESS）观察两组治疗后副反应;观察治疗前后血清ALT活性、乙型肝炎病毒DNA（hepatitis B virus DNA,HBV DNA）定量、5-羟色胺（5-hydroxytryptamine, 5-HT）、去甲肾腺上素（norepinephrine, NE）、脑源性神经营养因子（brain derived neurotrophic factor,BDNF）水平及内源性大麻素:N-花生四烯酸氨基乙醇（Arachidonoylethanolamine,AEA）、2-花生四烯酸甘油（2-arachidonoylglycerol,2-AG）及其受体（cannabinoid receptor,CBR）1、2指标变化。结果两组治疗中、治疗后的HAMD17评分较治疗前均明显降低（P0.05）。结论菖郁逍遥方具有显著改善CHB患者抑郁状态的临床疗效,作用机制与通过调控内源性大麻素水平来升高单胺类神经递质、BDNF相关。国家自然科学基金资助项目(No.81503529,No.81673660,No.81873242);;福建省科技计划引导性项目(No.2015D0007,No.2016D012);;厦门市科技计划项目(No.3502Z20174028);;厦门市重要重大疾病联合攻关项目(No.3502Z20179047);;福建省卫生计生医学创新科研人才培养项目(No.2018-CXB-28

慢性乙型肝炎患者肝组织HBcAg表达模式与中医证型的关系

目的:观察慢性乙型肝炎(CHB)患者肝组织乙型肝炎核心抗原(HBcAg)表达模式与中医证型及肝组织炎症、纤维化等指标的关系并进一步探讨相应的机制。方法:选择CHB患者556例,均进行中医辨证分型及肝组织活检,测定肝组织HBcAg表达模式、炎症、纤维化程度及ALT、AST、HBV DNA水平。结果:(1)556例患者按中医辨证标准分为6组:瘀血阻络证44例、肝肾阴虚证42例、肝郁气滞证48例、肝郁脾虚证282例、湿热蕴结证140例、脾肾阳虚证0例;按HBcAg表达模式分HBcAg阴性组42例、胞浆型HBcAg组186例、胞浆优势型HBcAg组290例、浆核各半型HBcAg组38例。(2)经Spearman秩相关分析,肝组织HBcAg表达模式与肝组织炎症活动度、纤维化程度均呈负相关;经Fisher确切概率检验,肝组织HBcAg表达模式与中医证型分布具有相关性。结论:肝组织HBcAg表达模式与中医证型、肝组织炎症、纤维化程度均有相关性;HBcAg阴性组、胞浆型HBcAg组及肝郁脾虚证、湿热蕴结证多处于慢性乙型肝炎活动期,正气未伤,免疫功能较强,为抗病毒治疗的较佳时机。国家自然科学基金项目(No.81503529,No.81673660);;福建省科技计划引导性项目(No.2015D007,No.2016D012);;厦门市科技惠民项目(No.3502Z20174028);;厦门市重要重大疾病联合攻关项目(No.3502Z20179047);;福建中医药大学基金项目(No.XB2016080)~

基于内毒素血症探讨皂术茵陈方对非酒精性脂肪性肝炎大鼠的影响

目的 基于内毒素血症探讨皂术茵陈方防治非酒精性脂肪性肝炎(NASH)大鼠的影响及其作用机制。方法 采用随机数字表法将40只大鼠分为5组,即正常组、模型组、皂术茵陈方组、盐酸吡格列酮组及培菲康组,每组8只。除正常组外,其余32只大鼠采用高脂饮食16周建立大鼠NASH模型,在造模第9周开始,皂术茵陈方组给予皂术茵陈方水提液60 mg/(kg·d)灌胃,盐酸吡格列酮组给予盐酸吡格列酮10 mg/(kg·d)灌胃,培菲康组给予培菲康210 mg/(kg·d)灌胃,正常组及模型组均给予双蒸水10 mL/(kg·d)灌胃,共治疗8周。第16周末经腹主动脉取血,生化法检测肝组织甘油三酯(TG)水平,苏木精—伊红(HE)染色观察肝组织病理学变化。终点显色法检测血浆内毒素(LPS)含量。ELISA法检测肝组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6等炎症因子的表达水平。结果 与正常组比较,模型组大鼠肝组织显示出典型的NASH组织学特征,出现重度脂肪变性,不同程度的炎细胞浸润和坏死灶。大鼠肝湿重、肝指数、肝脏TG含量、血浆LPS水平、肝组织TNF-α、IL-1β、IL-6等炎症因..

1978～2008年中国湿地类型变化

分别基于美国陆地卫星(Landsat MSS/TM/ETM+)和中巴资源卫星(CBERS-02B)影像数据,以人工目视解译为主,完成了中国1978～2008年4期(基准年分别为1978,1990,2000和2008年)湿地遥感制图,并进行了大量的室内外验证.在此基础上,对我国湿地现状及近30年来湿地变化进行了初步分析,得到以下主要结论:(ⅰ)截止2008年,中国湿地面积约为324097km2,其中以内陆沼泽(35%)和湖泊湿地(26%)为主.(ⅱ)1978～2008年,中国湿地面积减少了约33%,而人工湿地增加了约122%.过去30年里湿地减少的速度大幅降低,由最初5523km2/a(1978～1990年)降为831km2/a(2000～2008年).(ⅲ)减少的自然湿地(包括滨海湿地和内陆湿地),其类型变化由湿地向非湿地转化的比例逐渐降低.初期(1978～1990年)几乎全部(98%)转换为非湿地;在1990～2000年间减少的自然湿地约有86%转化为非湿地,而在2000～2008年,这一比例下降为77%.(ⅳ)气候变化和农业活动是中国湿地变化的主要驱动因素,湿地变化在中国分为三大不同特征区域,即西部三省/自治区(西藏、新疆和青海)、北部两省/自治区(黑龙江和内蒙古)和其他省市区.其中西部区域尤其是青藏高原,湿地变化的驱动因子以气候增温为主;新疆湿地由于气候增温和农业活动共同作用造成变化不大.北部省/自治区的湿地变化则主要由农业活动引起;而其他省市区的湿地变化几乎完全受控于人类的农业经济活动

Novel Method for Determination of Lysozyme Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-Mucopolysaccharides Association Complex Used as a Red Emitting Fluorogenic Substrate

在弱酸性介质中,具有红区发射特性的强荧光化合物阳离子铝酞菁(TTMAAlPC)在带磺酸基团的低浓度阴离子黏多糖(肝素,HP)的存在下,发生诱导聚集,导致酞菁荧光几乎完全猝灭。此聚集缔合物可作为溶菌酶的荧光底物,在溶菌酶的水解作用下,HP降解为小分子片段,破坏了TTMAAlPC的诱导聚集行为而使其释放,体系荧光显著恢复。据此现象,建立了测定溶菌酶的新方法。结合荧光光谱与荧光各向异性技术对反应机理进行了探讨。确定了最佳反应条件(醋酸缓冲体系,PH4.0、反应温度70$C、反应时间30 MIn),考察了共存物质的影响。在最佳条件下,方法的线性回归方程为y=#30.12121+214.65772X,r=0.99871,线性范围为0.2~2 Mg/l,检出限0.015 Mg/l。本研究操作简便且有较好的选择性和灵敏度。本方法用于溶菌酶实际样品的测定,并与常规的比浊法进行了比较,结果符合良好。本研究将阳离子金属酞菁荧光探针用于酶分析,开拓了酞菁荧光探针的应用范围。We developed a novel method for the rapid determination of lysozyme using a new fluorogenic substrate that consists of a cationic aluminum phthalocyanine( tetra( trimethylammonio) aluminum phthalocyanine,TTMAAlPc), and an anionic mucopolysaccharide( heparin, HP).We found that fluorescence from the cationic aluminum phthalocyanine,a red-region fluorescence probe,was quenched significantly in acidic media in the presence of low concentrations of anionic mucopolysaccharide heparin( HP) bearing anionic sulfonic acid groups,because of induced aggregation.The practically non-fluorescent substrate degraded into small molecular fragments upon the hydrolysis of lysozyme, and thus the phthalocyanine molecules aggregated in HP were released,resulting in significant fluorescence recovery in the reaction system.This phenomenon forms the foundation of the proposed method.The reaction mechanism was determined using fluorescence spectroscopy and fluorescence anisotropy techniques.Factors that affected the determination were investigated.Under optimal conditions,the linear range was 0.2- 2 mg /L,and the detection limit was 0.015 mg /L.The developed method is easy to operate and has good selectivity and sensitivity.This method was used in the analysis of practical samples of lysozyme,and the results were in agreement with those determined by a conventional turbidimetric method.国家自然科学基金重大研究计划项目(No.90206016); 福建省科技重点项目(No.2012Y0081); 福建省属公益类科研院所基本科研专项(No.2010R1039-3)~

Application of Cationic Aluminum Phthalocyanine,a Red-Emitting Fluorescent Probe,for Sensitive Quantitative Analysis of RNA at Nanogram Level

荷正电的四取代三甲基碘化铵铝酞菁(Tetra(trimethylammionio)aluminum phthalocyanine,TTMAAlPc)是新近出现的、很有应用潜力的新型红区荧光化合物。研究表明,弱碱性条件,RNA对TTMAAlPc的荧光具有高效猝灭作用,且猝灭程度与RNA含量呈线性正相关。据此发现建立了可测定纳克级RNA的高灵敏定量分析新方法,并对原理进行了讨论。考察了pH、缓冲体系、反应时间、温度、TTMAAlPc浓度的影响,确定了最佳反应条件(pH 8.0的广泛缓冲液、反应5 min、室温、浓度为2×10-6 mol·L-1的TTMAAlPc)。在最佳条件下,方法的线性回归方程为y=-15.0+0.51x,相关系数r=0.998 6,线性范围7.71~1 705.57ng·mL-1,检测限为1.55ng·mL-1。本法灵敏度高、线性范围宽、对RNA测定常见干扰物质包括阴、阳离子、表面活性剂和维生素等的抗干扰能力强,且操作简便。该法应用于实际样品RNA含量的测定,取得了满意的结果。还以参比法首次测定了不同pH下TTMAAlPc的荧光量子产率,结果显示TTMAAlPc具有较高的荧光量子产率,且对大范围的酸度稳定,表明TTMAAlPc是极具应用潜力的新型红色荧光探针,值得深入研究,开拓其应用。Tetrasubstituted trimethyl ammonium iodide aluminum phthalocyanine(TTMAAlPc),apositively charged phthalocyanine compound,is an emerging and potentially useful red-emitting fluorescence probe.The study showed that the fluorescence of TTMAAlPc could be quenched by RNA with high efficiency in weak alkaline media,and the degree of quenching has a linear relationship with RNA in a wide concentration range.The mechanism of quenching behavior of RNA on TTMAAlPc was discussed.It was attributed by the static interaction between RNA and TTMAAlPc,and the assembly of TTMAAlPc induced by RNA.Based on this new discovery,a novel method for quantitative determination of RNA at nanogram level has been established.The factors,including the pH of medium,buffer system,reaction time,reaction temperature,the usage of TTMAAlPc as well as the interferences,which affected the determination,were investigated and discussed.Under optimum conditions,the linear range of the calibration curve was 7.71~1 705.57ng·mL-1.The detection limit for RNA was 1.55ng·mL-1.This method has been applied to the analysis of practical samples with satisfied results.The constructed method is of high sensitivity and has a wide linear range,it also showed strong ability in the tolerance of foreign substances from anions,cations,surfactants and vitamins,all of which are common interferences encountered in the determination of RNA.Besides,it is the first report that the fluorescence quantum yield of TTMAAlPc has been measured at different pH by reference method in this work.The achieved data indicated that the fluorescence quantum yield of TTMAAlPc is larger than 20% and it keeps constant in a wide range of acidity,implying that TTMAAlPc is a high-quality red-emitting fluorescence probe,it has great potential for practical applications,thus is worthy of further study.This work expands the application of phthalocyanine compound in analytical sciences.国家自然科学基金重大研究计划项目(90206016);; 福建省科技重点项目(2012Y0081);; 省属公益类科研院所基本科研专项(2014R1102,2015R1101031-2);; 厦门市科技计划项目(3502Z20143015)资

Sensitive Determination of Chondroitin Sulfate by Fluorescence Recovery of an Anionic Aluminum Phthalocyanine-Cationic Surfactant Ion-Association Complex Used as a Fluorescent Probe Emitting at Red Region

硫酸软骨素的测定在生物医学领域有重要价值,但常规检测法在灵敏度、选择性或简易性方面尚存在不足。本文基于带正电基团的阳离子表面活性剂对具有红区发射特性的强荧光化合物—荷负电的四磺基铝酞菁具有高效荧光猝灭作用,而在生物多糖硫酸软骨素存在下,上述荧光猝灭体系荧光显著恢复的现象,提出酞菁-表面活性剂离子缔合物荧光恢复高灵敏测定硫酸软骨素的新方法,并用于实际样品分析。研究表明,中性介质中,红区荧光探针四磺基铝酞菁(TETrASulfOnATEd AluMInIuM PHTHAlOCyAnInE,AlS4PC)与阳离子表面活性剂十四烷基二甲基苄基氯化铵(TETrAdECyldIMETHylbEnzylAMMOnIuM CHlOrIdE,TdbAC)发生强烈的缔合作用,导致AlS4PC的荧光几乎完全猝灭,从而获得暗背景的荧光体系。在加入带有阴离子基团(磺酸基)的生物多糖硫酸软骨素(CHOndrOITIn SulfATE,CS)后,由于竞争结合作用,AlS4PC被释放而使体系的荧光大幅度恢复,且恢复程度与CS呈线性正相关。优化了反应条件,考察了共存物质的影响,结果表明本法具有较好的选择性。在最佳条件下,线性范围为0.20~10.0μg·Ml-1,检测限为0.070μg·Ml-1,工作曲线方程y=1.04X+2.09,r=0.999 5。该法操作简便,灵敏度、稳定性与准确性好,实际样品的分析结果令人满意。酞菁荧光化合物在分析科学中的应用尚不多见,本文工作进一步开拓了酞菁红区荧光探针的新应用。Determination of chondroitin sulfate in the biomedical field has an important value.The conventional methods for the assay of chondroitin sulfate are still unsatisfactory in sensitivity,selectivity or simplicity.This work aimed at developing a novel method for sensitive and selective determination of chondroitin sulfate by fluorimetry.We found that some kinds of cationic surfactants have the ability to quench the fluorescence of tetrasulfonated aluminum phthalocyanine(AlS4Pc),a strongly fluorescent compound which emits at red region,with high efficiency.But,the fluorescence of the above-mentioned fluorescence quenching system recovered significantly when chondroitin sulfate(CS)exits.Tetradecyl dimethyl benzyl ammonium chloride(TDBAC)which was screened from all of the candidates of cationic surfactants was chosen as the quencher because it shows the most efficient quenching effect.It was found that the fluorescence of AlS4 Pc was extremely quenched by TDBAC because of the formation of association complex between AlS4 Pc and TDBAC.Fluorescence of the association complex recovered dramatically after the addition of chondroitin sulfate(CS)due to the ability of chondroitin sulfate to shift the association equilibrium of the association,leading to the release of AlS4 Pc,thus resulting in an increase in the fluorescence of the reaction system.Based on this phenomenon,a novel method with simplicity,accuracy and sensitivity was developed for quantitative determination of CS.Factors including the reaction time,influencing factors and the effect of coexisting substances were investigated and discussed.Under optimum conditions the linear range of the calibration curve was 0.20~10.0μg·mL-1.The detection limit for CS was 0.070μg·mL-1.The method has been applied to the analysis of practical samples with satisfied results.This work expands the applications of AlS4 Pc in biomedical area.福建省科技重点项目(2012Y0081); 福建省公益类科研院所基本专项项目(2010R1101;2014R1102); 厦门市科技项目(3502Z20143015)资