转铁蛋白SA阳离子脂质体介导DNA转染肝癌细胞 SMMC-7721的实验研究

目的检测HTf(Fe)2SA阳离子脂质体是否具有靶向转染肝癌细胞的能力.方法在有血清和无血清两种条件下检测HTf(Fe)2SA阳离子脂质体和SA阳离子脂质体介导DNA进入肝癌细胞SMMC-7721和小鼠B16黑色素瘤细胞内的量.结果在有血清和无血清两种转染条件下HTf(Fe)2SA阳离子脂质体介导入SMMC-7721细胞内DNA的量明显高于SA阳离子脂质体,而对小鼠B16黑色素瘤细胞两者无明显差异.结论HTf(Fe)2SA阳离子脂质体在体外具有良好的靶向介导DNA转染肝癌细胞的能力.     

阳离子脂质体联合转铁蛋白对人肿瘤细胞的靶向基因转染

目的研究阳离子脂质体联合转铁蛋白(transferrin ,Tf)介导报告基因虫荧光素酶基因对人肿瘤细胞的靶向转染。方法比较阳离子脂质体Lipofectin、Lipofectin联合含双铁的人转铁蛋白[HTf(Fe) 2 ]介导质粒pDR2 luc转染人肝癌细胞株HepG2 、SMMC772 1、人肾癌细胞株GRC及非洲绿猴肾细胞COS7的转染效率,转染效率通过以液体闪烁计数仪单光子计数法测定荧光素酶活性估计。结果除COS7外,其余3株细胞以Lipofectin联合HTf(Fe) 2 介导pDR2 luc的转染效率均比单用Lipofectin的转染效率高(P <0 0 1) ,其中HepG2 与SMMC772 1中的转染效率增高8倍多,GRC中的转染效率增高4倍多。结论Lipofectin联合HTf(Fe) 2 介导质粒pDR2 luc转染细胞时对人肿瘤细胸株有一定的靶向作用。     

阳离子脂质体介导的虫荧光素酶基因表达

目的　研究阳离子脂质体Lipofectin介导虫荧光素酶基因在不同细胞株中的表达。方法　质粒pDR2 luc在大肠杆菌DH5α中扩增后用碱裂解法提取 ,并经Sepharose 2B凝胶过滤柱层析 ,通过凝胶电泳及紫外分光光度计分析纯度、定量 ,以Lipofectin分别转染人肝癌细胞株HepG2 、SMMC772 1、人肾癌细胞株GRC及非洲绿猴肾细胞COS7,然后以液体闪烁计数仪单光子计数法测定荧光素酶活性。结果　荧光素酶活性在 4种细胞株中均有表达 ,且均有显著性差异 (均P <0 0 5 )。结论　阳离子脂质体Lipofectin可用于多种真核细胞基因转染     

阳离子脂质体介导的虫荧光素酶基因表达

目的 研究阳离子脂质体Lipofectin介导虫荧光素酶基因在不同细胞株中的表达。方法 质粒pDR2luc在大肠杆菌DH5a中扩增后用碱裂解法提取,并经Sepharose 2B凝胶过滤柱层析,通过凝胶电泳及紫外分光光度计分析纯度、定量,以Lipofectin分别转染人肝癌细胞株HepG2、SMMC7721、人肾癌细胞株GRC及非洲绿猴肾细胞COS7,然后以液体闪烁计数仪单光子计数法测定荧光素酶活性。结果 荧光素酶活性在4种细胞株中均有表达,且均有显著性差异(均P<0.05)。结论 阳离子脂质体Lipofectin可用于多种真核细胞基因转染。     

转铁蛋白增强脂质体介导基因对肝癌细胞的转染和表达

目的：检测转铁蛋白能否增强脂质体介导外源基因对肝癌细胞的转染和表达。方法：应用本实验室构建的含小鼠白细胞介素（mIL)-12的真核表达质粒pcDNA3/mIL-12和绿色荧光蛋白（GFP）真核表达质粒pcDNA3/EGFP,观察转铁蛋白增强阳离子脂质体LipofectAMINE介导上述两种质粒DNA对小鼠和不同人肝癌细胞株的转染及表达效率。结果：LipofectAMINE加转铁蛋白介导pcDNA3/EGFP对小鼠肝癌细胞株Hep1-6的转染效率由2％－5％提高至20％,对4种不同人肝癌细胞株的转染效率由15％提高到65％－75％;但对小鼠肝癌细胞株MM45T.Li的转染效率并不增加。转铁蛋白可使LipofectAMINE介导pcDNA3/mIL-12在Hep1-6中的表达量增强18倍。结论：转铁蛋白能够与LipofectAMINE和质粒DNA形成复合物,显著增强脂质体介导外源基因对肝癌细胞的转染和表达,但对不同种肝癌细胞株的增强效率不同。     

转铁蛋白阿霉素脂质体的制备及其对人肝癌细胞系的杀伤活性

目的:利用正常肝细胞和肝癌细胞表面转铁蛋白受体以及亲和力的差异,用转铁蛋白修饰脂质体,使脂质体具有导向肝癌细胞的靶向性,分析其对肝癌细胞系的杀伤作用.方法:超声法制备阿霉素脂质体,暴露脂质体上的巯基,然后用双功能交联剂N-琥珀酰亚胺-3-(2-吡啶二硫代)丙酸酯(SPDP)交联HTf(Fe)_2和脂质体,制备成HTf(Fe)_2-阿霉素脂质体.用MTT法分析HTf(Fe)_2-阿霉素脂质体对人肝癌细胞系SMMC-7721的杀伤力.结果:实验检测HTf(Fe)_2与脂质体的交联率为73.5%,电镜观察修饰后的脂质体呈单层状,平均直径56±38 nm,未用HTf(Fe)_2修饰的脂质体直径平均为54±30 nm,两者无显著差异.SPDP修饰和脂质体交联不影响HTf(Fe)_2的活力.MTT法分析发现,当浓度为0.10 mg/L时HTf(Fe)_2阿霉素脂质体、无HTf(Fe)_2阿霉素脂质体及游离阿霉素对肝癌细胞的杀伤率分别为64.52%,22.12%和37.82%,前一组与后两组之间有显著差异(P<0.05).结论:HTf(Fe)_2阿霉素脂质体体外杀伤肝癌细胞系SMMc-7721具有用药量小、高效、特异性强等优点,为体内应用治疗肝癌提供了实验依据.     

转铁蛋白与细胞穿膜肽共修饰脂质体的制备及其抗肿瘤效果

目的制备转铁蛋白(TF)与细胞穿膜肽(TAT)共修饰载紫杉醇(PTX)脂质体(TF/TATLP-PTX),对其体外性质进行评价。方法采用薄膜分散法制备TF与TAT共修饰TF/TATLP-PTX,考察其粒径、电位、包封率等理化特征。通过MTT实验考察脂质体对肝癌HepG2细胞的毒性,流式细胞仪检测肿瘤细胞对脂质体的摄取能力。构建HepG2肝癌异位瘤模型,考察不同脂质体对肿瘤的生长抑制能力。结果制备的TF/TATLP-PTX粒径为(137.8±13.5)nm,Zeta电位(17.55±3.85)mV,紫杉醇的包封率为85.3%。MTT实验结果显示,给药72 h后,以生理盐水组为对照,未修饰紫杉醇脂质体(LP-PTX)、转铁蛋白修饰紫杉醇脂质体(TFLP-PTX)、TAT修饰紫杉醇脂质体(TATLP-PTX)和TF/TATLP-PTX肝癌HepG2细胞存活率分别为62.4%、45.5%、39.2%和17.7%,组间比较差异显著(P<0.01);体外细胞摄取实验表明,肝癌HepG2细胞对TF/TATLP的摄取效率分别是TFLP、TATLP和LP的2.7倍、2.2倍和3.9倍,组间比较差异显著(P<0.01);荷瘤裸鼠治疗实验以生理盐水组为对照,给药20 d后,生理盐水组体积变为给药前的2.55倍,LP-PTX、TFLP-PTX、TATLP-PTX和TF/TATLP-PTX组肿瘤体积分别增长到原体积的2.22、1.73、1.89、1.29倍,与其他组间差异显著(P<0.01)。结论 TF/TATLP-PTX制备工艺简单,与肝癌HepG2细胞具有良好的亲和性,是一种潜在高效的肿瘤靶向给药系统。     

磁性阳离子高分子脂质体结合野生型p53投递系统的构建

目的用合成的磁性阳离子高分子脂质体与野生型p53(WTp53)基因质粒结合,构建靶向纳米载体投递系统,为进一步行静脉内皮细胞转染奠定基础。方法应用大肠杆菌重组质粒扩增法扩增WTp53质粒DNA,合成的磁性阳离子高分子脂质体为羧甲基壳聚糖十八烷基季铵盐/胆固醇(OQCMC/Chol)包覆油溶性Fe3O4的载体粒子。在不同的pH值及不同的WTp53质粒DNA和载体粒子质量比的条件下,将WTp53质粒DNA与OQCMC/Chol超微磁性载体粒子进行混合,并分别进行DNA结合实验和沉淀实验。观察电泳结果,并用SPSS 13.0统计软件进行数据分析。在DNaseⅠ模仿体内酶的环境下,将其加入制备好的超微载体投递系统中,电泳后观察投递系统对DNA的保护作用。应用透析系统模拟体内环境,观察超微投递系统载体与基因分离情况,并利用紫外分光光度计测量超微载体投递系统累积缓释曲线。结果应用透射电镜观察OQCMC/Chol超微磁性载体粒子和WTp53结合后的投递粒子。在pH 7条件下,WTp53质粒DNA和载体粒子质量比为1∶0.6,p53质粒DNA与OQCMC/Chol超微磁性载体粒子经过直接静电作用几乎100%结合,并证实该投递系统对DNA具有较好的保护作用。超微载体投递系统在7～12 h左右有明显的突释波峰,到第54小时以后,超微载体释放浓度变化缓慢,并可持续释放9 d以上。结论成功构建磁性阳离子高分子脂质体结合WTp53投递系统,将为进一步行细胞转染奠定可靠的基础,并为构建血管内靶向定位基因投递新方法和新技术提供可靠理论依据。     

SA脂质体介导人白介素-10基因转染对大鼠局灶脑缺血再灌注损伤的影响

将48只SD大鼠随机分为正常对照组、缺血对照组（缺血组）、空质粒组和白介素（IL）-10基因转染组（转染组）,后三组采用Longa法建立局灶脑缺血再灌注损伤（MCAO）模型。造模成功后转染组及空质粒组分别于侧脑室注射SA脂质体/pcDNA3．1-IL-10,SA脂质体／pcDNA3,1（＋）。各组大鼠于预定时间处死制作脑组织标本,采用RT-PCR法检测IL-10基因表达情况;观察海马CA1区神经元损伤情况,测定脑梗死体积并进行神经行为学评分。结果转染组再灌注72h时大脑皮层及海马中均能检测到IL-10 mRNA表达,其余三组均无表达。转染组海马CA1区神经元损伤程度明显轻于缺血组及空质粒组,脑梗死体积和神经行为学评分亦低于缺血组及空质粒组（P均〈0．05）。证实SA脂质体介导人IL-10基因转染能减轻大鼠局灶脑缺血再灌注损伤。     

阳离子脂质体介导日本血吸虫组织蛋白酶L1基因的真核表达

目的真核表达日本血吸虫组织蛋白酶L1(SjCL1)以研究其生物学功能。方法采用阳离子脂质体复合质粒SjCL1/AD1-1 DNA并转染HeLa细胞,通过RT-PCR检测SjCL1基因在HeLa细胞中的转录,SDS-PAGE电泳法分析目的基因的蛋白表达产物,并进一步通过Western Blot法证实。结果RT-PCR检测到转染的细胞中有与目的基因相符大小约1000bp转录条带,SDS-PAGE电泳发现表达质粒SjCL1/AD1-1DNA转染的细胞上清液中存在大小约为31kDa的表达蛋白带,Western Blot法证实该表达蛋白产物可和日本血吸虫兔血清发生特异的反应。结论阳离子脂质体将SjCL1/AD1-1转染入HeLa细胞后可特异地表达一个大小约为31kDa的可分泌的日本血吸虫蛋白产物。     

Synthesis and Characterization of Fe(III) and Pb(II) Complexes with 3-Hydroxypyridine-2(1H)-Thiones

Two kinds of 3-hydroxypyridine-2(1H)-thiones were synthesized. The visible (VIS) spectroscopic analysis indicated that 3-hydroxy-1-methylpyridine-2(1H)-thione (4a) and 3-hydroxy-1-(2-hydroxyethyl)pyridine-2(1H)-thione (4b) formed stable 3:1 Fe(III) complexes. The stability constant of the 4b-Fe(III) complex was estimated from the competitive reaction with EDTA and was found to be 36.7 in logβ₃. Treatment of compound 4b with Ga(acac)₃ in D₂O:CD₃OD (9:1) solution afforded 3:1 Ga(III) complex, which was assigned by means of ¹H nuclear magnetic resonance (¹H NMR) spectroscopy. Treatment of compound 4b with Pb(NO₃)₂ gave 4b-Pb(II) complex. The Pb(II) selectivity over biologically relevant Mg(II) and Ca(II) was remarkably improved by adopting N-hydroxyethyl functionality instead of N-methyl group.     

Hepatocytes targeting of cationic liposomes modified with soybean sterylglucoside and polyethylene glycol

AIM: In this study, a hepatocyte-spedfic targeting technology was developed by modifying cationic liposomes with soybean sterylglucoside (SG) and polyethylene glycol (PEG) (C/SG/ PEG-liposomes). METHODS: The liposomal transfection efficiencies in HepG2 2.2.15 cells were estimated with the use of fluorescein sodium (FS) as a model drug, by flow cytometry. The antisense activity of C/SG/PEG-liposomes entrapped antisense oligonucleotides (ODN) was determined as HBsAg and HBeAg in HepG2 2.2.15 cells by ELISA. The liposome uptake by liver and liver cells in mice was carried out after intravenous injection of 3H-labeled liposomes. RESULTS: C/SG-liposomes entrapped FS were effectively transfected into HepG22.2.15 cells In vitro. C/SG/PEG-liposomes entrapped ODN, reduced the secretion of both HBsAg and HBeAg in HepG2 2.2.15 cells when compared to free ODN. After in vivo injection of 3H-labeled C/SG/PEG-liposomes, higher radiation accumulation was observed in the hepatocytes than non-parenchymal cells of the liver. CONCLUSION: C/SG/PEG-liposomes mediated gene transfer to the liver is an effective gene-delivery method for hepatocytes-specific targeting, which appears to have a potential for gene therapy of HBV infections.     

肝细胞DNA损伤诱导蛋白DDI2的单克隆抗体制备及鉴定

目的制备鼠抗DDI2蛋白单克隆抗体,并对其在细胞和组织中的定位进行初步研究。方法利用体外原核表达的DDI2蛋白,免疫BLAB/c小鼠,制备单克隆抗体。利用免疫组织化学技术明确该蛋白在组织中的定位。结果利用杂交瘤细胞技术,成功制备了两株DDI2单克隆抗体细胞系2H3、2B2。表达纯化的单克隆抗体具有较高的免疫活性。与正常对照组相比,肝损伤组织中该蛋白表达增强,且高表达于门脉周围肝实质细胞,并主要表达于细胞核。结论 DDI2的表达与四氯化碳诱导的肝损伤有关,损伤肝细胞主要表达于肝实质细胞的细胞核。     

Absorption of N-Nitroso-bis-(2-hydroxy-propyl)amine (BHP) from subcutaneous layers

Summary Nodules developing under the skin of rats after repeated injections of N-Nitroso-bis-(2-hydroxy-propyl)amine (BHP) are shown to contain no unchanged BHP     

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4]

The all-ferric [Fe₄S₄]⁴⁺ cluster [Fe₄S₄{N(SiMe₃)₂}₄] 1 and its one-electron reduced form [1]^- serve as convenient precursors for the synthesis of 3∶1-site differentiated [Fe₄S₄] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)₂C₆H₃, mesityl = 2,4,6-Me₃C₆H₂) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe₄S₄(SDmp)₃(THF)₃] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)₃(O)₃ coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1]^- with four equiv of HSDmp gave [Fe₄S₄(SDmp)₄]^- 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me₄Im) to give [Fe₄S₄(SDmp)₃(Me₄Im)] 4 modeling the [Fe₄S₄(Cys)₃(His)] cluster in hydrogenases, and its one-electron reduced form [4]^- was synthesized from the reaction of 3 with Me₄Im. The reversible redox couple between 3 and [3]^- was observed at E_1/2 = -820 mV vs. Ag/Ag⁺, and the corresponding reversible couple for 4 and [4]^- is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe₄S₄]⁴⁺ cluster, [Fe₄S₄(SDmp)₄].     

Plasma apolipoprotein (a) is increased in Type 2 (non-insulin-dependent) diabetic patients with microalbuminuria

Summary Patients with Type 2 (non-insulin-dependent) diabetes mellitus complicated by microalbuminuria or albuminuria, have an increased risk of developing macrovascular disease and of early mortality. Because lipoprotein abnormalities have been associated with diabetic nephropathy, this study tested the hypothesis that levels of apolipoprotein (a) are elevated in patients with Type 2 diabetes and increased levels of urinary albumin loss. Levels of apolipoprotein (a) in diabetic patients with microalbuminuria (n = 26, geometric mean 195 U/1, 95 % confidence interval 117–324) and albuminuria (n = 19, 281 U/1,165–479) were higher than in non-diabetic control subjects (n = 140,107 U/1, 85–134,p < 0.05), and in the albuminuric group than diabetic patients without urinary albumin loss (n = 58, 114 U/1, 76–169,p < 0.05). Patients with microalbuminuria and albuminuria had levels comparable with patients undergoing elective coronary artery graft surgery (n = 40,193 U/1,126–298). Apolipoprotein (a) levels were higher in diabetic patients with macrovascular disease than in those without (n = 49, 209 U/1, 143–306 vsn = 54, 116 U/1, 78–173,p < 0.05). These preliminary results suggest that raised apolipoprotein (a) levels of Type 2 diabetic patients with microalbuminuria and albuminuria may contribute to their propensity to macrovascular disease and early mortality.     

Preparation and purification of F(ab')(2) fragment from anti hepatoma mouse IgG(1) mAb

INTRODUCTION Since the advent of hybridoma technology[1], monoclonal antibodies have been widely used in basic studies and clinical application. F(ab')2 is a bivalent antibody fragment which is currently used for both diagnosis and treatment[2], and better than the original mAbs, because it does not retain complement-binding function due to lack of Fc regions and reduced interaction with non-specific proteins and the smaller molecular weight than the mAbs, furthermore, it can be digested by pepsin or papain and purified by size exclusion chromatography[4], ion-exchange chromatography[6] or hydrophobic interaction[7]. However, these methods are time-consuming, and cannot attain sufficient purity and recovery of F(ab')2 fragment. Development of efficient procedures for F(ab')2 preparation is an urgent necessity.     

Thermo-Exfoliated Graphite Containing CuO/Cu2(OH)3NO3:(Co2+/Fe3+) Composites: Preparation,Characterization and Catalytic Performance in CO Conversion

Thermo-exfoliated graphite (TEG)/CuO/Cu₂(OH)₃NO₃:(Co²⁺/Fe³⁺) composites were prepared using a wet impregnation method and subsequent thermal treatment. The physicochemical characterization of the composites was carried out by powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) and Ar temperature-desorption techniques. The catalytic efficiency toward CO conversion to CO₂ was examined under atmospheric pressure. Characterization of species adsorbed over the composites taken after the activity tests were performed by means of temperature programmed desorption mass-spectrometry (TPD MS). (TEG)/CuO/Cu₂(OH)₃NO₃:(Co²⁺/Fe³⁺) composites show superior performance results if lower temperatures and extra treatment with H₂SO₄ or HNO₃ are used at the preparation stages. The catalytic properties enhancements can be related to the Cu₂(OH)₃NO₃ phase providing reaction centers for the CO conversion. It has been found that prevalence of low-temperature states of desorbed CO₂ over high-temperature ones in the TPD MS spectra is characteristic of the most active composite catalysts.