顺铂对卵巢癌COC1细胞总蛋白表达的影响研究

对卵巢癌COC1细胞进行体外培养, 当培养至10⁹/mL浓度时裂解细胞, 获得细胞总蛋白, 利用二维电泳对顺铂诱导前后的细胞内总蛋白质进行分离, 利用PDQuest 7.1.1专业分析软件对二维凝胶图谱进行差异比较, 利用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)进行质量分析, 最后进行生物信息学检索, 以寻找卵巢癌细胞发生、发展和凋亡的分子标记物. 经二维电泳图谱和质谱分析, 鉴定了11种蛋白, 4种蛋白表达量有差异, 7种蛋白在诱导前后表现为有和无的关系. 有5种是“分子伴侣”蛋白, 有4种蛋白与人体能量代谢有关. 蛋白功能分析提示卵巢癌细胞的蛋白质组表达存在差异, 这些差异蛋白对于寻找卵巢癌生物标志物, 开发新药提供很好的理论依据.     

聚乙烯亚胺介导siRNA和顺铂协同抗肿瘤治疗

用聚乙烯亚胺(PEI)为载体,介导siRNA(siSurvivin)沉默肿瘤细胞抗凋亡基因survivin,并与抗癌药物(顺铂)进行协同抗肿瘤治疗.凝胶阻滞电泳实验显示,PEI能够对siRNA进行有效复合,在PEI/siRNA质量比为0.4时实现完全阻滞.细胞耐药性实验证明了耐顺铂细胞(A549DDP细胞)的survivin基因过度表达且耐顺铂能力是顺铂敏感细胞(A549细胞)的8倍.RT-PCR实验验证了PEI担载siSurvivin后对survivin基因实现了有效沉默,与顺铂药物共同作用后不影响基因沉默效果.细胞凋亡实验验证了基因与药物协同作用后细胞的凋亡率达到60.9％,而单独药物或PEI/siSurvivin复合物分别作用后的细胞凋亡率仅分别为30.2％和19.8％.细胞增殖实验进一步验证了PEI介导siSurvivin与顺铂联合治疗能够实现有效地协同抗肿瘤效果.     

海洋珍珠生物提取液单独与顺铂联合对人宫颈癌Siha细胞的生长抑制作用

为探讨海洋珍珠生物提取液对人宫颈癌Siha细胞体外生长的抑制作用,以及海洋珍珠生物提取液增加Siha细胞对顺铂的敏感性,以期对治疗宫颈癌及其它肿瘤寻找新的治疗药物,体外培养人宫颈癌Siha细胞,分别用海洋珍珠生物提取液、顺铂、海洋珍珠生物提取液加顺铂处理24、48、72 h,采用四甲基偶氮唑蓝(MTT)比色法检测海洋珍珠生物提取液和顺铂及两者联合应用Siha细胞的存活率。结果表明,海洋珍珠生物提取液对Siha细胞的增殖有明显的抑制作用(P0.01),其处理,24、48、72 h的IC50分别为56.26、51.92、48.92μg/mL,顺铂对Siha细胞的24、48、72 h的IC50分别为16.74、4.68、6.69μg/mL;两者合用可大大提高对Siha细胞的抑制作用,比单用海洋珍珠生物提取液或单用顺铂均有明显的提高(P0.01)。提示海洋珍珠生物提取液能抑制Siha细胞的生长,同时能增加Siha细胞对顺铂的敏感性。     

IL-24重组载体的构建及其联合化疗药物对SKOV3细胞中TUBB3与ERCC-1基因表达的影响

通过基因重组技术将白介素24(IL-24)基因片段分别克隆成pc DNA3.0-OSP-1-IL-24和pc DNA3.0-IL-24载体,利用逆转录PCR(RT-PCR)测定2种载体的表达.将2种化疗药物紫杉醇(PTX)与顺铂(DDP)分别作用于正常SKOV3细胞及pc DNA3.0,pc DNA3.0-IL-24,pc DNA3.0-OSP-1和pc DNA3.0-OSP-1-IL-24稳定转染SKOV3细胞(卵巢癌细胞株),通过噻唑蓝(MTT)法检测化疗药物联合IL-24基因对细胞的杀伤效果及细胞的增殖能力,应用实时荧光定量PCR技术检测人β-微管蛋白3(TUBB3)与核苷酸切除修复交叉互补基因位1(ERCC1)基因在各组细胞中的表达.RT-PCR检测结果显示,通过基因重组技术克隆了pc DNA3.0-IL-24与pc DNA3.0-OSP-1-IL-24载体,IL-24基因能提高SKOV3细胞对化疗药物顺铂和紫杉醇的敏感性.其联合化疗药物对肿瘤药物的IC50值分别下降了10倍和6倍,TUBB3和ERCC1基因在IL-24基因转染的SKOV3细胞内的表达水平与在正常SKOV3细胞内相比,分别下降4倍和10倍多.上述结果表明,IL-24联合化疗药物可明显下调TUBB3与ERCC1基因的表达,该实验为临床治疗卵巢癌提供了重要的理论依据.     

石墨炉原子吸收光谱法测定奥沙利铂中银量

奥沙利铂又名草酸铂(L-OHP),是继顺铂和卡铂之后问世的铂类第三代抗癌药物。具有抗瘤谱广,对大肠癌、卵巢癌疗效好,对胃癌、非霍奇金氏淋巴瘤、非小细胞肺癌和头颈部肿瘤有一定疗效,对5氟脲嘧啶(5-FU)治疗无效的大肠癌病人、对铂类耐药者有效。奥沙利铂中的银为杂质,其最大限量为5,0mg·kg~(-1)。准确测定银含量对该药的     

硝普钠分光光度法测定头孢他啶

建立了硝普钠分光光度法测定头孢他啶的方法。实验结果表明,在碱性条件下,头孢他啶与硝普钠以1∶2的化学计量比反应形成一种红褐色产物,其最大吸收波长为528 nm。 头孢他啶在2.50~810 mg/L浓度范围内与吸光度呈良好线性关系,线性回归方程为A=0.02704+0.00218ρ(mg/L),线性相关系数r＝0.9987,表观摩尔吸收系数ε528=1.2×103 L/(mol·cm),检测限(3σ/k)为1.38 mg/L。 本方法操作简便、快速,成功用于药品中头孢他啶含量的测定。     

反向流动注射化学发光法测定姜黄素

铁氰化钾氧化鲁米诺在碱性介质中产生化学发光,姜黄素对该体系化学发光具有强烈的抑制作用。因此,利用该化学发光的抑制体系,结合反向流动注射技术,建立了测定大黄类药物姜黄素含量的新方法。在优化的条件下,化学发光抑制信号强度ΔI与姜黄素的浓度分别在1×10-7~1×10-6和1×10-6~1×10-5mol/L范围内呈良好的线性关系,检出限为1×10-9mol/L。对2.0×10-6mol/L的姜黄素进行平行测定10次,得相对标准偏差(RSD)为1.8%。方法应用于中药姜黄中姜黄素(总姜黄素计)的含量测定。     

人卵巢癌耐药细胞株的比较蛋白质组分析

本文采用高分辨二维凝胶电泳分离技术对人卵巢癌细胞株COC1及其耐药细胞株COC1/DDP中的蛋白质进行分离和差异表达分析, 应用基质辅助激光解吸电离-飞行时间质谱对酶解多肽进行测定[即测定蛋白质的肽质量指纹图(Peptide mass fingerprinting, PMF)], 并通过相应的数据库搜索来鉴定蛋白质. 为获得更准确的检索结果, 采用串联质谱技术对各肽段进行氨基酸测序, 并应用IPI-HUMAN数据库对上述检索结果进一步加以确认.      

气相色谱法测定尿液中可卡因及其代谢物爱冈宁甲基酯

建立了尿样中可卡因（COC）及其代谢物爱冈宁甲基酯（EME）的气相色谱检测方法。 采用液液萃取法提取尿样中可卡因和爱冈宁甲基酯,考察了萃取剂种类和用量、试样pH值以及萃取时间等因素对提取效果的影响。 结果表明,尿样中COC和EME的最佳液液萃取条件是：以V(氯仿)∶V(异丙醇)=9∶1为提取溶剂,调节样品溶液pH=9.5,在40 ℃ 水浴振荡提取6 min。 COC和EME日内精密度分别为1.73%和1.44%,日间精密度分别为2.57%和2.89%,最低检出限（LOD）为0.040 mg/L。 此法无需衍生化、快速、准确、灵敏度高,可同时检测尿样中COC和EME的含量。     

一个共配的铜基配合物的合成、晶体结构及抗肿瘤活性

合成了一个以N-{[2-羟基-4-甲氧基苯基\]甲亚胺}苯丙氨酸阴离子和2,2′-联吡啶共配位的铜基配合物[Cu(C₁₇H₁₅NO₄)(C₁₀H₈N₂)]₂•CH₃OH（1）,其结构经IR、 元素分析、X-射线单晶衍射及Hirshfeld表面分析表征。用MTT法考察了配合物对耐顺铂的人肺癌细胞株（A549/DDP,DDP=顺铂）和人宫颈癌细胞株（C33A）的细胞毒性,用流式细胞术分析了配合物〖STHZ〗1〖STBZ〗诱导A549/DDP细胞凋亡和线粒体膜电位下降,初步考察了配合物1的抗肿瘤活性。结果表明：配合物1（CCDC:1913991）属于三斜晶系,Pī空间群,晶体学参数为：a = 10.1770(5) Å, b, c, α = 104.576(4)°,β = 101.151 (4)°,γ = 101.151(4)°,V = 1197.71(11) ų, Z =1,R₁ = 0.0344, wR₂,S = 1.061a = 10.1770(5) Å, b = 10.2030(5) Å, c = 12.5857(6) Å, α = 104.576(4)°,β = 101.151 (4)°,γ = 101.151(4)°,V = 1197.71(11) ų, Z =1,R₁ = 0.0344, wR₂ = 0.0913,S = 1.061;配合物1对肿瘤细胞有显著细胞毒性,对A549/DDP和C33A细胞作用24 h后,IC₅₀值分别为(9.75±0.39) μmol·L^-1和(28.13±1.27) μmol·L^-1;其通过诱导A549/DDP细胞的线粒体膜电位下降而导致细胞凋亡,配合物1浓度为40.0 μmol·L^-1时,与A549/DDP细胞孵育36 h,诱导A549/DDP细胞总凋亡率为41.0%,与A549/DDP细胞孵育30 h,使A549/DDP细胞的线粒体膜电位降低了47.4%。 = 0.0913 = 12.5857(6) Å = 10.2030(5) Å     

Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3⁺ LSCs), EoL-1 (FLT3⁺ LCs), and U937 (FLT3⁻ LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC₁₀–IC₈₀ values and each concentration of Cur at the IC₂₀, IC₃₀, IC₄₀, and IC₅₀ values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox–Cur increased cytotoxicity in leukemic cells. Dox–Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox–Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox–Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.     

Validated LC–MS/MS method for simultaneous determination of doxorubicin and curcumin in polymeric micelles in subcellular compartments of MCF‐7/Adr cells by protein precipitation–ultrasonic breaking method

A specific and sensitive LC–MS/MS with protein precipitation– ultrasonic breaking method has been developed and validated for simultaneous determination of doxorubicin (DOX) and curcumin (Cur) in DOX and Cur co‐loaded hyaluronic acid–vitamin E succinatemicelles [(DOX + Cur)–polymeric micelles (PMs)] in subcellular compartments of resistant MCF‐7/Adr cells. Sequential extraction of four subcellular protein fractions (cytosolic, membrane/organelle, nucleic and cytoskeleton) was performed directly from MCF‐7/Adr cells after incubation with (DOX + Cur)–PMs. An ultrasonic breaking–methanol precipitation method was used for extraction of the fractions, and the micelle breaking efficiency with methanol was 98.1 and 97.6% for DOX and Cur, respectively. The analytes were analyzed using positive electrospray ionization coupled with multiple reaction monitoring. The calibration curves were linear over a concentration range of 0.5–400 ng/mL for DOX and 2–2000 ng/mL for Cur, and the recovery for the two analytes were >85% with negligible matrix effect. The intra‐day and inter‐day precision was <10.80% and relative error was within ±7.70%. The developed method was successfully applied for subcellular determination of DOX and Cur in MCF‐7/Adr cells. Moreover, Cur and (DOX + Cur)–PMs had a marked promoting effect on the distribution of DOX in the nucleic protein fraction.     

Determination of acidity constants of curcumin in aqueous solution and apparent rate constant of its decomposition

The stability of curcumin (H3Cur) in aqueous media is improved when the systems in which it is present are at high pH values (higher than 11.7), fitting a model describable by a pseudo-zero order with a rate constant k' for the disappearance of the Cur3- species of 1.39 (10(-9)) Mmin(-1). There were three acidity constants measured for the curcumin as follows: pKA3 = 10.51 +/- 0.01 corresponding to the equilibrium HCur2- = Cur3- + H+, a pKA2 = 9.88 +/- 0.02 corresponding to the equilibrium H2Cur- = HCur-(2) + H+. These pKA values were attributed to the hydrogen of the phenol part of the curcumin, while the pKA1 = 8.38 +/- 0.04 corresponds to the equilibrium H3Cur = H2Cur- + H+ and is attributed the acetylacetone type group. Formation of quinoid structures play an important role in the tautomeric forms of the curcumin in aqueous media, which makes the experimental values differ from the theoretically calculated ones, depending on the conditions adopted in the study.     

硒化壳聚糖抑制人早幼粒白血病细胞增殖的研究

为探讨硒化壳聚糖对体外培养人早幼粒白血病细胞增殖的抑制作用,用SRB法和集落形成法检测了药物对细胞增殖的抑制作用,流式法检测了细胞周期阻断作用。结果表明,25、50、100mg／L硒化壳聚糖作用HL60细胞48h对细胞有增殖抑制作用（P〈0．01）;50、100mg／L硒化壳聚糖作用细胞48h后,G0／G1期细胞数较对照组增加了14．9％-22．0％（P〈0．05）,S期细胞减少了14．3％～20．1％（P〈0．05）。可见硒化壳聚糖对人早幼粒白血病细胞增殖具有抑制作用。     

A Nanomedicine-Enabled Ion-Exchange Strategy for Enhancing Curcumin-Based Rheumatoid Arthritis Therapy

Curcumin (Cur) has been clinically used for rheumatoid arthritis treatment by the means of reactive oxygen species (ROS) scavenging and immune microenvironment regulation. However, this compound has a poor water solubility and moderate antioxidative activity, favoring no further broadened application. Metal complexes of curcumin such as zinc-curcumin (Zn−Cur) features enhanced water solubilities, while copper-curcumin (Cu−Cur) shows a higher antioxidant activity but lower solubility than Zn−Cur. Based on their inherent biological properties, this work proposes a nanomedicine-based ion-exchange strategy to enhance the efficacy of Cur for rheumatoid arthritis treatment. Copper silicate nanoparticles with hollow mesoporous structure were prepared to load water-soluble Zn−Cur for constructing a composite nanomedicine, which can degrade in acidic microenvironment of arthritic region, releasing Cu²⁺ and Zn−Cur. Cu²⁺ then substitute for Zn²⁺ in Zn−Cur to form Cu−Cur with a significantly enhanced antioxidative effect, capable of efficiently scavenging ROS in M1 macrophages, promoting their transition to an anti-inflammatory M2 phenotype. In addition, the silicate released after nanocarrier degradation and the Zn²⁺ released after ion exchange reaction synergistically promote the biomineralization of osteoblasts. This work provides a new approach for enhancing the antiarthritic effect of Cur via an ion-exchange strategy.     

Wound healing improvement by curcumin‐loaded electrospun nanofibers and BFP‐MSCs as a bioactive dressing

Wound healing, one of the most complex processes of the body involving the cooperation of several important biomolecules and pathways, is one of the major therapeutic and economic issues in regenerative medicine. The present study aimed to introduce a novel electrospun curcumin (Cur)‐incorporated chitosan/polyvinyl alcohol/carbopol/polycaprolactone nanofibrous composite for concurrent delivery of the buccal fat pad‐derived mesenchymal stem cells (BFP‐MSCs) and Cur to a full‐thickness wound on the mouse model. Scaffolds were characterized structurally using scanning electron microscopy (SEM), fluorescence microscopy imaging and Fourier‐transform infrared spectroscopy, and toxicity of the scaffolds was also evaluated after BFP‐MSC seeding by SEM imaging and 3‐(4,5 dimethyiazol‐2‐1)‐2‐5‐diphenyl tetrazolium bromide (MTT) assay. Then, its influence on the wound‐healing process was investigated as a wound dressing for a full‐thickness skin defect in mouse model. Results demonstrated that the designed composite scaffolds have the capability for cell seeding and support their growth and proliferation. Macroscopic and histopathological characteristics were evaluated at the end of the 7 and 14 days after surgery, and their results showed that our designed scaffold groups accelerated the wound‐healing process compared with the control group. Among those, scaffold/Cur, scaffold/Cur/BFP‐MSC and scaffold/BFP‐MSC groups demonstrated more wound repair efficacy. These results indicated that the combined grafts can be used to improve the wound‐healing process, and therefore, the electrospun nanofibers presented in this study, Cur and BFP‐MSC together, were demonstrated to have promising potential for wound‐dressing applications.     

Cold atmospheric plasma modification of curcumin loaded in tri‐phosphate chitosan nanoparticles enhanced breast cancer cells apoptosis

The anti‐cancer mechanisms of curcumin have been reported to include suppressions of angiogenesis and tumor proliferation. The main goal of this research is to increase the solubility of curcumin by cold atmospheric plasma (CAP) and assess the effects of modified curcumin by charging with tri‐polyphosphate chitosan nanoparticles for MCF‐7, MDA‐MB‐231 breast cancer cells, and human fibroblast cells. Curcumin modification was done by CAP and its solubility was evaluated by spectrophotometry. After loading modified curcumin into nano‐chitosan‐TPP, nanocurcumin was characterized by scanning electron microscopy. Cellular viability and apoptosis of treated cells were assessed by MTT and Annexin V. The changes of messenger RNA expression of TP5353 and VEGF genes were analyzed by real‐time PCR. CAP was able to transform the curcumin to possess hydrophilic characteristics after 90 seconds. The mean diameter of Curcumin loaded chitosannanoparticles (NPs) were determined as 48 nm. MTT results showed that the IC₅₀ of nano Cur‐chitosan‐TPP was effectively decreased compared to free curcumin in MCF‐7 (15 μg/mL at 72 hours vs 20 μg/mL at 48 hours). Additionally, nano Cur‐chitosan‐TPP had no significant effect on normal cells (Human dermal fibroblas: HDF), whereas it also decreased the viability of triple negative breast cancer cell line (MDA‐MB‐231). Real‐time PCR results showed that expression level of TP53 gene was upregulated (P = .000), whereas VEGF gene downregulated (P = .000) in treated MCF‐7 cells. Curcumin loaded chitosan nanoparticles have led to an induction of apoptosis (79.93%) and cell cycle arrest (at S and G2M). Modified‐curcumin‐tri‐polyphosphate chitosan nanoparticles using CAP can be considered as a proper candidate for breast cancer treatment.     

Topical application of zein-silk sericin nanoparticles loaded with curcumin for improved therapy of dermatitis

Atopic dermatitis is characterized by leukocyte migration into the skin dermis and typically driven by excessive chemokine production at the site of inflammation. Conventional topical formulations such as gels, creams, and ointments are insufficient for this treatment because of low penetration of drug molecules into the targeted skin tissues. Herein, using a simple, green, sustainable strategy, we have developed novel primary zein nanoparticles embedded in curcumin (Cur) and coated with silk sericin (ZHSCs) for the topical delivery of Cur to penetrate into the dermis and exercise anti-dermatitis effects on the lesion with minimal side-effects. Transdermal delivery experiments and porcine skin fluorescence imaging indicated that ZHSCs facilitate the penetration of Cur across the epidermis layer of skin to reach deep-seated sites. Notably, ZHSCs = 1:0.25 (zein-to-silk sericin mass ratios of 1:0.25) markedly elevated the skin permeability and cumulative turnover of Cur transferred, which were provided a greater than a 3.8-fold increase relative to free Cur. The special nanoparticles of ZHS = 1:0.25 possessed the deepest localization depth and experience a transition of the particle structure and core-shell separation after penetrating into the dermis of skin. In a cell model of dermatitis induced by tumor necrosis factor α/interferon γ co-stimulation, compared with free Cur, Cur-loaded ZHS nanoparticles down-regulated the generation of inflammatory cytokines and chemokines in keratinocytes through suppression of the nuclear translocation of NF-κBp65 and hence exerted an anti-dermatitis effect. This strategy may provide new avenues and direction for the demanding issues of valid topical delivery systems.     

Study of a water-soluble supramolecular complex of curcumin and β-cyclodextrin polymer with electrochemical property and potential anti-cancer activity

As a member of the curcuminoid compound family, curcumin (Cur) has many interesting therapeutic properties. However, its low aqueous solubility and stability have resulted in poor bioavailability and restricted clinical efficacy. Based on size matching, β-cyclodextrin polymer (β-CDP), with its hydrophilic polymer chains and hydrophobic cavities, can form an inclusion complex with Cur. To improve the water solubility and stability of Cur, a simple and eco-friendly grinding method was designed to form β-CDP inclusion complexes. According to the Boltzmann–Hamel's method and Job's method, the molar ratio of the β-CD unit in β-CDP to Cur was determined to be 1:1. The diffusion coefficient and diffusion activation energy of Cur-β-CDP were calculated in an electrochemical study. This supramolecular complex worked well in vitro to inhibit the proliferation of hepatoma carcinoma cells HepG2. Remarkably, this method visibly reduced the undesirable side effects on normal cells, without weakening the anti-cancer activity of the drugs. We expect that the obtained host–guest complex will provide a new approach for delivering natural drug molecules, having low water solubility.