低分子量肝素对骨肉瘤细胞生长与凋亡的影响研究

目的 研究低分子量肝素（LMWH）体外抗肿瘤生长及诱导肿瘤细胞凋亡的作用,初步探讨低分子量肝素抗肿瘤作用机制,为该药的抗肿瘤作用开发和临床应用提供试验依据.方法在体外培养的骨肉瘤系（MG-63）细胞培养基中加入不同浓度的LMWH,对照组加完全培养基,用MTT法检测不同浓度LMWH在干预不同时间后对骨肉瘤细胞生长的影响,流式细胞仪检测不同浓度干预组细胞凋亡率的变化.结果LMWH可抑制骨肉瘤细胞系MG-63生长,并具有时间剂量依赖性,在LMWH浓度为200 U•mL^-1时,对细胞生长有明显的抑制作用. 流式细胞仪检测显示,随着LMWH剂量的增加,MG-63细胞凋亡率明显增加. 结论LMWH在体外具有抗肿瘤活性,其作用可能通过抑制细胞增殖和促进肿瘤细胞凋亡而实现.     

抗MRSA小链霉菌NIM521发酵工艺的优化

小链霉菌NIM521能产生对耐甲氧西林金黄色葡萄球菌(MRSA)有强烈抑制作用的活性物质.利用单因素多浓度和正交实验法对其发酵培养基组分及发酵条件进行了优化,并在优化后的条件下进行了20 L罐发酵放大实验.实验表明该菌的最适培养条件为:培养基含麦芽汁9%、蛋白胨6%、L-甲硫氨酸0.02%、维生素C 0.02%;培养温度30℃、转速250 r/min、接种量15%、发酵时间6 d.优化发酵条件下,20 L发酵罐发酵5 d时发酵液抑菌圈直径可达37.5 mm.     

基于人肝癌细胞系SMMC-7721的三维培养条件研究

目的 基于人肝癌细胞系SMMC-7721考察合适的海藻酸钠、氯化钙浓度,形成良好的海藻酸钙凝胶,优选三维条件下肿瘤细胞的培养条件。方法 在96孔板中培养SMMC-7721细胞,基于L₉（3⁴）正交表设计实验,以MTT法检测并计算氯化钙溶液、柠檬酸钠溶液不同浓度与作用时间下的细胞存活率;结合正交优选结果,考察适宜的海藻酸钠浓度,筛选适合的成胶、溶胶条件,并在三维培养模式下进行细胞活力确证。结果 最佳凝胶应用条件为1%氯化钙溶液与1%海藻酸钠溶液作用15 min内用于成胶,10%柠檬酸钠溶液作用凝胶10 min内用于溶胶,MTT法检测细胞存活率为94.97%。该三维培养条件下的肝癌细胞72 h内生长状态良好,存活率可达92.10%,细胞堆积紧密,出现肿瘤细胞球样聚集体。结论 本实验筛选出适合肿瘤细胞三维培养的成胶、溶胶条件。在该条件下培养肝癌细胞SMMC-7721,细胞生长状态良好,且细胞生长与聚集形态与传统二维培养存在差异,更接近细胞体内生存环境。基于该方法进行肿瘤细胞三维培养,可为深入研究肿瘤细胞生长状态,为抗肿瘤药物筛选提供更有利的方式。     

人绒毛膜促性腺激素对人PBMC产生MIF的影响

目的:探讨人绒毛膜促性腺激素(hCG)对人外周血单核细胞(PBMC)产生巨噬细胞移动抑制因子(MIF)的影响.方法:分离正常人PBMC,体外加入不同浓度的hCG,在37 ℃ 5%CO_2条件下培养一定时间收获细胞或加入一定浓度的hCG,同样条件下培养,在不同时间收获细胞,用荧光定量PCR方法检测MIF mRNA转录水平.结果:在实验的hCG浓度范围内,MIF的转录水平随着hCG浓度增加而明显提高.在100 U/mL的hCG作用下,MIF mRNA表达在培养的1～2 h达到峰值,随后很快下降,至8 h基本回到刺激前水平.结论:hCG在一定浓度范围内有促进MIF mRNA转录的作用,且在本实验筛选浓度下,MIF mRNA转录水平与作用时间相关,提示hCG可通过促进PBMC分泌细胞因子参与一定的免疫调节作用.     

阿卡波糖产生菌原生质体的制备与再生

目的研究阿卡波糖产生菌原生质体制备与再生的条件。方法通过溶菌酶破壁的方法制备原生质体,考察影响原生质体制备与再生的因素。结果和结论确认原生质体的制备条件为:一级培养采用SM2液体培养基,培养时间为33 h,转二级培养的转种体积分数为15%;二级培养采用R2YE培养基,培养时间为20 h,甘氨酸质量分数为0.7%;溶菌酶作用质量浓度为3 g.L-1,作用时间100 min,最大原生质体制备量达到8×1010个.L-1。考察了原生质体再生培养基的组成,在优化的再生培养基上,再生率达到9.2%。     

罗丹明123联合MDR1抑制剂维拉帕米鉴别肝细胞和胆管细胞

目的 罗丹明123(Rhodamine123,Rdm123)染料通常作为染色胆管细胞一种标记染料,本实验旨在探究:a)Rdm123可否染色肝细胞,并判断多药耐药性的蛋白质(MDR1)抑制剂维拉帕米(Verapamil,Vrp)对其染色的影响;b)如何通过Rdm123染色法判断胆管细胞和肝细胞.方法 100μM的Rdm123分别染色加入或不加入Vrp孵育的人肝癌细胞系HepG2、原代大鼠正常肝细胞和原代人肝细胞原代肝细胞,对比细胞在特定时间的荧光强度变化.大鼠来源的胆管细胞的诱导采用Katsuda报道CLiP技术制作.诱导的胆管细胞分别染色加入或不加入Vrp孵育,对比细胞在特定时间的荧光强度变化.结果 Rdm123可以染色肝细胞,包括肝癌细胞(HepG2)和正常肝细胞(PHH和PRH),Vrp对其摄入和排泄无明显影响.肝细胞之间形成胆小管(BC)结构,肝细胞摄取Rdm123染料不会排泄后存留胆小管结构中.Rdm123可以染色诱导的胆管细胞结构,Vrp可以抑制其摄入排泄.结论 罗丹明123可以染色肝细胞和胆管细胞,其结合MDR1抑制剂维拉帕米可以用于鉴别肝细胞和胆管细胞.     

Study on Effects of Different Concentrations of Ozone Solution on the HBV Virus in Dental Unit Waterlines

目的:研究不同质量浓度臭氧溶液对口腔综合治疗台水道系统(DUWLs)中乙肝病毒(HBV)的灭活作用,探讨限时灭活HBV的最低臭氧溶液浓度.方法:常规培养2215细胞,在室温5 ℃、震荡混合、密闭等条件下配制不同浓度的臭氧溶液.将臭氧溶液加入2215细胞培养瓶中,作用不同时间后,取其上清液,提取HBV-DNA,聚合酶链式反应(PCR)扩增,荧光定量(FQ-PCR)检测HBV-DNA.结果:水温、密闭条件对臭氧溶液的质量浓度有影响,在上述条件下配置的臭氧溶液浓度稳定,臭氧溶液能抑制2215细胞表达HBV-DNA,臭氧浓度越高,其对HBV-DNA的灭活作用越强.臭氧浓度为0.6 mg/L 10 min及0.5 mg/L15min能将设定浓度的HBV-DNA灭活.结论:0.5～0.6 mg/L臭氧水溶液可用于杀灭口腔综合治疗台水系统中的HBV.     

荧光偏振免疫法测定血中依替米星的浓度

目的:探索应用荧光偏振免疫法测定依替米星血药浓度的实验方法,考察该方法测定依替米星的可行性.方法:使用庆大霉素荧光偏振免疫试剂盒,用TDx对依替米星进行测定.结果:本方法能够检测依替米星的血药浓度,回归曲线为Y=0.439X+0.1772,r=0.9981,在0.5～20μg·mL-1,浓度范围内线性关系良好.日内、日间标准偏差均小于10%,低、中、高不同浓度的加样回收率分别为(93.12～9,74)%,(102.41～2.73)%,(89.50～1.45)%.结论:本方法灵敏度高,操作简便,检测时间短,可以用于临床快速检测依替米星的血药浓度.     

The Effect of Silencing Nanog Gene Expression on Sensitization to Cisplatin in Esophageal Cancer Cell-Line

目的:检测干细胞基因Nanog在食管癌细胞系TE-1中的表达及其对顺铂敏感性的影响.方法:通过细胞免疫荧光检测食管癌细胞系TE-1中Nanog蛋白的表达;应用Lipofectamine 2000将荧光染料标记的Nanog siRNA转染进细胞中,通过荧光显微镜观察转染效率,再通过逆转录聚合酶链反应(RT-PCR)和Western blot检测小干扰RNA (siRNA)的沉默效率;转染后24 h加入顺铂,继续孵育48 h后测得不同转染组的半数抑制浓度(IC50),后加入相同浓度的顺铂5 mg/L,48 h后测得不同转染组的生存率.结果:Nanog蛋白在食管癌细胞系TE-1中高表达,以核表达为主;Nanog siRNA细胞转染率为(67.57±16.90)％,沉默效率可高达85％以上;转染24 h后加入顺铂,继续孵育48 h后测得Nanog siRNA组的IC50较阴性对照组和空白组相比均降低,差异有统计学意义.加入相同浓度顺铂5 mg/L,48 h后测得Nanog siRNA组细胞生存率明显低于阴性对照组和空白对照组,差异有统计学意义.结论:Nanog在食管癌细胞系TE-1中高表达,降低Nanog的表达后能够提高食管癌细胞对顺铂的敏感性.     

聚乙烯吡咯烷酮预处理培养细胞促进TAT穿膜效率

目的:探讨PVP预处理细胞对TAT穿膜效率的影响及其作为促渗剂的安全性,以优化TAT穿膜条件。方法:人工合成荧光标记短肽TAT-FITC和作为阴性对照的无意义肽NCO-FITC作用于PVP预处理各种细胞株,荧光显微镜观察TAT-FITC的穿膜效率及其定位;荧光酶标仪定量检测不同预处理的各种细胞摄取荧光标记短肽;MTT检测PVP对各种细胞的存活率及溶血实验检测其对细胞膜结构完整性的影响。结果:经PVP预处理细胞后,TAT-FITC可有效穿膜进入细胞内,MTT结果显示适当浓度的PVP对细胞活力几乎没有影响;溶血实验也证实,PVP不影响红细胞的细胞膜完整性。结论:本实验观察显示,PVP可以明显提高TAT对培养细胞的穿膜效率,但对细胞活力影响很小。     

反义核酸药物脂质体肝靶向性的研究

目的研究反义核酸药物脂质体给药系统的肝主动靶向性。方法采用薄膜分散法制备脂质体,以花青染料荧光标记脂质体,以荧光分光光度法定量分析;利用肝组织的体内冷冻切片和小鼠体内组织分布研究肝靶向性;检测大鼠肝非实质细胞与肝实质细胞的荧光强度(INPC和IPC)。结果主动靶向脂质体在肝脏中的荧光强度明显强于非主动靶向脂质体,相对摄取率(re)为5.64;非主动靶向脂质体的荧光值比(IPC/INPC)为1.16±0.89,主动靶向脂质体的IPC/INPC为8.24±1.37。结论主动靶向脂质体具有良好的肝实质细胞靶向性和肝靶向性。     

荧光染色法测定重组酵母乙型肝炎疫苗原液中残留DNA的影响因素分析

目的: 对荧光染色法测定重组酵母乙肝疫苗原液中残留DNA的影响因素进行探索分析,以了解该方法对本疫苗残留DNA检定的适用性。方法: 参照探针杂交法对乙肝疫苗原液残留DNA的测定结果,对乙肝疫苗原液中可能存在Tween-20、PEG、蛋白等物质对荧光染色法测定残留DNA含量的影响进行分析,对该方法的线性范围、用于乙肝疫苗原液残留DNA检定的准确性和重复性进行了研究,并对不同来源DNA标准存在的差异进行比较。结果: 荧光法测定酵母乙肝疫苗残留DNA线性范围为2.5 ng.mL-1~80 ng.mL-1;发现Tween-20、PEG等对残留DNA检测影响较小,加标回收率均在80%-120%之间;而蛋白质对检测影响较大,经酚-三氯甲烷抽提后可有效去除蛋白质干扰,回收率达到90%左右,CV小于10%;同时发现不同来源的DNA标准品存在荧光标记效率的差异。 结论: 乙肝疫苗原液经处理去除蛋白干扰后可采用荧光染色法进行残留DNA含量的测定,但应注意使用与疫苗表达系统相同宿主来源的DNA标准品。     

Studies on the fluorescence labeling of human red blood cell membrane ghosts with 4'-(9-acridinylamino)methanesulfon-m-anisidide

4'-(9-Acridinylamino)methanesulfon-m-anisidide (mAMSA) interacts with red cell membranes, resulting in the formation of fluorescent protein adducts. The mAMSA-membrane protein adducts exhibited an emission fluorescence maximum at 445 nm, with two shoulders at approximately 425 and 470 nm. The major labeled proteins were identified as spectrins 1 and 2 and bands 3, 4.1, 4.2 and 5. The fluorescence intensity increased with increasing mAMSA concentrations (0.03 to 1.5 mM), time (15-120 min), and temperature of the reaction. Results from sodium dodecyl sulfate gel electrophoresis show that mAMSA caused no detectable change in the molecular weight of membrane proteins. This indicates that mAMSA is a monofunctional, noncrosslinking agent. Other acridine analogs, 9-aminoacridine and acridine, did not fluorescently label membrane proteins, suggesting that the presence of the acridine nucleus is not sufficient for labeling. Addition of 2-mercaptoethanol to the mAMSA-membrane reaction mixtures reversed the fluorescence labeling. Furthermore, pretreatment of membrane proteins with N-ethylmaleimide or iodoacetamide prevented the formation of fluorescent mAMSA-membrane protein adducts. These data suggest that mAMSA interacts with sulfhydryl groups of the membrane proteins. When the membrane sulfhydryl groups were assayed by labeling with N-[ethyl-2-3H]ethylmaleimide, it was shown that the accessible membrane sulfhydryl groups were reduced after the mAMSA treatment. The above results suggest that mAMSA covalently binds to the sulfhydryl groups in the red cell membrane, with the production of fluorescent mAMSA-protein adducts.     

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3＇-processing reaction

Aim： To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3＇-processing reaction in vitro and apply it to inhibitor screening. Methods： The recombinant human irnmunodeficiency virus （HIV）-I integrase （IN） is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV- 1 long terminal repeats （LTR）. Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5＇ end and a quencher at the 3＇ end. IN cleaves the terminal 3＇-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. Results： The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3＇-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV- 1 IN, 400 nmol/L substrate, and 10 mmol/L Mn^2＋. The IN 3＇-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. Conclusion： Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3＇-processing reaction and that it can be used for inhibitor screening.     

2,3-吲哚醌对多巴胺能细胞氧化损伤的保护作用

目的采用过氧化氢(H2O2)作为损伤因素,检测2,3-吲哚醌(isatin,ISA)对MES 23.5细胞的保护作用及其机制。方法 MTT比色法检测H2O2的毒性作用及ISA的保护作用。细胞分为:对照组,H2O2 20μmol.L-1组,和ISA 100μmol.L-1+H2O2 20μmol.L-1组,采用流式细胞技术(FCM)检测线粒体膜电位(△ψm)的变化;激光扫描共聚焦显微镜(LSCM)检测细胞内Ca2+浓度([Ca2+]i)变化。结果 H2O2组细胞存活率明细降低。ISA 100μmol.L-1及以上浓度处理的细胞存活率均高于H2O2组(P<0.05)。H2O2组细胞内R123平均荧光强度较对照组明显降低而ISA组明显增强(P<0.01)。H2O2组细胞内Fluo-3荧光强度明显高于对照组(P<0.01),而ISA组细胞内荧光强度明显低于H2O2组(P<0.01)。结论 ISA可减轻H2O2导致的DA能MES 23.5细胞损伤,其机制与减轻△ψm异常,降低[Ca2+]i水平有关。     

Lead-induced changes in human erythrocytes and lymphocytes

In the present work we studied, by chemiluminescence measurements, the influence of lead on the production of reactive oxygen species (ROS) in haemolysates obtained from human erythrocytes incubated in the presence of different concentrations of lead acetate. Moreover, we evaluated the modification of proteins and lipids in human erythrocyte and lymphocyte membranes by using the fluorescence probes N-(1-pyrene)maleimide (PM), laurdan and pyrene. No significant changes in chemiluminescence were detected for erythrocytes incubated with 1-10 microM lead acetate for 3 h at 37 degrees C. By increasing the lead acetate concentration in cell suspensions up to 50 microM for the same incubation time, the percentage of chemiluminescence inhibition was ca. 20%. It was shown that, after incorporating fluorescence probes in the membrane lipid bilayer of erythrocytes and lymphocytes treated with 10 and/or 50 microM lead acetate, the total fluorescence intensity and the excimer to monomer intensity ratio of PM decreased and the generalized fluorescence polarization of laurdan decreased by 10-15%. The pyrene excimerization coefficient (kappa(ex)) increased by 20% (in comparison with a magnitude of kappa(ex) for white membranes isolated from intact erythrocytes) with 6-10 microM lead acetate for 3 h at 37 degrees C. The data obtained suggest that the effect of low concentrations of lead acetate does not cause production of ROS in erythrocytes in vitro, but can change the physicochemical state of proteins and lipids in erythrocyte and lymphocyte membranes. This effect is important because it influences the enzymatic activity and the functionality of receptors and channels present at the plasma membrane level, thus modulating the molecular composition of the intracellular space and cell functions.     

3M Covilon液体敷料在放疗体表标记应用的可行性研究

目的 利用3M液体敷料的特性,探讨其用于放疗体表标记的可行性.方法 将15例接受放射治疗的肿瘤患者随机分为观察组（n=10）和对照组（n=5）.观察组在体表标记处喷上3M液体敷料,待液体敷料完全干燥后在原位置上做记号,再喷洒3M液体敷料后待干.此操作重复2次,此后每日观察标记区的清晰程度,若模糊再补上并喷涂敷料,观察至治疗结束,记录喷涂的次数和喷涂间隔的时间,同时每日观察患者的皮肤状况;对照组标记后叮嘱患者对照射野皮肤要保持清洁、干燥,不予任何药物涂用,此后观察至标记线模糊,记录标记线保留时间,同时观察皮肤反应.结果 观察组平均耐久性为8天（范围4～ 20天）,平均需要标记的次数为4次（1～6次）.对照组平均耐久性为4天（范围2～5天）.观察组、对照组均没有不良的皮肤反应.结论 3M液体敷料对于皮肤油脂较少、年龄较低的患者效果较好.     

Real‐time visualization of oxidative stress in a floating macrophyte Lemna minor L. exposed to cadmium,copper, menadione,and AAPH

An ultra‐sensitive digital imaging system was employed to visualize oxidative stress in intact L. minor plants exposed to Cd, Cu, menadione, AAPH, and ascorbate in real time. The increase of ROS production was assessed by measuring the rate of fluorescence intensity increases of the test medium supplemented with a fluorescing probe (dichlorofluorescein diacetate). The addition of 100 μM CdCl₂ or 100 μM CuSO₄ to the growth medium resulted in a significant increase of medium fluorescence. Additionally, CuSO₄ caused a significantly higher fluorescence intensity than CdCl₂ did. A strong positive correlation (R² = 0.99) between menadione concentration and fluorescence intensity was observed. The positive correlation between AAPH concentration and fluorescence intensity was not as strong as in the case of menadione (R² = 0.81). Menadione induced a stronger oxidative stress than similar concentration of AAPH. The addition of 100 μM ascorbate to L. minor treated with 50 μM menadione significantly reduced the fluorescence intensity increase. A linear trend of the fluorescence increase was observed in all treatments, indicating that chemical‐induced oxidative stress is a gradual process and that the applied concentrations of the chemicals caused a constant increased production of ROS with different intensities, depending on the treatment. This is the combined result of a gradual diminishing of antioxidant reserves and accumulating oxidative damage. The observed rates of ROS production were slower than those in the studies using cell cultures. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 573–580, 2010.     

功能性量子点作为荧光探针测定环磷酸腺苷的含量

在温和的水溶液中合成水溶性的Mn2＋参杂的L-半胱氨酸包裹的ZnS量子点。该量子点在水溶液中能稳定存在,并与环磷酸腺苷有较强的亲和性。量子点与环磷酸腺苷相互作用产生荧光淬灭。在最优条件下,环磷酸腺苷在0.25×10-7~4.0×10-7mol.L-1浓度范围内与量子点有较好的线性关系,检出限为2.2×10-8mol.L-1。本文对常见干扰离子进行了考察,结果显示量子点对环磷酸腺苷有较强的选择性。该方法简便,快速,灵敏度高。     

Effect of ultrafiltrate volume on determination of free phenytoin concentration

Monitoring free phenytoin concentration is clinically useful for patients with uremia, hepatic disease, hypoalbuminemia, and related conditions. Free phenytoin is commonly measured by immunoassay in the protein-free ultrafiltrate prepared by centrifuging serum for 20-30 minutes, using an appropriate ultrafiltration device. We studied the effect of centrifugation time (15-40 minutes) and protein concentrations on ultrafiltration volume, and the related effects on measured free phenytoin concentrations. Temperature was ambient for all studies. The ultrafiltration volumes were directly proportional to centrifugation time and were inversely proportional to the protein concentrations. Although ultrafiltration volume significantly increased with longer centrifugation time, the measured free phenytoin concentrations did not increase proportionately. The concentration of phenytoin in the residual serum retained in the ultrafiltration device did not change proportionally either. Therefore, equilibrium of phenytoin concentrations between the ultrafiltrate and retentate was maintained, regardless of centrifugation time or protein concentration.