Fluorescent intracellular imaging of reactive oxygen species and pH levels moderated by a hydrogenase mimic in living cells

The catalytic generation of H₂ in living cells provides a method for antioxidant therapy. In this study, an [FeFe]-hydrogenase mimic [Ru + Fe₂S₂@F127(80)] was synthesized by self-assembling polymeric pluronic F-127, catalytic [Fe₂S₂] sites, and photosensitizer Ru(bpy)₃²⁺. Under blue light irradiation, hydrated protons were photochemically reduced to H₂, which increased the local pH in living cells (HeLa cells). The generated H₂ was subsequently used as an antioxidant to decrease reactive oxygen species (ROS) levels in living cells (HEK 293T, HepG2, MCF-7, and HeLa cells). Our findings revealed that the proliferation of HEK 293T cells increased by a factor of about six times, relative to that of other cells (HepG2, MCF-7, and HeLa cells). Intracellular ROS and pH levels were then monitored using fluorescent cell imaging. Our study showed that cell imaging can be used to evaluate the ability of Ru + Fe₂S₂@F127 to eliminate oxidative stress and prevent ROS-related diseases.     

Effect of Yizhitongxuan decoction on learning and memory ability,Gαq/11 expression and Na+−K+-ATP enzyme activity in rat models of Alzheimer's Disease

ObjectiveTo study the effects of Yizhitongxuan decoction on learning and memory abilities, Gαq/11 expression and Na⁺−K⁺−ATP enzyme activity in rat models of Alzheimer's disease (AD) caused by injecting Aβ_25-35 into the hippocampus.MethodsNinety male Wistar rats (age ≤10 months) were selected and injected with Aβ_25-35 into their hippocampi to establish model animals, which were randomly divided into six groups including a sham-operated group (blank group), a model group, a donepezil HCL group (Western Medicine group), and a high/general/dilute concentrations of Yizhitongxuan decoction groups (TCM I II III group). The Morris water maze was used to examine the learning and memory abilities of rats in each group by place navigation and spatial probe tests. Then, the rats were sacrificed to collect the hippocampi for biochemical tests, using western blotting to detect the expression of Gαq/11 and an ultramicro Na ⁺−K⁺−ATP enzyme kit to measure Na⁺−K⁺−ATP enzyme activity.ResultsYizhitongxuan decoction improved model rats' learning and memory abilities, and increased the expression of Gαq/11 in the hippocampus and the level of Na⁺−K⁺−ATP enzyme activity in brain tissue.ConclusionsYizhitongxuan decoction could improve model rats' learning and memory abilities, and had a regulating effect on the expression of Gαq/11 and Na⁺−K⁺−ATP enzyme activity.     

两种叶酸偶联物的合成及体外靶向性研究

合成叶酸偶联物并将其应用于叶酸靶向脂质体制备，考察其在体外肝癌HepG2细胞中的靶向性。通过酰胺反应将叶酸、胆固醇琥珀酸单酯与两种不同相对分子质量的聚氧乙烯二胺材料连接，合成两种两亲性的叶酸-聚乙二醇-胆固醇琥珀酸单酯（Folate-PEG₂₀₀₀-CHEMS和Folate-PEG₄₀₀₀-CHEMS），并利用核磁共振氢谱（¹H NMR）和超高分辨率复杂体分离鉴定质谱系统对其进行了结构表征。选取钙黄绿素为模型药物，采用薄膜分散法分别利用Folate-PEG₂₀₀₀-CHEMS和Folate-PEG₄₀₀₀-CHEMS制备了钙黄绿素脂质体FA-PEG₂₀₀₀-L与FA-PEG₄₀₀₀-L。利用激光粒度仪检测FA-PEG₂₀₀₀-L与FA-PEG₄₀₀₀-L的粒径、Zeta电位；利用流式细胞仪和激光共聚焦扫描显微镜，考察了脂质体FA-PEG₂₀₀₀-L与FA-PEG₄₀₀₀-L在HepG2细胞体外摄取实验中的药物递送效果。结果显示，钙黄绿素脂质体的平均粒径为（205.8 ± 10.2） nm，经电位测试脂质体的Zeta电位为-（1.19 ± 0.31） mV。FA-PEG₄₀₀₀-L靶向脂质体的荧光强度分别约为普通脂质体、FA-PEG₂₀₀₀-L的3.6和3.1倍（P < 0.01），FA-PEG₄₀₀₀-L在HepG2细胞中的递送效率明显高于FA-PEG₂₀₀₀-L与非靶向组，说明Folate-PEG₄₀₀₀-CHEMS可应用于脂质体制备，并可促进体外HepG2细胞对脂质体的摄取。     

Separation and expansion of tumor infiltrating lymphocytes of digestive systemin Vitro and their cytotoxicity

Summary Primary tumor tissues in the digestive system were harvested form 15 patients. By mincing, enzymatic digestion and gradient density separation, sufficient TILs (>5 × 10⁶) were obtained from 13 of 15 (88. 7 %) patientsin vitro in the presence of 500 μ/ml of recombinant interleukin-2 and 5 % fetal calf serum after one month culture. 92. 3 % (12/13) of TILs proliferated wellin vitro (92. 3%). TILs expanded from 10²-folds to 10³-folds after being cultured for one month. CD₂₅ ⁺ cell of the most fresh TILs was more than that of peripheral blood lymphocytes. CD₂₅ ⁺ cells of TILs during 4th week of the culture was significantly greater (P <0. 01) than that of fresh TILs. CD₄ ⁺/CD₈ ⁺ ratio was decreased during four culture weeks because of increase of CD₈ cells. By using modified colonmetric MTT assay for measuring activity of TILs against various tumor cells the results showed that cytotoxicity of gastro-intestinal TILs amtologous tumor cells is greater than on the other tumor cells.     

Effect of Tiotropium Bromide on Expression of CD_8~＋CD_（25）~＋FoxP_3~＋ Regulatory T Cells in Patients with Stable Chronic Obstructive Pulmonary Disease

The expression of CD8+CD25+FoxP3+ regulatory T cells(CD8+Tregs) in the peripheral blood of patients with stable chronic obstructive pulmonary disease(COPD),and the effect of muscarinic cholinergic receptor antagonist tiotropium bromide on the expression of CD8+Tregs were investigated.Twenty-three patients with moderate to severe stable COPD were enrolled in this study.All patients inhaled tiotropium bromide(18 μg daily) for 3 months.Before and after inhalation of tiotropium bromide,peripheral blood samples were collected from the patients,and T cells were labeled by three-color labeled monoclonal antibodies.Flow cytometry was used to detect the quantity and percentage of CD8+T cells,CD8+CD25+T cells,CD8+Tregs,CD4+T cells,CD4+CD25+T cells and CD4+CD25+FoxP3+ regulatory T cells(CD4+Tregs) respectively.The percentage of CD4+T cells was increased from(27.82±2.18)% to(35.53±1.3)%(t=3.20,P=0.004) in the peripheral blood of patients with stable COPD after inhalation of tiotropium bromide for 3 months,that of CD4+CD25+T cells was decreased from(10.03 ±1.42)% to(4.21 ±0.65)%(t=3.78,P=0.001),and that of CD8+Tregs was increased from(8.41 ±1.68)% to(21.34 ±4.20)%(t=2.72,P=0.013).At baseline,CD8+T cells,CD8+CD25+T cells and CD4+Tregs were detectable in the peripheral blood,but no significant changes were observed after treatment.Linear correlation analysis revealed that the difference before and after treatment in CD4+T cells and CD4+CD25+T cells was negatively correlated with the ratio of change in CD8+Tregs before and after treatment(r=-0.61,P=0.013;r=-0.72,P=0.001 respectively).In the peripheral blood of patients with stable COPD,there was the expression of CD8+Tregs and CD4+Tregs.Muscarinic receptor antagonist,tiotropium bromide,can promote the amplification of CD4+T cells,inhibit the expression of CD25+T cells,and enhance the expression of CD8+Tregs.CD8+Tregs and CD4+Tregs can be used as new indicators to understand the immune status of patients.They are helpful in judging the treatment efficacy and disease immunophenotype.     

K<Superscript>+</Superscript> channels and their effects on membrane potential in rat bronchial smooth muscle cells

Summary In order to investigate the K⁺ channels and their effects on resting membrane potential (Em) and excitability in rat bronchial smooth muscle cells (BSMCs), the components of outward K⁺ channel currents and the effects of K⁺ channels on Em and tension in rat bronchial smooth muscle were observed by using standard whole-cell recording of patch clamp and isometric tension recording techniques. The results showed that under resting conditions, total outward K⁺ channel currents in freshly isolated BSMCs were unaffected by ATP-sensitive K⁺ channel blocker. There were two types of K⁺ currents: voltage-dependent delayed rectifier K⁺ channel (K_v) and large conductance calcium-activated K⁺ channel (BK_Ca) currents. 1 mmol/L 4-aminopyridine (4-AP, an, inhibitor of K_V) caused a significant depolarization (from −8.7±5.9 mV to −25.4±3.1 mV,n=18,P<0.001). In contrast, 1 mmol/L tetraethylammonium (TEA, an inhibitor of BK_Ca) had no significant effect on Em (from −37.6±4.8 mV to −36.8±4.1 mV,n=12,P>0.05). 4-AP caused a concentration-dependent contraction in resting bronchial strips. TEA had no effect on resting tension, but application of 5 mmol/L TEA resulted in a left shift with bigger pD₂ (the negative logarithm of the drug concentration causing 50% of maximal effect) (from 6.27±0.38 to 6.89±0.54,n=10,P<0.05) in the concentration-effect curve of endothine-1, and a right shift with smaller pD₂ (from 8.10±0.23 to 7.69±0.08,n=10,P<0.05) in the concentration-effect curve of isoprenaline. It was suggested that in rat BSMCs there may be two types of K⁺ channels, K_v and BK_Ca, which serve distinct roles. K_v participates in the control of resting Em and tension. BK_Ca is involved in the regulation of relaxation or contraction associated with excitation. LIU Xiansheng, male, born in 1969, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30270583).     

Mitogenic factor for T inducer/helper cells in Entamoeba histolytica extracts

Summary In order to investigate mitogenic effect of Entamoeba histolytica extracts (EHE) on T cell subpopulations, monoclonal antibodies of the OKT system were used to separate T inducer/helper (OKT₄ ⁺) and T suppressor/cytotoxic (OKT₈ ⁺) cells. Only OKT₄ ⁺ cells were found to respond to EHE stimulation as indicated by incorporation of³HTdR into cultured cells. Determination of interleukin-2 (IL-2) activities revealed that the response of OKT₄ ⁺ cells to EHE was due to the action of IL-2.     

姜黄素与顺铂联合对人肺腺癌A549细胞增殖和凋亡作用

目的:研究姜黄素、顺铂和两者联合应用对人肺癌A549细胞增殖、凋亡和细胞周期的影响。方法:采用MTT法观察姜黄素和顺铂对人肺癌A549细胞增殖的影响;采用流式细胞仪检测姜黄素、顺铂和两者联合应用对人肺癌A549凋亡和细胞周期的影响。结果:①姜黄素及顺铂能抑制人肺腺癌A549细胞的增殖,并呈浓度及时问依赖性;姜黄素10,15,20μmol/L分别与顺铂1,2 mg/L联合24,48,72 h的抑制率显著高于单用顺铂、姜黄素组(均为P<0.05),两者呈现出相加的抗瘤效果。②姜黄素和顺铂均可在一定程度上诱导细胞凋亡,并且凋亡率与浓度呈正相关。两种药物联用后,诱导细胞凋亡作用显著增强。③姜黄素将细胞周期阻滞在G2/M期(P<0.01),顺铂将细胞周期阻滞在S期(P<0.01)。结论:姜黄素、顺铂均可抑制人肺癌A549细胞的增殖、诱导细胞的凋亡,在一定浓度范围内,呈量-效关系,而且两者联合应用具有相加或协同作用。     

Anti-oxidative and anti-inflammatory effects of Tagetes minuta essential oil in activated macrophages

Objective

To investigate antioxidant and anti-inflammatory effects of Tagetes minuta (T. minuta) essential oil.

Methods

In the present study T. minuta essential oil was obtained from leaves of T. minuta via hydro-distillation and then was analyzed by gas chromatography-mass spectrometry. The anti-oxidant capacity of T. minuta essential oil was examined by measuring reactive oxygen, reactive nitrogen species and hydrogen peroxide scavenging. The anti-inflammatory activity of T. minuta essential oil was determined through measuring NADH oxidase, inducible nitric oxide synthase and TNF-α mRNA expression in lipopolysacharide-stimulated murine macrophages using real-time PCR.

Results

Gas chromatography-mass spectrometry analysis indicated that the main components in the T. minuta essential oil were dihydrotagetone (33.86%), E-ocimene (19.92%), tagetone (16.15%), cis-β-ocimene (7.94%), Z-ocimene (5.27%), limonene (3.1%) and epoxyocimene (2.03%). The T. minuta essential oil had the ability to scavenge all reactive oxygen/reactive nitrogen species radicals with IC₅₀ 12-15 µg/mL, which indicated a potent radical scavenging activity. In addition, T. minuta essential oil significantly reduced NADH oxidase, inducible nitric oxide synthaseand TNF-α mRNA expression in the cells at concentrations of 50 µg/mL, indicating a capacity of this product to potentially modulate/diminish immune responses.

Conclusions

T. minuta essential oil has radical scavenging and anti-inflammatory activities and could potentially be used as a safe effective source of natural anti-oxidants in therapy against oxidative damage and stress associated with some inflammatory conditions.     

不同麻醉方式对腹腔镜全子宫切除2型糖尿病患者细胞免疫及血清炎症因子水平的影响

目的 探讨不同麻醉方式对腹腔镜全子宫切除2型糖尿病（T2DM）患者细胞免疫及血清炎症因子水平的影响。方法 选取2016年1月—2019年1月邯郸市中心医院收治的120例拟行腹腔镜全子宫切除术的T2DM患者,采用随机数字表法分为A、B组,每组60例。A组采用靶控输注丙泊酚和瑞芬太尼全身麻醉,B组在A组基础上复合吸入七氟醚全身麻醉。比较两组T淋巴细胞（CD3⁺、CD4⁺及CD8⁺）、NK细胞（CD3^-CD16⁺CD56⁺）、血清白细胞介素-6（IL-6）、超敏C反应蛋白（hs-CRP）、降钙素原（PCT）、IL-10水平及术中不良反应。结果 两组麻醉前（T₀）、建立气腹前（T₁）、建立气腹后90 min（T₂）、术后第1天（T₃）及术后第3天（T₄）CD3⁺、CD4⁺、CD8⁺、CD3^-CD16⁺CD56⁺和IL-6、hs-CRP、PCT、IL-10水平比较,在不同时间、不同组间及变化趋势上有差异（P <0.05）。B组苏醒时间、拔管时间短于A组（P <0.05）。两组麻醉维持时间、术中恶心呕吐、牵拉反应及低血压发生率比较,差异无统计学意义（P >0.05）。结论 靶控输注丙泊酚和瑞芬太尼复合七氟醚吸入全身麻醉可降低全子宫切除术T2DM患者围手术期血清炎症因子水平,保护细胞免疫功能,且不增加术中不良反应,具有一定的临床应用价值。     

Changes in T cell subsets and T suppressor cell function and their relationship in human schistosomiasis japonica

Summary The function of spontaneous T suppressor cell (STs) of peripheral blood was examined in 56 patients with schistosomiasis japonica at various stages. The subsets of T cell were simultaneously phenotyped in 46 cases. The percentages of CD₃ ⁺(pan T cell), CD₄ ⁺ (helper/inducer T cell) and CD₈ ⁺ (suppressor/cytotoxic T cell) in patients with acute schistosomiasis japonica were significantly higher than those in the normal controls. In patients with chronic and advanced schistosomiasis japonica, the percentage of CD₈ ⁺ T cell and the function of STs were greatly increased, but the percentage of CD₃ ⁺ T cell and the ratio of CD₄ ⁺/CD₈ ⁺ were obviously reduced. All of these markers changed more significantly in patients with advanced schistosomiasis japonica. The percentage of CD₈ ⁺ T cell in patients with acute schistosomiasis japonica was negatively correlated with the function of STs. In patients with chronic and advanced schistosomiasis japonica the percentage of CD₈ ⁺ T cell was correlated positively and the ratio of CD₄ ⁺/CD₈ ⁺ negatively with the function of STs. The results indicated that the cellular immunity was significantly increased in cases of acute schistosomiasis japonica and decreased in those with chronic or advanced schistosomiasis japonica. The increased CD₈ ⁺ T cell may be principally cytotoxic T cells in patients with acute schistosomiasis japonica, but suppressor T cells in patients with chronic and advanced schistosomiasis japonica. The subsets of T cells and the function of T suppressor cells may play an important role in the immunoregulation of schistosomiasis japonica.     

Use of dentritic cells pulsed with HLA-A2-restricted MAGE-A1 peptide to generate cytotoxic T lymphocytes against malignant glioma

This study developed a novel approach of targeting malignant glioma with pMAGE-A1278-286-specific cytotoxic T lymphocytes (CTLs) induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen (HLA)-A2-restricted pMAGE-A1278-286 peptide-pulsed dentritic cells.Cytotoxic assays were performed by the colorimetric CytoTox 96 assay to analyze cytotoxic activity of the induced CTLs against various target cells.The induced CTLs showed approximately 45% specific lysis against T2pMAGE-A1278-286 (pMAGE-A1278-286 peptide pulsed T2 cells) and U251 (HLA-A2+, MAGE-A1+) at an effector:target ratio of 40:1, and approximately 5% cytolysis against T2pHIV, A172 (HLA-A2-, MAGE-A1+), K562 and T2 cells without being pulsed with peptide at any effector:target ratio.The specific killing activity of the induced CTLs against T2pMAGE-A1278-286 and U251 was much more obvious than in any other control group (P<0.05).The cytotoxic activity against the T2pMAGE-A1278-286 and U251 was significantly eliminated by anti-HLA class Ⅰ mAb W6/32.These results suggest that pMAGE-A1278-286 epitope may serve as a surrogate tumor antigen target of specific immunotherapy for treating HLA-A2 patients with malignant glioma.     

罗丹宁衍生物的合成及抗肿瘤活性

在罗丹宁衍生物WL-276的基础上设计并合成一系列新的罗丹宁衍生物,并对这些化合物的抗肿瘤活性进行测定。以氨基酸为原料,经环合和缩合反应,合成了化合物Ⅱ_1-4,然后分别与硫化氢供体ADT-OH偶联得到化合物Ⅲ_1-4,共合成了8个目标化合物,其中4个未见报道,其结构均经¹H NMR、IR和HR-MS确证。然后用MTT法筛选其抗肿瘤活性。初步研究表明,化合物Ⅱ_1,3,4和Ⅲ_1-4对HepG2肿瘤细胞和DU145肿瘤细胞的增殖均具有较强的抑制作用,且化合物Ⅲ的活性比Ⅱ强;化合物Ⅲ_2,4对HepG2细胞和化合物Ⅲ_1,2,4对DU145细胞的抗增殖活性均高于阳性对照5-氟尿嘧啶。     

小鼠肝脏中细胞角蛋白19阳性细胞的肝向分化特性

目的 明确小鼠肝脏中的细胞角蛋白19 (cytokeratin 19,CK19)阳性(CK19+)细胞是否可分化为成熟肝细胞.方法 利用CK19CreERT小鼠和Rosa26-GFP小鼠杂交得到CK19CreERT/Rosa26-GFP双转基因小鼠,注射他莫昔芬(tamoxifen,TM)后检测小鼠肝脏中CK19+细胞的GFP标记情况.在此基础上分别构建3,5-二乙氧基羰基-1,4-二氢-2,4,6-三甲基吡啶和四氯化碳(carbon tetrachloride,CCl4)肝脏损伤模型,采用肝脏组织冰冻切片结合免疫荧光染色检测肝脏中GFP标记的CK19+细胞的分化情况.结果 获得CK 19CreERT/Rosa26-GFP双转基因小鼠,免疫荧光染色结果显示CK19+细胞可被GFP标记.在DDC肝脏损伤模型小鼠中检测到增生性小胆管内有GFP+细胞,这些GFP+细胞表达胆管上皮细胞标志物CK19,且DDC肝损伤模型小鼠中GFP+胆管细胞比例高于未损伤对照组[(63.5±6.3)％ vs (53.6±4.8)％,P＜0.05];在CCl4肝损伤模型组小鼠中检测到肝脏实质细胞中有GFP+细胞,这些GFP+细胞表达成熟肝细胞标志物白蛋白(ALB),且CCl4肝损伤模型组小鼠肝脏中GFP+细胞比例高于未损伤对照组[(0.15±0.02)％vs (0.008±0.003)％,P＜0.01].结论 小鼠肝脏内的CK19+细胞群体中存在具有肝向分化潜能的前体细胞,可能为真正的肝干细胞的识别鉴定提供一个新线索.     

钌配合物[Ru（MeIm）4（bpy）]2+的合成及对人肝癌细胞株增殖的影响

目的合成钌配合物[Ru(MeIm)4(bpy)]2+并初步探讨其体外抗肝癌活性。方法利用元素分析、电喷雾质谱(ESI-MS)、核磁共振(NMR)等对目标化合物进行表征并通过溴化乙啶(EB)竞争结合实验检测其与DNA的结合能力;采用MTT法检测其对3株人肝癌细胞株HepG2、HCC-LM3和MHCC-97L细胞增殖的影响,用Hoechst33342染色法观察细胞凋亡的形态学改变,以流式细胞仪观察药物作用后细胞的凋亡率和细胞周期的变化。结果合成并表征了金属钌配合物[Ru(MeIm)4(bpy)]2+,同时竞争结合实验表明其能取代EB结合DNA。该化合物可抑制HepG2、HCC-LM3和MHCC-97L的增殖,呈浓度和时间依赖性。该化合物对HepG2细胞的增殖抑制作用最为明显,且浓度依赖性地诱导HepG2细胞凋亡,并随着作用时间的延长Sub-G1凋亡峰越增加。31.25～125μg/ml钌配合物阻滞细胞于G0/G1期,而浓度达250μg/ml时则阻滞细胞于G2/M期。结论合成的目标产物钌配合物[Ru(MeIm)4(bpy)]2+在体外可抑制人肝癌HepG2细胞的生长并能诱导其发生凋亡。     

Gene transfection mediated by ultrasound and Pluronic P85 in HepG2 cells

Summary In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irradiated by ultrasound at 1 MHz, 0.4–2.0 W/cm² and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm² for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm² for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound. This project was supported by a grant from National Natural Sciences Foundation of China (No.30670620).     

Peripheral blood and bone marrow lymphocyte subsets in patients with multiple myeloma

Summary A panel of monoclonal antibodies (McAb) from the Wu series was used to characterize lymphocyte subsets of peripheral blood (PB) from 9 patients with multiple myeloma (MM) by means of indirect immunofluorescence assay. In 7 of these patients bone marrow (BM) cells were also studied. Abnormal distribution of T lymphocyte subsets was observed in these patients, and the alterations were quite similar in both PB and BM. A significant increase in the proportion of WUT ₈ ⁺ cells (P<0.00l) and a reduced WuT₄/WuT₈ ratio (P<0.001) were found in patient group. In both PB and BM of patients, WuT ₁₀ ⁺ cell counts were significantly higher than normal (P<0.00l). There was a good correlation between BM WuT ₁₀ ⁺ cells and BM myeloma cells (r= 0.76, P<0.05), whereas no correlation was found between PB WuT ₁₀ ⁺ cells and BM myeloma cells. The significance of abnormal T-cell subset distribution and elevated WuT ₁₀ ⁺ cells in MM is discussed.     

HIF-1α516对人肝癌HepG2细胞侵袭转移能力的影响

目的观察HIF-1α516对人肝癌HepG2细胞侵袭转移能力的影响。方法人肝癌HepG2细胞分为空白组(同步培养,不做任何处理)、对照组(转染siRNA)和siRNA干扰组(转染HIF-1α516 siRNA)。siRNA和HIF-1α516 siRNA通过脂质体转染HepG2,转染18h后Western blot检测HepG2细胞中HIF-1β核蛋白和全细胞蛋白的表达、ELISA法检测细胞培养上清中MMP-2、MMP-9的含量,Transwell小室观察HepG2的转移侵袭能力。结果 HIF-1α516 siRNA能显著降低HepG2细胞HIF-1α516mRNA的表达(降低70.0%)(P<0.01);增强HepG2细胞的侵袭转移能力(增强71.10%)(P<0.01)。HIF-1α516siRNA对全细胞HIF-1β蛋白的表达无影响,但能促进HIF-1β蛋白的核转移(增强64.1%)(P<0.01)。HIF-1α516 siRNA能明显升高HepG2细胞培养上清中MMP-2、MMP-9蛋白表达,分别升高37.72%、55.46%(P<0.01)。结论 HIF-1α516能抑制HepG2的转移侵袭,其机制可能与抑制HIF-1β的核转移有关。     

Analysis of the inhibitory effect of gypenoside on Na+, K+-ATPase in rats’ heart and brain and its kinetics

Objective： To study the effects of gypenoside （Gyp） on the activity of microsomalNa^＋, K^＋-ATPase in rat＇s heart and brain in vitro. Methods： The microsomal Na^＋, K^＋-ATPase was prepared from rat＇s heart and brain by differential centrifugation. The activity of microsomal Na^＋, K^＋-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na^＋, K^＋-ATPase of rats. Results： Gyp reversibly inhibited the brain and heart＇s microsomal Na^＋, K^＋-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07± 6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na^＋, or K^＋-concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na^＋, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K^＋. Cenclusien： Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain＇s microsomal Na^＋, K^＋-ATPase activities in rats.     

Targeted MR Imaging Adopting T1-Weighted Ultra-Small Iron Oxide Nanoparticles for Early Hepatocellular Carcinoma: An in vitro and in vivo Study

ObjectiveThe purpose of this study was to produce an arginylglycylaspartic acid (RGD) peptide-modified ultra-small superparamagnetic iron oxide (Fe₃O₄) nanoparticles (NPs) for targeted magnetic resonance (MR) imaging of hepatocellular carcinoma (HCC) cells and verify its utility as a T1 positive MRI imaging contrast agent in vitro and in vivo.MethodsThe carboxylated Fe₃O₄ NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD nanoparticles to form a novel target contrast agent Fe₃O₄-PEG-RGD NPs. The specificity of Fe₃O₄-PEG-RGD to bind RGD receptor was investigated in vitro by HepG2 cellular uptake and cell MR imaging, and in vivo by MR imaging of subcutaneous HepG2 tumors of nude mice.ResultsThe formed Fe₃O₄-PEG-RGD NPs displayed good biocompatibility, and the ultrahigh r1 relaxivity was 1.37 mM⁻¹S⁻¹. The synthesized Fe₃O₄-PEG-RGD NPs were demonstrated spherical-like with an approximate diameter of 2.7 nm in similar size. The targeting effect to HepG2 cells was confirmed by in vitro cellular uptake and cell MR imaging. The in vivo MR imaging of nude mice demonstrated that the MR signal intensity enhancement of HepG2 tumor in Fe₃O₄-PEG-RGD NPs treated mice was significantly higher than in mice treated with non-targeted Fe₃O₄-mPEG NPs at the same post-administration time point.ConclusionThe results indicate that the Fe₃O₄-PEG-RGD particles have potential utility as T1 positive contrast agent in targeted MR imaging.