TFR和VEGF基因双启动子调控的慢病毒载体构建及其表达研究

目的:构建含有转铁蛋白受体(TFR)和血管内皮生长因子(VEGF)的双启动子慢病毒载体,体外转染中华小型猪骨髓间充质细胞并检测其表达.方法:PCR法分别扩增TFR和VEGF基因,分别克隆人pLenti-GFP-Neo慢病毒载体的CMV启动子和SV40启动子后,构建出双启动子调控的慢病毒表达载体pLenti-TG-VEGF.利用Lipofectin2000试剂将pRsv-REV、pMDlg-pRRE、pMD2G共转染293T细胞进行慢病毒包装,72 h后收集病毒上清,感染中华小型猪骨髓间充质细胞,并通过Western blot法检测TFR和VEGF的表达情况.结果:成功构建了含有TFR和VEGF基因的双启动子慢病毒载体;包装好的慢病毒颗粒可成功感染中华小型猪骨髓间充质细胞;Western blot法检测TFR和VEGF高水平的表达.结论:成功构建了双启动子调控的慢病毒载体pLenti-TG-VEGF,并建立其慢病毒表达系统,从而为进一步探讨活体内观察移植细胞携带治疗基因的MRI基因成像应用的可行性奠定了基础.     

Dietary fat level affects tissue iron levels but not the iron regulatory gene HAMP in rats

Because dietary fats affect the regulation and use of body iron, we hypothesized that iron regulatory and transport genes may be affected by dietary fat. A model of early-stage I to II, nonalcoholic fatty liver was used in which rats were fed standard (35% energy from fat) or high-fat (71% energy from fat) liquid diets with normal iron content (STD/HF groups). In addition, intraperitoneal injections of iron dextran were given to iron-loaded (STD+/HF+ groups) and iron-deficient diets to STD−/HF− groups. Plasma osmolality, hemoglobin level, and mean corpuscular hemoglobin concentration were increased in all STD diet groups compared with all HF diet groups. Plasma iron and transferrin saturation were affected by an interaction between dietary fat and iron. They were high in the STD group (normal iron) compared with their respective HF group. Similarly, this group also showed a 4-fold increase in the messenger RNA expression of the hepatic hemochromatosis gene. Spleen iron was high in the iron-loaded STD+ group compared with all other groups. Hepatic iron and messenger RNA expression of peroxisome proliferator–activated receptor-γ, CCAAT/enhancer binding protein α, interleukin-6, and iron transport genes (transferrin receptor 2, divalent metal transporter 1 iron-responsive element, and divalent metal transporter 1 non–iron-responsive element) were increased, whereas tumor necrosis factor α was decreased in the HF diet groups. The expression of iron regulatory gene HAMP was not increased in the HF diet groups. Iron regulatory and transport genes involved in cellular and systemic iron homeostasis may be affected by the macronutrient composition of the diet.     

映山红总黄酮对脑缺血大鼠ERK通路的影响

目的:观察映山红总黄酮(TFR)对大鼠全脑缺血-再灌注损伤是否具有保护作用,以及保护作用是否与ERK信号转导通路有关。方法:采用四血管结扎法建立大鼠全脑缺血-再灌注损伤模型。记录脑缺血前、缺血10min和再灌注后5、10、15、30、45、60 min的脑电图改变;采用蛋白免疫印迹方法,测定大鼠脑皮质和海马组织中ERK1/2和p-ERK1/2在脑缺血-再灌注后表达的变化,以及TFR对此变化的影响。结果:脑缺血时大鼠的脑电图幅度迅速下降,再灌注后脑电幅度逐渐恢复。再灌注45 min和60 min时,映山红总黄酮各剂量组脑电图幅度与模型组比较差异显著(P0.05或P0.01)。蛋白印迹结果显示,与模型组比较,映山红总黄酮各剂量组p-ERK1/2的表达显著性升高(P0.05)。结论:TFR对大鼠全脑缺血-再灌注损伤有保护作用,其机制可能与增加大鼠脑组织中ERK1/2的激活有关。     

Iron overload and prolonged ingestion of iron supplements: clinical features and mutation analysis of hemochromatosis-associated genes in four cases

We evaluated and treated four white adults (one man, three women) who had iron overload associated with daily ingestion of iron supplements for 7, 15, 35, and 61 years, respectively. We performed HFE mutation analysis to detect C282Y, H63D, and S65C in each patient; in two patients, HFE exons were sequenced. In two patients, direct sequencing was performed to detect coding region mutations of TFR2, HAMP, FPN1, HJV, and ALAS2. Patients 1-4 ingested 153, 547, 1,341, and 4,898 g of inorganic iron as supplements. Patient 1 had hemochromatosis, HFE C282Y homozygosity, and beta-thalassemia minor. Patient 2 had spherocytosis and no HFE coding region mutations. Patient 3 had no anemia, a normal HFE genotype, and no coding region mutations in HAMP, FPN1, HJV, or ALAS2; she was heterozygous for the TFR2 coding region mutation V583I (nt 1,747 G-->A, exon 15). Patient 4 had no anemia and no coding region mutations in HFE, TFR2, HAMP, FPN1, HJV, or ALAS2. Iron removed by phlebotomy was 32.4, 10.4, 15.2, and 4.0 g, respectively. There was a positive correlation of log(10) serum ferritin and the quantity of iron removed by phlebotomy (P = 0.0371). Estimated absorption of iron from supplements in patients 1-4 was 20.9%, 1.9%, 1.1%, and 0.08%. We conclude that the clinical phenotypes and hemochromatosis genotypes of adults who develop iron overload after ingesting iron supplements over long periods are heterogeneous. Therapeutic phlebotomy is feasible and effective, and would prevent complications of iron overload.     

Novel model of antigen-specific induction of bile duct injury

BACKGROUND & AIMS: Biliary-directed inflammation is an important cause of acute and chronic liver disease. We developed and characterized a transgenic mouse model of immune-mediated hepatobiliary injury. METHODS: Ovalbumin (OVA)-BIL mice were developed using 3.0 kilobase of the rat apical sodium-dependent bile acid transporter promoter to drive aberrant expression of a membrane form of ovalbumin (OVA) on biliary epithelium. Liver inflammation resulted from adoptive transfer of OVA-specific T cells. Liver immune cells were characterized to determine the mechanism of the response by assessing activation, proliferation, and intracellular cytokine expression. RESULTS: OVA-BIL transgenic mice were tolerant to OVA, without evidence of liver disease. Adoptive transfer of OVA-specific CD4+ and CD8+ T cells into na?ve OVA-BIL mice led to biliary-centered necroinflammatory damage in a dose-dependent manner. This inflammation absolutely required CD8+ T cells and was augmented by CD4+ T cells. Adoptively transferred OVA CD8+ cells homed to and proliferated in the liver but not the spleen. These activated, adoptively transferred cytotoxic T lymphocytes produced elevated levels of tumor necrosis factor alpha and interferon gamma. CONCLUSIONS: T-cell recognition of antigen aberrantly expressed on bile duct epithelium induced an acute necroinflammatory response specific to the liver, with activation, proliferation, and cytokine production predominantly by the OVA-specific cytotoxic T cells. Thus, OVA BIL represents an antigen-specific animal model of inflammatory bile duct injury.     

Early onset hereditary hemochromatosis resulting from a novel TFR2 gene nonsense mutation (R105X) in two siblings of north French descent

The molecular basis of hereditary hemochromatosis (HH) is more complex than previously expected. More than 80% of hemochromatosis probands of Northern European descent are homozygous for the C282Y HFE gene mutation. However, five novel non-related-HFE HH forms have now been identified. The transferrin receptor(TFR2)-linked form is inherited in an autosomal recessive pattern and is considered to be an adult-onset syndrome. Until now, it has been associated with five mutations that have only been detected in Japanese and southern European patients. Here, we report the identification of a novel TFR2 nonsense mutation in two related French adolescents. We discuss the phenotype of this sibling pair from precedent biological and clinical findings as well as the expected role of TFR2 in iron homeostasis. Finally, we suggest that iron overload phenotypes associated with mutations in TFR2 may be intermediate between those related to mutations in HFE and those related to mutations in juvenile hemochromatosis genes.     

Spectral pattern similarity analysis: Tutorial and application in developmental cognitive neuroscience

The human brain encodes information in neural activation patterns. While standard approaches to analyzing neural data focus on brain (de-)activation (e.g., regarding the location, timing, or magnitude of neural responses), multivariate neural pattern similarity analyses target the informational content represented by neural activity. In adults, a number of representational properties have been identified that are linked to cognitive performance, in particular the stability, distinctiveness, and specificity of neural patterns. However, although growing cognitive abilities across childhood suggest advancements in representational quality, developmental studies still rarely utilize information-based pattern similarity approaches, especially in electroencephalography (EEG) research. Here, we provide a comprehensive methodological introduction and step-by-step tutorial for pattern similarity analysis of spectral (frequency-resolved) EEG data including a publicly available pipeline and sample dataset with data from children and adults. We discuss computation of single-subject pattern similarities and their statistical comparison at the within-person to the between-group level as well as the illustration and interpretation of the results. This tutorial targets both novice and more experienced EEG researchers and aims to facilitate the usage of spectral pattern similarity analyses, making these methodologies more readily accessible for (developmental) cognitive neuroscientists.     

杜鹃花总黄酮对豚鼠心室肌细胞生物电的影响

目的观察杜鹃花总黄酮(total flavones of rhododen-dron,TFR)对离体豚鼠右心室乳头肌细胞生物电的影响,以探讨其抗心肌缺血的作用机制。方法采用标准玻璃微电极技术记录心肌细胞的静息电位以及动作电位。结果25、50mg·L-1的TFR可明显缩短动作电位复极50%、90%时程(APD50、APD90),但100、200、400mg·L-1却可明显延长APD50和APD90,200、400mg·L-1的TFR可明显降低0期的动作电位峰值(APA)及动作电位去极化最大速率(Vmax),400mg·L-1的TFR使静息电位减小,200mg·L-1TFR能延长心室肌细胞有效不应期(ERP)。结论低浓度的TFR可能有钙阻滞作用,高浓度的TFR可能对钾通道有一定的阻断作用,200mg·L-1TFR能延长心室肌细胞有效不应期。     

滤泡辅助性T细胞和滤泡调节性T细胞在骨肉瘤患者中的数量变化及临床意义

目的探讨滤泡辅助性T细胞(TFH)和滤泡调节性T细胞(TFR)在骨肉瘤患者外周血中的数量变化及与预后的关系。方法收集2015年1月至2018年12月20例骨肉瘤患者和20例健康体检者的静脉血,采用流式细胞术检测骨肉瘤患者和健康体检者外周血循环TFH细胞和TFR细胞频率,分析骨肉瘤患者不同年龄段、不同性别以及不同分期上述指标及TFH/TFR细胞频率比值。将外周血循环TFH/TFR细胞频率比值按中位值对骨肉瘤患者进行分层,并根据随访数据分析总生存期(OS)。结果流式细胞术检测结果显示,20例骨肉瘤患者外周血TFH细胞频率[(6.916±1.778)%vs.(2.310±0.874)%,P=0.004)]和TFR细胞频率[(3.016±0.760)%vs.(0.472±0.174)%,P=0.002)]均显著高于健康体检者。骨肉瘤患者TFH/TFR细胞频率比值为2.679±1.246,低于健康体检者的5.830±2.464(P=0.005)。10~25岁患者的外周血TFH细胞和TFR细胞频率明显高于其他年龄段(P<0.05),Ⅲ期患者外周血TFH细胞和TFR细胞频率明显高于Ⅰ期和Ⅱ期患者(P<0.05)。男性和女性骨肉瘤患者外周血TFH细胞和TFR细胞频率的差异无统计学意义(P>0.05)。以20例骨肉瘤患者外周血循环TFH/TFR细胞频率比值的中位数2.362为界值,分为低比值组(≤2.362)和高比值组(>2.362)各10例。20例骨肉瘤患者的中位OS为27个月。其中低比值组中位OS为19个月,低于高比值组的32个月,差异有统计学意义(P=0.036)。结论在骨肉瘤致病过程中,TFH细胞和TFR细胞频率显著升高,与年龄和分期有关。