肝硬化患者红细胞Ⅰ型补体受体基因点突变与数量表达的变化

目的：动态观察肝炎肝硬化患者红细胞Ⅰ型补体受体基因点突变与数量表达及其天然免疫黏附功能(erythrocyte cell nature-immune-adhesion function,RNIAF)的变化，探讨它们在评估肝炎肝硬化病情严重程度中的应用价值。方法：分别检测134例肝炎肝硬化不同分级的Ⅰ型补体受体基因点突变与数量表达及其RNIAF的变化。结果：与正常人群相比较，肝硬化患者I型补体受体基因点突变率无统计学差异，而肝硬化Child分级各组的CRI数量表达及RNIAF都明显低于正常人群组，且随着分级病情严重，CRI、RNIAF逐渐降低。结论：红细胞Ⅰ型补体受体活性及天然免疫黏附功能可灵敏地反映肝炎肝硬化患者病情的发展，红细胞CRI及RNIAF。可作为判断肝炎肝硬化病情严重程度的重要参考指标。     

Efficacy and safety of apatinib combined with whole-brain radiation therapy with a simultaneous integrated boost for brain metastases from non-small cell lung cancer: a multicenter retrospective study

BackgroundBrain metastases (BMs) develop in 20–65% of non-small cell lung cancer (NSCLC) patients and are associated with a poor prognosis. Apatinib, a tyrosine kinase inhibitor (TKI) that selectively inhibits the vascular endothelial growth factor receptor 2, is safe and significantly prolongs the survival of chemotherapy-refractory gastric cancer patients. This retrospective study evaluated the safety and efficacy of apatinib combined with concurrent brain radiotherapy in NSCLC patients with BMs.MethodsThis trial enrolled patients with non-recurrent BM from histologically-confirmed NSCLC without any limits regarding the BM size/quantity. Eligibility criteria were patients 18–75 years old with measurable BM from histologically-confirmed NSCLC (including both newly-diagnosed and previously treated NSCLC) and expected survival time greater than 3 months. Oral apatinib (500 or 250 mg/day) was started within 1 week prior to commencing whole brain radiotherapy with simultaneous integrated boost (WBRT-SIB) and continued until one week after radiotherapy completion. In addition to toxicities, analyzed outcomes included intracranial overall response rate (iORR), intracranial disease control rate (iDCR), intracranial progression free survival (iPFS), and overall survival (OS).ResultsFrom July 2016 to January 2020, 16 patients were enrolled in this retrospective study. After 3 months of brain radiotherapy, the iORR was 75%, the iDCR was 100%, and the brain edema index (EI) was significantly reduced compared to that before brain radiation therapy (4.2 vs. 1.9; P=0.02). The median iPFS was 16.5 months [95% confidence interval (CI): 15.1–37.4 months]. The median OS was 26 months (95% CI: 17.0–54.0 months). Most of the patients tolerated apatinib well, but 7 patients had side effects, most commonly grade 1 or 2. Only 2 patients experienced grade 3 adverse events (hypertension and oral mucositis), and no grade 4 or 5 toxicities were observed.ConclusionsApatinib combined with WBRT-SIB appears to be safe and effective in treating BMs in NSCLC patients.     

Dietary Linolenic Acid Increases Sensitizing and Eliciting Capacities of Cow’s Milk Whey Proteins in BALB/c Mice

α-Lactalbumin (BLA) and β-lactoglobulin (BLG) are the major whey proteins causing allergic reactions. Polyunsaturated fatty acids (PUFAs) stand among the extrinsic factors of the food matrix that can bind BLA and BLG and change their bioactivities, but their contribution to change the allergenic properties of these proteins has not been investigated. Here, we aimed to determine how PUFAs influence BLA and BLG to sensitize and trigger allergic responses in BALB/c mice. First, tricine–SDS–PAGE and spectroscopic assays identified that α-linolenic acid (ALA, as a proof-of-concept model) can induce BLA and BLG to form cross-linked complexes and substantially modify their conformation. Then, BALB/c mice (n = 10/group) were orally sensitized and challenged with BLA and BLG or ALA-interacted BLA and BLG, respectively. Allergic reactions upon oral challenge were determined by measuring clinical allergic signs, specific antibodies, levels of type-1/2 cytokines, the status of mast cell activation, and percentage of cell populations (B and T cells) in different tissues (PP, MLN, and spleen). Overall, systemic allergic reaction was promoted in mice gavage with ALA-interacted BLA and BLG by disrupting the Th1/Th2 balance toward a Th2 immune response with the decreased number of Tregs. Enhanced induction of Th2-related cytokines, as well as serum-specific antibodies and mast cell activation, was also observed. In this study, we validated that ALA in the food matrix promoted both the sensitization and elicitation of allergic reactions in BALB/c mice.     

阻断磷脂酶C-γ1信号通路对大肠癌细胞株LoVo细胞增殖与凋亡的影响

背景与目的:磷脂酶C-γ1(phospholipase C gamma 1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系.本研究主要探讨阻断PLC-γ1信号通路后对大肠癌LoVo细胞增殖、凋亡的影响,及其上述影响的信号机制.方法:以人大肠癌LoVo细胞作为研究模型,利用PLC-γ1特异的化学阻断剂U73122处理以阻断LoVo细胞中PLC-γ1信号通路,通过绘制细胞生长曲线、PI单染的流式细胞仪检测细胞周期而评估对其细胞增殖的影响,通过细胞形态观察及DNA片段琼脂糖凝胶电泳评估是否启动细胞凋亡,同时检测阻断PLC-γ1信号通路后HSP70、Caspase-3表达水平的变化来探讨可能的信号机制.结果:阻断PLC-γ1信号通路明显减缓大肠癌LoVo细胞的生长,其细胞增殖抑制率随药物作用时间和浓度的增加逐渐增高,10 μmol/L U73122作用24、48 h后其抑制效果可分别达到35%和45%,使LoVo细胞G1期细胞比例增加,而S期细胞比例降低,延缓细胞从G1期向S期的过渡,即抑制细胞周期的进行,阻断PLC-γ1信号通路后LoVo细胞未能出现凋亡特征性的形态学改变,DNA琼脂糖凝胶电泳未能检测到凋亡特征的梯状带的出现,不能引起Caspase-3的激活,同时PLC-γ1信号通路的阻断可上调HSP70的表达水平,HSP70的分子伴侣作用可能是其抑制大肠癌细胞周期进行的机制.结论:阻断磷脂酶C-γ1信号通路能够抑制大肠癌LoVo细胞的过度增殖、抑制其细胞周期的进行,其机制可能是通过上调热休克蛋白70的表达水平而实现,但不能启动LoVo细胞凋亡,磷脂酶C-γ1不是调控LoVo细胞凋亡的关键信号分子.     

不同转移潜能鼻咽癌细胞钙电流特征与细胞迁徙能力的相关性

背景与目的:肿瘤转移活性与瘤细胞以迁徙运动潜能为代表的生物力学特性有关,而其迁移运动的生物力学机制又受制于细胞内的钙离子活动和钙电流特征.本文探讨不同转移潜能鼻咽癌单克隆亚系细胞钙电流特征,及与细胞迁徙运动潜能的相关性.方法:人鼻咽癌高转移潜能细胞株5-8F及低转移潜能细胞株6-10B分别与10%含药血清(由黄芪、党参、白花蛇舌草等组成)共同培养,MTT法检测含药血清对细胞增殖的影响,Western blot检测nm23-H1蛋白表达水平.利用全细胞膜片钳制方法观察细胞钙电流特征(药物作用20 min),划痕实验观察细胞迁移能力(药物作用24 h),比较分析其相关性.结果:6-10B细胞的nm23-H1表达水平(2.9±0.4)较5-8F细胞(2.3±0.21)高(P＜0.05).5-8F细胞内钙释放激活的钙电流ICRAC为(-1.39±0.36)nA,与6-10B细胞lCRAC[(-0.66±0.40)nA]相比差异有统计学意义(P＜0.05).通过划痕区的5-8F细胞数(350±3)也显著高于6-10B细胞(246±1)(P＜0.05).应用含药血清干预后,5-8F和6-10B细胞增殖活性差异无统计学意义(P＞0.05),5-8F细胞的nm23-H1表达水平(3.9±0.1)明显高于6-10B细胞(1.0±0.1)(P＜0.05).干预后,5-8F细胞ICRAC降至(-1.27±0.35)nA,平均抑制幅度为(1.90±0.47)%;6-10B细胞则降至(-0.37±0.23)nA,平均抑制幅度为(0.46±0.12)%,两者差异有统计学意义(P＜0.05),并出现与钙电流变化特点平行的细胞迁移能力变化,通过划痕线的5-8F细胞数明显减少(94±6),而6-10B细胞则受影响甚小(229±6,P＜0.05).干预前后5-8F细胞迁移能力变化具有显著性(P＜0.05),6-10B细胞则无显著性差异(P＞0.05).结论:不同转移潜能鼻咽癌细胞钙电流特征及细胞迁移能力以及nm23-H1表达存在明显差异.应用含药血清干预后,高转移潜能细胞5-8F出现了与细胞钙电流ICRAC降低幅度相平行的细胞迁移能力抑制,药物血清可增加5-8F细胞的nm23-H1活性.     

Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection

The ability to measure many single molecules simultaneously in large and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM³) to circumvent the above limits. In SSM³, single-molecule binding processes were dynamically recorded by surface plasmon resonance microscopy in a nanoparticle-mediated sandwich scheme. The binding kinetics between analyte and probes are fine-tuned by nanoparticle micromanipulations to promote the repetitive binding and dissociation. Quantifying the heterogeneous lifetime of each molecular complex allows the discrimination of specific binding from nonspecific background noise. By digitally counting the number of repetitive specific binding events, we demonstrate the direct detection of microRNAs and amyloid-β proteins with the limit of detection at the subfemtomolar level in buffer and spiked human serum. Together with the nanoparticle micromanipulation to promote the transportation rate of analyte molecules, the assay could be performed within as short as 15 min without the need for preincubation. The advantages over other single-molecule sensors include short assay time, compatible with common probes and ultrasensitive detection. With further improvement on the throughput and automation, we anticipate the proposed approach could find wide applications in fundamental biological research and clinical testing of disease-related biomarkers.

The analytical methods have converged from ensemble measurements of numerous entities to quantized measurements at the single-molecule level. Single-molecule measurements could reveal heterogeneities and stochastic processes within biological systems (1, 2) and set the ultimate detection limit of chemical and biological sensors. By reducing the measurement volume to a few femtoliters, the detection of a single molecule has been realized in various forms [i.e., single-molecule fluorescence (3, 4), nanopores (5, 6), localized surface plasmon resonance (7, 8), and surface-enhanced Raman scattering (9, 10)]. These measurements typically require quantifying many single-molecule events to gain new molecular and mechanistic insights or to achieve better analytical performance. However, it has been difficult to perform quantitative analysis with sufficient efficiency and statistical accuracy because of the concentration limit from mass transportation (11, 12) and the thermodynamic limit from probe affinity (13). For quantification of biomarkers in biological media, in which the required concentrations are usually at the femtomolar level or even lower (14), the single-molecule measurements could take inordinately long, and the nonspecific binding of unwanted species degrades the accuracy.In the past two decades, several single-molecule approaches for biomarker detection have been developed to surpass the above limits by biasing the equilibrium and driving binding reactions (15, 16). A typical scheme involves the usage of nanoparticles to collect the analyte followed by a digital measurement of single molecules at a confined space (17), such as the commercialized, single-molecule enzyme-linked immunosorbent analysis (digital ELISA) (18). The digital ELISA uses the antibody-modified magnetic beads to capture the analyte in solution and loads them into femtoliter-sized reaction chambers termed single-molecule arrays. It effectively improves the sensitivity of conventional ELISA by three orders with a limit of detection (LoD) at the subfemtomolar level but requires sophisticated devices and excessive operation to remove free analyte molecules. Besides, the performance is still limited by the probe affinity and false positive arising from detection antibodies that bind nonspecifically to assay surface.A distinct yet effective strategy is to explore the in-depth heterogeneous information of single-molecule interaction (19). Walter et al. first demonstrated a kinetic fingerprinting approach to perform highly specific and sensitive detection of biomarkers via single-molecule fluorescence microscopy (20–22). This single-molecule recognition through equilibrium Poisson sampling technique surpasses the thermodynamic limit by exploiting the repetitive binding of fluorescently labeled, low-affinity probes to the analyte (23) and discriminating specific binding from background noise by a kinetic signature. The detection limits of microRNAs (miRNAs) and proteins also reach the subfemtomolar level, but screening probes with unique kinetic property is not compatible with current pipelines, and the concentration limit implies long incubation time before detection.Herein, we present the integration of single-molecule manipulation and dynamic sensing to allow rapid and ultrasensitive detection of biomarkers beyond the concentration and thermodynamic limits. In this Sensing Single Molecule under MicroManipulation (SSM³) approach, an external force is applied on the molecular bound between analyte and probes through tethered nanoparticles to actively tune the binding kinetics. This strategy, together with a dynamic sensing approach to exploit the heterogeneity at the single-molecule level, is able to beat the limits in both assay time and sensitivity. We show the principle and realization of the SSM³ technique and demonstrate 15-min assays to directly measure miRNAs and proteins at the subfemtomolar concentration.     

声门下型喉癌10例临床回顾性分析并文献复习

背景与目的:声门下型喉癌发病率低,且解剖部位隐匿,故极易漏诊、误诊,确诊时常为临床晚期;因缺乏大宗病例对照研究,其治疗方法目前尚存在争议.本研究旨在探讨声门下型喉癌的临床特征、治疗方法及疗效.方法:回顾分析1981年1月至2005年10月中山大学肿瘤防治中心收治的10例声门下型喉癌患者临床资料.结果:10例中4例喉内侵犯超过半周,4例侵犯喉外器官,1例颈部淋巴结转移;随访15年,1例失访.总的5年生存率为55.5%(5/9),经过治疗5年生存率提高至71.4%(5/7),5年无瘤生存率为42.9%(3/7).行单纯放疗、单纯手术、手术 辅助放疗患者的5年无瘤生存分别为0/2、1/2和2/3.结论:声门下型喉癌早期诊断困难,易在喉内扩散及向周围器官浸润,预后较差.全喉切除术仍为主要手术方式,采用手术与放疗综合治疗疗效满意.     

Maternal Zinc,Copper, and Selenium Intakes during Pregnancy and Congenital Heart Defects

The effects of zinc, copper, and selenium on human congenital heart defects (CHDs) remain unclear. This study aimed to investigate the associations of the maternal total, dietary, and supplemental intakes of zinc, copper, and selenium during pregnancy with CHDs. A hospital-based case-control study was performed, including 474 cases and 948 controls in Northwest China. Eligible participants waiting for delivery were interviewed to report their diets and characteristics in pregnancy. Mixed logistic regression was adopted to examine associations and interactions between maternal intakes and CHDs. Higher total intakes of zinc, selenium, zinc to copper ratio, and selenium to copper ratio during pregnancy were associated with lower risks of total CHDs and the subtypes, and the tests for trend were significant (all p < 0.05). The significantly inverse associations with CHDs were also observed for dietary intakes of zinc, selenium, zinc to copper ratio, selenium to copper ratio, and zinc and selenium supplements use during pregnancy and in the first trimester. Moreover, high zinc and high selenium, even with low or high copper, showed a significantly reduced risk of total CHDs. Efforts to promote zinc and selenium intakes during pregnancy need to be strengthened to reduce the incidence of CHDs in the Chinese population.     

Genetic analysis combined with 3D‐printing assistant surgery in diagnosis and treatment for an X‐linked hypophosphatemia patient

BackgroundHypophosphatemia is mainly characterized by hypophosphatemia and a low level of 1alpha,25‐Dihydroxyvitamin D2 (1,25‐(OH)₂D2) and/or 1alpha,25‐Dihydroxyvitamin D3 (1,25‐(OH)₂D3) in the blood. Previous studies have demonstrated that variants in PHEX and FGF23 are primarily responsible for this disease. Although patients with variants of these two genes share almost the same symptoms, they exhibit the different hereditary pattern, X‐link dominant and autosome dominant, respectively. Three‐dimensional (3D) printing is a method which can accurately reconstruct physical objects, and its applications in orthopedics can contribute to realizing a more accurate surgical performance and a better outcome.MethodsAn X‐linked hypophosphatemia (XLH) family was recruited, with four patients across three generations. We screened candidate genes and filtered a duplication variant in PHEX. Variant analysis and co‐segregation confirmation were then performed. Before the operation of our patient, a digital model of our patient''s leg had been rebuilt upon the CT scan data, and a polylactic acid (PLA) model had been 3D‐printed.ResultsA novel duplication PHEX variant c.574dupG (p.A192GfsX20) was identified in a family with XLH. Its pathogenicity was confirmed by the co‐segregation assay and online bioinformatics database. The preoperative plan was made with the help of the PLA model. Then, arch osteotomy and transverse osteotomy were performed under the guidance of the previous simulation. The appearance of the surgical‐intervened leg was satisfactory.ConclusionsThis study identified a novel PHEX variant and showed that 3D printing tech is a very promising approach for corrective osteotomies.     

搏动灌注对体外循环期间血流剪切力及血管内皮糖萼功能的影响

目的 研究应用搏动灌注对体外循环期间血流剪切力(FSS)及血管内皮糖萼(VEG)功能的影响.方法 选取体外循环下瓣膜置换手术患者40名,随机分为搏动灌注组及平流灌注组,每组患者20名,比较两组患者临床预后指标、FSS、VEG及炎症反应标志物[多配体蛋白聚糖-1(syndecan-1)、硫酸乙酰肝素及趋化因子C-C型配体...