大鼠脂肪间充质干细胞的成骨分化

目的:观察大鼠脂肪间充质干细胞经成骨诱导向成骨细胞分化的生物学特性,探讨其作为骨组织工程种子细胞的可行性。方法:实验于2004-07/2006-03在中南大学湘雅医院中心实验室完成主要工作。①取健康SD大鼠双侧腹股沟区脂肪垫,消化法分离出脂肪间充质干细胞,接种入含有体积分数为0.1的新生牛血清的低糖DMEM培养基进行原代培养。②取第3代的脂肪间充质干细胞,用含有体积分数为0.1的新生牛血清、0.1μmol/L地塞米松、50μmol/L抗坏血酸、10mmol/Lβ-甘油磷酸钠的高糖DMEM培养基诱导其向成骨细胞分化。③于3,5,7,10,12,14,21d分别采用倒置显微镜观察细胞形态及增殖情况、Gomori改良钙钴法碱性磷酸酶染色、茜素红S钙结节染色和Ⅰ型胶原免疫细胞化学染色检测脂肪间充质干细胞成骨分化的情况。结果:①脂肪间充质干细胞原代细胞呈成纤维细胞样长梭形外观,传代稳定,细胞形态均一。②经成骨诱导,脂肪间充质干细胞体积增大,呈多角形;成骨诱导14d,Gomori改良钙钴法碱性磷酸酶染色,细胞胞浆内可见浅棕色至棕黑色的颗粒,平均染色阳性率为80%;碱性磷酸酶活性随时间的延长而逐渐增高[3,5,7,10,12,14d依次为(2.43±0.09),(3.60±0.08),(5.01±0.09),(7.75±0.07),(9.59±0.09),(10.94±0.10)μkat/L];成骨诱导21d,钙结节形成明显,茜素红S染色,呈红色结节;成骨诱导7d,Ⅰ型胶原免疫细胞化学染色,细胞胞浆呈棕黄色,胞核经苏木精复染为蓝色。结论:大鼠脂肪间充质干细胞经成骨诱导具有成骨细胞的生物学特性,可作为骨组织工程的种子细胞。     

Survey on pretransfusion testing

BACKGROUND: Hospitals and blood centers throughout the United States use a variety of reagents and methods to perform pretransfusion testing. A survey was developed to determine the reagents and methods in use and their relative prevalence in different work settings. STUDY DESIGN AND METHODS: A national survey on pretransfusion testing was conducted. Surveys were distributed to state and regional blood bank associations, which then distributed them to hospitals and blood centers within their region. In most instances, the blood centers distributed the survey to the local hospitals. Completed surveys were returned to the authors for review, and all information was entered into a database for analysis. RESULTS: Analysis of the data shows that the majority of blood banks use monoclonal reagents for ABO testing and monoclonal-polyclonal blended reagents for Rh testing. The data show that anti-IgG and polyclonal antihuman globulin reagents are used almost equally for antibody screening (detection) tests and that most blood banks use a three-cell antibody-screening test. Slightly more than 50 percent of hospitals use an immediate-spin crossmatch in the absence of unexpected antibodies. CONCLUSION: A number of approved reagents and methods are used by blood bank laboratories for pretransfusion testing. Facility size (number of beds) and type tend to influence the choice of methods and reagents employed. This survey provides an opportunity for blood bank laboratories to compare their current practices with those of their peers.     

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

白介素与溃疡性结肠炎

近年来对白介素(interleukin,IL)和溃疡性结肠炎(ulcerative colitis,UC)的研究取得了很大进展,我们通过总结整理以前有关IL和UC的文献,概括出IL的产生和在UC发病及病理变化中的作用机制:IL-1直接介导了UC初期阶段炎症的发生:IL-8、IL-6直接促进炎性细胞过度分泌和/或抑制了炎性细胞的凋亡,IL-2分泌减少导致免疫系统内细胞间网络调节失衡, 使局部炎症介质和自由基释放,引起细胞毒作用,IL主要通过影响机体整体和/或局部免疫系统的功能介导UC的产生,并与UC的迁延难愈和反复发作有关．     

Nanostructured gellan and xanthan hydrogel depot integrated within a baghdadite scaffold augments bone regeneration

Controlled delivery of biological cues through synthetic scaffolds to enhance the healing capacity of bone defects is yet to be realized clinically. The purpose of this study was development of a bioactive tissue‐engineered scaffold providing the sustained delivery of an osteoinductive drug, dexamethasone disodium phosphate (DXP), encapsulated within chitosan nanoparticles (CN). Porous baghdadite (BD; Ca₃ZrSi₂O₉) scaffolds, a zirconia‐modified calcium silicate ceramic, was coated with DXP‐encapsulated CN nanoparticles (DXP–CN) using nanostructured gellan and xanthan hydrogel (GX). Crosslinker and GX polymer concentrations were optimized to achieve a homogeneous distribution of hydrogel coating within BD scaffolds. Dynamic laser scattering indicated an average size of 521 ± 21 nm for the DXP–CN nanoparticles. In vitro drug‐release studies demonstrated that the developed DXP–CN–GX hydrogel‐coated BD scaffolds (DXP–CN–GX–BD) resulted in a sustained delivery of DXP over the 5 days (78 ± 6% of drug release) compared with burst release over 1 h, seen from free DXP loaded in uncoated BD scaffolds (92 ± 8% release in 1 h). To estimate the influence of controlled delivery of DXP from the developed scaffolds, the effect on MG 63 cells was evaluated using various bone differentiation assays. Cell culture within DXP–CN–GX–BD scaffolds demonstrated a significant increase in the expression of early and late osteogenic markers of alkaline phosphatase activity, collagen type 1 and osteocalcin, compared to the uncoated BD scaffold. The results suggest that the DXP‐releasing nanostructured hydrogel integrated within the BD scaffold caused sustained release of DXP, improving the potential for osteogenic differentiation. Copyright © 2015 John Wiley & Sons, Ltd.     

Prospective epidemiological study of the prevalence of human leukocyte antigen (HLA)‐B*5701 in HIV‐1‐infected UK subjects

Objectives

Human leukocyte antigen (HLA)‐B^*5701 is strongly associated with developing a hypersensitivity reaction to abacavir (ABC) in White and Hispanic subjects. Across the UK, limited data exist on HLA‐B^*5701 prevalence in HIV‐1‐infected subjects. We determined HLA‐B^*5701 prevalence in the general HIV‐1‐infected population and in specific ethnic groups, particularly Black Africans who, in general, exhibit greater genetic diversity. We also compared HLA‐B^*5701 results obtained from local laboratories with those from a central provider.

Design and methods

Multi‐centre, observational study. All HIV‐1‐infected adult individuals receiving care at participating centres were eligible, irrespective of treatment status or prior exposure to ABC. Subjects provided samples for HLA‐B^*5701 assessment by both local (blood) and central laboratories (buccal swabs). HLA‐B^*5701 prevalence was adjusted to represent the ethnic group composition of the general UK population, and by main ethnic group.

Results

From eight UK centres, 1494 subjects [618 (41%) White, 770 (52%) Black] were recruited. Eighty‐nine per cent of Black subjects reported an immediate country of origin in Africa. Overall adjusted HLA‐B^*5701 prevalence was 4.55% [95% confidence interval (CI) 3.49% to 5.60%]. Among White subjects, prevalence was 7.93% (CI 5.80% to 10.06%). Among Black subjects, only two (both Ugandan) were HLA‐B^*5701 positive giving a rate of 0.26% (CI 0.07% to 0.94%).

Conclusions

HLA‐B^*5701 prevalence was similar to previously reported rates in White HIV‐infected subjects but considerably lower than that reported in Black HIV‐1‐infected subjects, as a result of the large proportion of Black African subjects.     

肿瘤干细胞和抗肿瘤药物研发思路

目前有些抗肿瘤药物尽管有较好的近期疗效,然而肿瘤患者单纯依赖化疗通常难以达到长期生存的目的。本文试图从肿瘤干细胞理论解释抗肿瘤药物近期疗效和远期生存之间存在的矛盾,并探讨抗肿瘤药物的研究发展方向。