BST-2参与HCMV感染诱导的恶性胶质瘤细胞增殖和迁移

为探讨BST-2蛋白是否参与人巨细胞病毒(HCMV)感染导致的恶性胶质瘤细胞增殖和迁移,以HCMV AD169株感染U251细胞,通过细胞划痕愈合实验检测HCMV感染对U251迁移的影响;通过Western-blot方法检测HCMV感染对BST-2蛋白表达的影响;通过CCK-8、细胞划痕愈合和transwell方法检测HCMV感染后下调BST-2对U251细胞增殖和迁移能力的影响。结果显示,HCMV感染可促进U251细胞迁移并高表达BST-2,沉默BST-2后可抑制由HCMV感染诱导的细胞增殖和迁移。结果证实HCMV感染可促进胶质瘤细胞U251增殖迁移,BST-2参与了HCMV感染导致的恶性胶质瘤细胞增殖和迁移。     

天然药物抗HIV 机制的研究进展

艾滋病是威胁人类健康的重大传染性疾病,目前仍未有令人满意的治疗方法。近年来的研究发现,天然药物具有广泛的抗HIV药理学活性,如免疫调节、抑制HIV复制中相关功能酶、抗氧化等。本文就天然药物抗HIV的相关作用机制展开综述。     

HIV介导的细胞凋亡

细胞凋亡是多细胞生物的一种生理性细胞死亡 ,它和坏死性细胞死亡是两种不同的细胞死亡形式 ,细胞凋亡是细胞内在的有规律的机制引起的 ,它可由细胞内部因素和外部刺激所诱导 ,细胞凋亡有其固有的形态特点 ,这种“自杀”行动对机体的正常发育和新陈代谢都是必要的 ,有助于维持组织稳态 ,对于机体是有利的。如细胞凋亡异常将会给机体带来各种病理后果。另外由于细胞凋亡的可诱导性 ,就为疾病的治疗提供了一条新思路。近年来发现 ,ＨＩＶ病毒也参与了细胞凋亡的诱导和抑制 ,在这些过程中相关的病毒基因的表达和相应蛋白质的合成起着关键性的作…     

HIV Vpr蛋白诱导细胞凋亡研究进展

Vpr蛋白是HIV的一个辅助蛋白,可以诱导多种细胞的凋亡。目前的研究表明Vpr蛋白引起细胞凋亡主要是通过线粒体途径实现的。Vpr蛋白通过直接而且特异地与结合在PTPC中的ANT相互作用,改变线粒体膜通透性,导致凋亡诱导因子(AIF)和细胞色素C的释放,激活Caspase、DNases等的级联反应,引起核染色质的固缩,最终引起细胞凋亡。     

ADCC效应在抗HIV感染中的作用

抗体依赖性细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity,ADCC)是一种固有免疫和适应性免疫相结合的免疫学效应。ADCC效应主要是通过效应细胞膜表面的受体IgG Fc受体(Fc receptor,FcR)如FcγRIIIa(CD16)、FcγRIIc(CD32)、FcγRI(CD64)识别靶细胞膜表面抗原,结合相应IgG抗体的Fc段而促发效应细胞脱颗粒和细胞因子分泌的一类细胞毒效应。对人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染已有研究证实了,ADCC效应在控制HIV感染中发挥着重要作用。现对ADCC效应在抗HIV感染中的作用作一综述。     

中国HIV阳性参比品库的建立以及HIV不同生物标志物的意义

为建立HIV阳性样品库并分析HIV不同生物标志物的意义,从我国不同地区以及不同人群中收集HIV感染者或可疑感染者血浆,对其进行HIV抗体、抗原、核酸以及基因型的检测,并用WB试剂对其进行抗体的确认检测。结果显示,该样品库共有样品190份,均为HIV阳性,含有我国流行的主要基因型,即B′、BC、AE和B亚型;HIV抗体S/CO值小于10者占11.1%,在10~15之间者占63.2%,大于15的占25.8%;病毒载量在50~103copies/mL者占7.9%,在103~105copies/mL者占82.2%,大于105copies/mL者占10.0%。而且抗体S/CO值大于10者,均为HIV抗体确认阳性,小于10者仅有61.9%为抗体确认阳性,但核酸均大于50copies/mL,而且抗体不确定样品的病毒载量均大于105copies/mL,但抗体不确定的8份样品中仅有4份样品为P24抗原阳性。结果提示该样品库样品来自于HIV感染的不同时期,可用于对HIV的不同试剂进行评价;而且核酸的检测可有助于对HIV早期感染的明确诊断。     

整合蛋白信号转导研究进展

细胞外基质受体整合蛋白全方位影响细胞的形态、运动、存活和增殖。它通过介导复杂的信号通路,将细胞外信息内传,调控细胞的生命活动。本文综述了整合蛋白的活化过程、信号转导过程及其与生长因子受体信号通路相互关联研究的最新进展。     

整细胞酶促合成抗HIV新药 D-D4FC

D-D4FC(β-D-2′,3′-双脱氢双脱氧-5-氟胞嘧啶核苷)是一种新型抗HIV病毒的核苷类药物,目前正在美国、法国和德国进行Ⅱ期临床。利用乳酸杆菌提取的粗制N-脱氧核糖转移酶实现了由D4T(β-D-2′,3′-双脱氢双脱氧-胸苷,司他夫定)和5-FC(5-氟胞嘧啶)合成D-D4FC,转化率达到25%。现在,发现利用乳酸杆菌整细胞也可实现此反应,其转化率经过12.5h可达到50%,更有利于可能的工业化连续生产。研究了整细胞催化合成D-D4FC反应中,pH值、缓冲液类型、底物浓度、加菌量、反应时间等条件的影响并进行了优化,探讨了反应中乳酸杆菌整细胞催化的可能机理。     

Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells

Background aimsMultipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed.MethodsHuman bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red–positive adipocytes after automated image acquisition.ResultsMSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences.ConclusionsWe measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.     

HFCL细胞对U937细胞的诱导分化作用

为了观察正常人骨髓成纤维样细胞系HFCL对急性单核细胞白血病U937细胞促分化作用,及其对经典诱导分化剂TPA诱导分化作用的影响,先建立U937细胞和HFCL细胞共培养体系,以细胞形态学改变、硝基四氦唑蓝(NBT)、流式细胞仪检测细胞周期和CD11b、CD13、CD14、CD33细胞表面抗原作为诱导分化指标;Western印迹检测P38蛋白的表达变化。结果发现,与HFCL细胞共培养后,U937细胞出现分化成熟的形态学改变,且与HFCL细胞直接接触组的诱导分化作用大于用transwell组。同时发现U937细胞与HFCL细胞共培养后,G1期细胞增高,S期细胞减少;CD11b、CD13、CD14和CD33表达增高;且NBT阳性细胞增高至46、3％。Western印迹检测结果显示,直接接触组总P38蛋白表达增加。而且HFCL细胞还能增强TPA对U937的诱导分化作用。     

骨髓基质细胞的特征及其在细胞和基因治疗中的应用

骨髓基质细胞是一类独特的间质干细胞,可分化为多种非造血系的组织。骨髓基质细胞具有贴壁生长的特性,因而易于在体外分离和扩增;另外骨髓基质细胞可在体内外表达多种治疗性的外湖目的基因。因此,骨髓基质细胞被认为是一种理想的治疗性细胞的基因治疗中的靶细胞。本文对骨髓基质细胞的研究进展及其在细胞和基因治疗中的应用作一综述。     

Stereotypic culture systems for liver and bone marrow: evidence for the development of functional tissue in vitro and following implantation in vivo

Stromal cell-associated liver cell and bone marrow (BM) culture on three-dimensiional nylon screen or polyglycolic acid (PGA) felt templates conveys certain functional advantages to the parenchyma of these tissues. Hepatic parenchymal cells (PC) manifest long-term ( approximately 2 month) expression of liver-specific activities including cytochrome P450 enzyme activity and the synthesis of albumin, fibrinogen, transferrin, and other proteins. PC also undergo proliferation in association with stromal cells that were pre-established on these templates. PC mitoses are directly proportional to available space within the template for their expansion indication that geometric or sterotypic parameters influence the growth of these cells in vitro. BM cultured on a similar template exhibits long-term multilineage hematopoietic expression and limited expansion of progenitor cell numbers. Progenitor cell concentration within the cultures can be substantially enhanced if these cells are liberated from co-culture and reseeded onto a template containing fresh stromal cells. BM and liver cel cultures established on felt composed of bioresorbable PGA filaments was grafted into various sites in rats. Liver co-cultures generated sinusoids and other liver-like structures in situ; active hematopoietic blasts were observed at sites of BM co-culture grafts. Biodegradable polymer constructs may prove useful for certain clinical applications as vehicles for the delivery of tissues that were engineered in culture.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Endocytic activity of HIV‐1 Vpu: Phosphoserine‐dependent interactions with clathrin adaptors

HIV‐1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune‐evasion, triggering ubiquitin‐mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP‐1 and AP‐2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α‐chain. CD8‐VpuCD was rapidly endocytosed in a clathrin‐ and AP‐2‐dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β‐TrCP binding site and a leucine‐based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV‐dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP‐2 and showed that this is enhanced by serine‐phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP‐2 and AP‐1; this interaction was dependent on serine‐phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.      

The construction of an in vitro three-dimensional hematopoietic microenvironment for mouse bone marrow cells employing porous carriers

Spatial development of mouse bone marrow cellsemploying porous carriers was investigated in order todesign a bioreactor with a three-dimensionalhematopoietic microenvironment. Three types of porouscarriers were used for examining the spatialdevelopment of anchorage-dependent primary stromalcells as feeder cells. Stromal cells were found tospread well at a high density on a polyester nonwovendisc carrier (Fibra cel (FC)) under a scanningelectron microscope, while cells on porous cellulosebeads (Microcube (MC), 500 m pore diameter)spread at a low density; cells on another type ofcellulose porous beads (CPB, 100 m pore diameter)were globular. Mouse bone marrow cells wereinoculated to dishes containing three types of porouscarriers which shared more than 30% of the bottomsurface in a dish. The concentration of stromal cellsin the well containing FC was lower than that on theother two carriers. However, the weekly output oftotal hematopoietic cell (suspension cells) increasedbetween day 21 and 28 in the culture using FC while itdecreased monotonously in the cultures by use of theother two carriers. The proportion of progenitorcells (BFU-E, CFU-GM) in the total hematopoietic cellpopulation, after showing an initial decrease,increased after 1 week in the culture using FC whilethe proportion decreased monotonously to zero in thecultures using MC and CPB.