Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions

Certain inflammatory stimuli render cultured human vascular endothelial cells hyperadhesive for neutrophils. This state is transient and reversible, in part because activated endothelial cells secrete a leukocyte adhesion inhibitor (LAI). LAI was identified as endothelial interleukin-8 (IL-8), the predominant species of which is an extended amino-terminal IL-8 variant. At nanomolar concentrations, purified endothelial IL-8 and recombinant human IL-8 inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and protect these monolayers from neutrophil-mediated damage. These findings suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood.     

口蹄疫病毒非结构蛋白3D原核表达、纯化和反应原性分析

采用RT-PCR技术扩增获得口蹄疫病毒(FMDV)细胞毒O/Akesu/58的非结构蛋白3D(NSP 3D)基因编码区,并定向克隆到pProexHTb原核表达载体上,再将重组pProex-3D转化至大肠杆菌BL21,经IPTG诱导,表达产物用亲和层析法纯化,经SDS-PAGE鉴定和Western-blot分析抗原性;以纯化的表达蛋白免疫豚鼠制备其多克隆抗体,ELISA测定抗体效价,间接免疫荧光法检测制备的豚鼠抗NSP 3D多克隆抗体与细胞毒天然抗原的反应原性.结果表明:表达的目的蛋白大小为53ku左右;ELISA结果表明,制备的多克隆抗体效价达1∶1 024以上;Western-blot分析表明,纯化后的蛋白能与FMDV感染动物血清发生特异性反应;间接免疫荧光检测发现,豚鼠抗3D蛋白多克隆抗体可与细胞毒天然抗原反应,与阴性对照未见反应.     

大鼠不同部位内皮细胞的体外培养

为了建立一种能有效分离、培养和获取较高纯度的大鼠心肌膜微血管内皮细胞(RMMECs)、肠黏膜微血管内皮细胞(RIMECs)、主动脉内皮细胞(RAECs)、后腔静脉内皮细胞(RPVECs)的模型,自7dSD大鼠分别取左心室壁、空肠、主动脉、后腔静脉,采用II型胶原酶消化、差速贴壁、差速消化法获得较纯的RMMECs、RIMECs、RAECs、RPVECs。结果表明:通过形态学特征及微血管内皮细胞和血管内皮细胞相关特异性抗原检测证实,得到高纯度RMMECs、RIMECs、RAECs、RPVECs。结论:利用胶原酶II型消化法成功分离并培养大鼠的RMMECs、RIMECs、RAECs、RPVECs,为进一步开展内皮细胞的生物学特性的相关研究提供有效的方法。     

枸杞多糖组分-Ⅳ对有机磷农药所致血管内皮功能损伤的保护作用

[目的]探讨枸杞多糖组分-Ⅳ（LBP-Ⅳ）对对氧磷（PARA）所致血管内皮功能损伤的保护作用及其机制。[方法]以大鼠离体胸主动脉血管环（EVAPVR）和培养的人脐静脉内皮细胞（HUVEC）为试验对象,以PARA为损伤药物,以LBP-Ⅳ为保护药,检测血管内皮依赖性舒张反应（EDRR）、内皮细胞单层通透性（ECMP）及细胞培养液生化指标。[结果]LBP-Ⅳ剂量依赖性（0.1、1、10mg/ml）地显著减轻了PARA（3.63μmol/l）对血管EDRR的抑制作用,降低了ECMP的增加,保护了超氧化物歧化酶（SOD）活性,阻滞了丙二醛（MDA）浓度的升高以及一氧化氮（NO）浓度的降低（P〈0.05）。[结论]LBP-Ⅳ对PARA所致的血管内皮功能损伤有明显的保护作用,其机制可能与LBP-Ⅳ的抗氧化作用有关。     

黄芪多糖组分-A1对甲基异柳磷所致血管内皮功能损伤的保护作用

[目的]研究黄芪多糖组分-A1(APS-A1)对甲基异柳磷所致血管内皮功能损伤的保护作用。[方法]以大鼠离体胸主动脉血管环(EVAPVR)和培养的人脐静脉内皮细胞(HUVEC)为试验对象,以甲基异柳磷为损伤药物,以APS-A1为保护药,检测血管内皮依赖性舒张反应(EDRR)、内皮细胞单层通透性(ECMP)及细胞培养液生化指标。[结果]APS-A1能明显减轻甲基异柳磷(7μmol/L)对血管EDRR的抑制作用,且呈剂量依赖性;APS-A1也能降低ECMP的增加,保护超氧化物歧化酶(SOD)的活性,并阻滞丙二醛(MDA)浓度的升高以及一氧化氮(NO)浓度的降低。[结论]APS-A1对甲基异柳磷所致的血管内皮功能损伤有明显的保护作用。     

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

The aim of the study was to investigate whether phosphorus (P) transporters, type IIb sodium-dependent phosphate cotransporter (NaP-IIb) and inorganic phosphate transporter 2 (PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^–1 of P as KH₂PO₄ to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaP-IIb and PiT2 expressions, respectively. Supplemental P increased (P=0.065) the protein abundance of PiT2 and enhanced (P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaP-IIb silencing decreased (P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect (P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.     

棉花种子特异性启动子的克隆及序列分析

启动子是基因表达的重要顺式调控元件,在基因工程中,种子特异性启动子可以调控外源基因在种子中特异表达,提高表达效率,增强转基因的效果。本试验根据棉花LEA蛋白D34基因序列设计引物,以海岛棉基因组DNA为模板,通过touch down PCR技术,获得D34基因的种子特异性启动子片段。测序结果表明该片段长1 384 bp,与已发表的D34基因序列相似度为94.87%。该片段除了含有启动子的基本元件TATA框、CAAT框外,还含有种子特异性启动子元件E-box、G-box、B-box、AACA基序等。此外,对其顺式元件做了生物学功能分析。     

Heparan sulfate degradation: relation to tumor invasive and metastatic properties of mouse B16 melanoma sublines

After transport in the blood and implantation in the microcirculation, metastatic tumor cells must invade the vascular endothelium and underlying basal lamina. Mouse B16 melanoma sublines were used to determine the relation between metastatic properties and the ability of the sublines to degrade enzymatically the sulfated glycosaminoglycans present in the extracellular matrix of cultured vascular endothelial cells. Highly invasive and metastatic B16 sublines degraded matrix glycosaminoglycans faster than did sublines of lower metastatic potential. The main products of this matrix degradation were heparan sulfate fragments. Intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization degraded purified heparan sulfate from bovine lung at higher rates than did B16 cells with a poor potential for lung colonization. Analysis of the degradation fragments indicated that B16 cells have a heparan sulfate endoglycosidase. Thus the abilities of B16 melanoma cells to extravasate and successfully colonize the lung may be related to their capacities to degrade heparan sulfate in the walls of pulmonary blood vessels.     

Semaphorin 3E and plexin-D1 control vascular pattern independently of neuropilins

The development of a patterned vasculature is essential for normal organogenesis. We found that signaling by semaphorin 3E (Sema3E) and its receptor plexin-D1 controls endothelial cell positioning and the patterning of the developing vasculature in the mouse. Sema3E is highly expressed in developing somites, where it acts as a repulsive cue for plexin-D1-expressing endothelial cells of adjacent intersomitic vessels. Sema3E-plexin-D1 signaling did not require neuropilins, which were previously presumed to be obligate Sema3 coreceptors. Moreover, genetic ablation of Sema3E or plexin-D1 but not neuropilin-mediated Sema3 signaling disrupted vascular patterning. These findings reveal an unexpected semaphorin signaling pathway and define a mechanism for controlling vascular patterning.     

Luteolin prevents f MLP-induced neutrophils adhesion via suppression of LFA-1 and phosphodiesterase 4 activity

Luteolin is an active ingredient found early from Folium perillae and Flos lonicerae, and has a specific inhibition on phosphodiesterase 4(PDE4) activity in vitro. Researches show luteolin has pharmacological effects of anti-inflammation, anti-anaphylaxis, antitumor, antioxidant, protection of nervous system and so on, and has mainly been used for the treatment of respiratory inflammatory diseases, cancer and cardiovascular disease in clinic. PDE4, specific to hydrolyze cyclic AMP(c AMP), is considered to be a new anti-inflammatory target due to the decisive role on c AMP signal in inflammatory cells such as neutrophils. In order to explore the anti-inflammatory mechanism, we further studied the effects of luteolin on the activity and expression of PDE4, the expression of lymphocyte function-associated antigen-1(LFA-1) and macrophage-1(MAC-1) in neutrophils, and the adhesion of neutrophils and endothelial cells. The results showed that luteolin had a dose-dependent inhibition on both bare PDE4 activity and PDE4 in cultured neutrophils, and had an obviously promotive effect on gene expressions of PDE4 A, 4B and 4D in later period. Luteolin had a significant inhibitory effect on neutrophils adhesion and LFA-1 expression in early stage, and had no obvious effect on MAC-1 expression. Therefore, luteolin can inhibit LFA-1 expression of neutrophils, then inhibit the adhesion of neutrophils and endothelial cells, and the mechanism is at least related with the inhibition of PDE4 activity.     

Vascular endothelial growth factor is a secreted angiogenic mitogen

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells, respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins, in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF.     

猪瘟病毒E2基因的克隆及其初步鉴定

[目的]为了研究猪瘟病毒E2蛋白的抗原性质和进一步筛选保护性抗原表位。[方法]克隆猪瘟病毒E2蛋白的部分基因片段,通过自行设计的一对引物,应用RT-PCR从猪瘟兔化弱毒株细胞培养物,扩增出一DNA片段,并对其进行鉴定。[结果]经初步鉴定,该片段大小约703 bp,核苷酸序列测定结果表明该片段为E2蛋白基因。[结论]该研究为筛选CSFV抗原奠定了基础。     

FSH对体外培养仔猪睾丸支持细胞cyclin D1 mRNA和 cyclin E1 mRNA表达的影响

【目的】确定FSH是否能调节支持细胞cyclin D1 mRNA和cyclin E1 mRNA的表达及可能的机制。【方法】以培养的仔猪睾丸支持细胞为试验材料,通过添加各种信号通路的抑制剂,应用实时荧光定量PCR 检测cyclin D1 mRNA和cyclin E1 mRNA的相对表达量。【结果】不同浓度的FSH（0—100 ng•mL-1）均可促进cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,在FSH浓度为50 ng•mL-1时两种基因的表达量最大（P＜0.05）;FSH（50 ng•mL-1）也以时间依赖的方式促进了cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,在作用30 min时其表达量达到高峰（P＜0.05）。不同浓度的Foskolin （0—20 μmol•L-1）均可以促进cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,但均低于FSH单独作用时两种基因的表达量;Rp-cAMP（0—40 μmol•L-1）、H-89（0—30 μmol•L-1）和Verapamil（0—100 μg•mL-1）以剂量依赖的方式抑制了FSH诱导的cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,而且Rp-cAMP和Verapamil联合作用对FSH的抑制效果高于单独的抑制效果（P＜0.05）,但低于两者之和。此外,不同浓度的U0126（0—10 μmol•L-1）降低了FSH诱导的cyclin D1 mRNA和cyclin E1 mRNA的表达（P＜0.05）,而Rp-cAMP、H-89、Verapamil和U0126单独作用时,cyclin D1 mRNA和cyclin E1 mRNA的表达与对照相比没有显著差异（P＞0.05）。【结论】FSH以剂量依赖和时间依赖的方式诱导了cyclin D1 mRNA和cyclin E1 mRNA的表达;cAMP-PKA、Ca2+和ERK1/2参与了FSH对cyclin D1 mRNA和cyclin E1 mRNA表达的调节。     

心脉隆注射液对动脉粥样硬化大鼠血管内皮细胞的保护作用

目的探讨心脉隆注射液对动脉粥样硬化（AS）大鼠血管内皮细胞的保护作用.方法采用高脂饲料加维生素D3喂养8周的方法建立动脉粥样硬化大鼠的模型.将大鼠随机分为正常组、模型组、辛伐他汀组、心脉隆治疗组四组,高脂饲料喂养8周后取血测一氧化氮（NO）、内皮素（ET）、前列环素（PGI2）和血栓素（TXA2）的含量.结果心脉隆注射液给药8周后能明显的升高AS大鼠血清NO和血浆PGI2水平（P〈0.05）,显著降低血浆ET的含量（P〈0.05）.结论心脉隆注射液能保护AS大鼠血管内皮细胞,减轻动脉粥样硬化的进程,对动脉粥样硬化有治疗作用.     

桃果实中脱落酸受体ABAR/CHLH结合区蛋白的原核表达、纯化及复性

为了剖析桃果实中脱落酸受体ABAR/CHLH蛋白的结合活性,从桃果实中提取总RNA,采用RT-PCR方法,以桃果实RNA逆转录的cDNA为模板,扩增出ABAR/CHLH基因的结合区功能片段C369,回收目的片段并测序,基因片段长度为1 121 bp,编码369个氨基酸残基,分子量约为40 kD。利用BamHⅠ和NotI酶切位点将该片段编码区插入原核表达载体pET-28a（＋）中,构建ABAR/CHLH基因片段原核表达载体pET28a-C369,经菌落PCR和测序确证后,转化E.coliRosetta（DE3）,通过IPTG诱导其表达His-CHLH融合蛋白。通过SDS-PAGE检测及Ni-NTA琼脂糖树脂亲和层析柱纯化目的蛋白,并用纯化复性的His-CHLH C369融合蛋白制备抗体。     

Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4+ T cells

Studies of the biology and pathogenesis of Kaposi's sarcoma (KS) have been hampered by the inability to maintain long-term cultures of KS cells in vitro. In this study AIDS-KS-derived cells with characteristic spindle-like morphology were cultured with a growth factor (or factors) released by CD4+ T lymphocytes infected with human T-lymphotropic virus type I or II (HTLV-I or HTLV-II) or with human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2). Medium conditioned by HTLV-II-infected, transformed lines of T cells (HTLV-II CM) contained large amounts of this growth activity and also supported the temporary growth of normal vascular endothelial cells, but not fibroblasts. Interleukin-1 and tumor necrosis factor-alpha stimulated the growth of the KS-derived cells, but the growth was only transient and these could be distinguished from that in HTLV-II CM. Other known endothelial cell growth promoting factors, such as acidic and basic fibroblast growth factors and epidermal growth factor, did not support the long-term growth of the AIDS-KS cells. The factor released by CD4+ T cells infected with human retroviruses should prove useful in studies of the pathogenesis of KS.     

缢蛏六群体16S rRNA基因片段序列的差异分析

采用PCR技术扩增了缢蛏线粒体DNA的16SrRNA基因片段，PCR产物经纯化、测序、同源序列比对获得长度为440bp的核苷酸序列。利用16SrRNA基因片段分析了江浙沪地区三个野生群体（江苏射阳、上海崇明、浙江象山）和三个养殖群体（江苏射阳、上海奉贤、浙江象山）的遗传多样性，共检测到了19个单倍型和41个核苷酸多态位点。序列分析结果显示，三个野生群体之间出现了明显的遗传分化，其中崇明群体遗传多样性最高，其次为射阳群体，象山群体遗传多样性最低，表明崇明群体未受到养殖群体基因的污染。在养殖群体之间则没有达到遗传分化，且单倍型混杂，聚类结果显示与象山野生群体亲缘关系最近，这表明长期的养殖过程在一定程度上对野生群体产生了影响，降低了种质资源的丰富度。     

缢蛏六群体16S rRNA基因片段序列的差异分析

采用PCR技术扩增了缢蛏线粒体DNA的16SrRNA基因片段，PCR产物经纯化、测序、同源序列比对获得长度为440bp的核苷酸序列。利用16SrRNA基因片段分析了江浙沪地区三个野生群体（江苏射阳、上海崇明、浙江象山）和三个养殖群体（江苏射阳、上海奉贤、浙江象山）的遗传多样性，共检测到了19个单倍型和41个核苷酸多态位点。序列分析结果显示，三个野生群体之间出现了明显的遗传分化，其中崇明群体遗传多样性最高，其次为射阳群体，象山群体遗传多样性最低，表明崇明群体未受到养殖群体基因的污染。在养殖群体之间则没有达到遗传分化，且单倍型混杂，聚类结果显示与象山野生群体亲缘关系最近，这表明长期的养殖过程在一定程度上对野生群体产生了影响，降低了种质资源的丰富度。     

A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells

E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells.     

Functional arteries grown in vitro

A tissue engineering approach was developed to produce arbitrary lengths of vascular graft material from smooth muscle and endothelial cells that were derived from a biopsy of vascular tissue. Bovine vessels cultured under pulsatile conditions had rupture strengths greater than 2000 millimeters of mercury, suture retention strengths of up to 90 grams, and collagen contents of up to 50 percent. Cultured vessels also showed contractile responses to pharmacological agents and contained smooth muscle cells that displayed markers of differentiation such as calponin and myosin heavy chains. Tissue-engineered arteries were implanted in miniature swine, with patency documented up to 24 days by digital angiography.