截断型人可溶性CD40L的表达与纯化

目的　表达相对分子质量 180 0 0的截断型人可溶性CD4 0L。方法　针对人CD4 0L(Glu10 8 Leu2 6 1)设计引物 ,以全长人CD4 0L为模板 ,扩增目的基因 ,经pGEM T中间载体进一步连接到pQE 4 0载体 ,构建pQE sCD4 0L表达载体。转化E .coliM15后经IPTG诱导表达 ,分离、洗涤包涵体 ,并利用Ni NTA亲和层析纯化目的蛋白 ,经复性后检测对骨髓瘤细胞SP2 0细胞株的抑制作用。结果　克隆的目的基因为 4 80bp ,经测序与文献报道一致 ,所构建的菌株 (M15 pQE sCD4 0L)经诱导表达目的蛋白量为 2 6 7% ,相对分子质量为 180 0 0 ,纯化后蛋白纯度为96 5 % ,对SP2 0细胞有生长抑制作用。结论　原核表达的截断型人可溶性CD4 0L具有抑制肿瘤细胞生长作用。     

藁本内酯对血管紧张素Ⅱ诱导损伤的人脐静脉内皮细胞中miR-122表达的影响及其作用机制

目的 探讨藁本内酯对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)中miR-122表达的影响及其作用机制.方法 将HUVECs随机分为6组:空白组(不给药)、模型组(1×10-5 mol/L AngⅡ...     

丹参三七不同配比对缺氧复氧损伤人脐静脉内皮细胞的保护作用

研究了丹参、三七及其以不同质量比例配伍对缺氧复氧(H/R)人脐静脉内皮细胞(HUVECs)的保护作用。采用全自动生化分析仪测定了培养液中乳酸脱氢酶(LDH)漏出率。实验结果显示,正常对照组、H/R模型组的LDH漏出率分别为0.212与0.309,说明H/R引起HUVECs的LDH漏出率增加。而丹参、三七按质量比10∶0、10∶1、5∶1、5∶3、1∶1、3∶5、1∶5、1∶10、0∶10配伍后,使H/R损伤HUVECs的LDH漏出率分别降低为0.218、0.240、0.247、0.239、0.230、0.241、0.247、0.242、0.227,其中只有丹参三七质量比为10∶0、5∶3、1∶1及0∶10与模型组比有显著性差异,因此,丹参、三七及丹参三七质量比为5∶3、1∶1配伍,均可保护H/R诱导血管内皮损伤。     

人内皮抑素基因对人脐静脉内皮细胞周期变化的影响

目的 研究人内皮抑素基因对人脐静脉内皮细胞增殖能力的影响.方法 RT-PCR获取人内皮抑素基因(hEndostain,hES),通过酶切、测序鉴定获取的hES,MTT法检测该基因对人脐静脉内皮细胞(HUVEC)增殖的影响.结果 获得的hES基因经测序证实成功克隆人内皮抑素基因;hES基因转染HUVEC细胞24h,48h和72h,细胞增殖率均较正常组明显降低(P＜0.05),而脂质体对照与载体对照与对照组无明显变化.结论 hES基因能够抑制HUVEC增殖.     

人C-反应蛋白重组真核表达质粒的构建及其对人脐静脉内皮细胞LOX-1和TF表达的影响

目的构建人C-反应蛋白(C-reactive protein,CRP)重组表达质粒pTracer CMV2-CRP,并观察其在人脐静脉内皮细胞(Human umbilical vein endothelical cells,HUVEC)中的表达及其对凝集素样氧化型低密度脂蛋白受体-1(Lectin-type oxidizedLDL receptor-1,LOX-1)、组织因子(Tissue factor,TF)表达的影响。方法以质粒pCR-BluntⅡ-TOPO-CRP为模板,PCR扩增CRP基因CDS序列,克隆至pTracer CMV2载体中,转化感受态大肠杆菌DH5α,构建重组表达质粒pTracer CMV2-CRP,转染HUVEC,设实验组(转染pTracer CMV2-CRP)、阴性对照组(转染pTracer-CMV2)及正常对照组,各组细胞经G418抗性筛选,RT-PCR及Western b1ot检测CRP基因的过表达效应及CRP的表达对HUVEC中LOX-1和TF转录及蛋白水平的影响。结果重组真核表达质粒pTracer CMV2-CRP经双酶切鉴定及测序证明构建正确。实验组细胞中,CRP基因过表达,且LOX-1和TF基因的转录及蛋白水平明显高于正常对照组和阴性对照组(P<0.05)。结论已成功构建人CRP基因重组真核表达质粒pTracerCMV2-CRP,并在HUVEC中过表达CRP,且明显上调HUVEC中LOX-1和TF的表达,为进一步阐述CRP在动脉粥样硬化形成过程中的作用提供了新的实验依据。     

p38 MAPK抑制剂SB203580对缺氧致人脐静脉内皮细胞损伤的保护作用

目的观察p38MAPK特异性抑制剂SB203580对缺氧致人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)损伤的保护作用。方法将HUVECs分为正常对照组、缺氧培养组、SB203580+正常对照组和SB203580+缺氧培养组,培养24h后,流式细胞术检测各组细胞的凋亡率;Western blot分析各组细胞p38MAPK蛋白及其磷酸化水平;Transwell小室模型检测各组细胞的迁移率;ELISA法检测各组细胞培养上清中可溶性血管内皮生长因子受体-1(sFlt-1)及可溶性Endoglin(sEng)的含量。结果与正常对照组相比,缺氧培养组细胞的凋亡率、p38MAPK的磷酸化水平、sFlt-1及sEng含量显著增加(P<0.01),细胞体外迁移能力下降;SB203580+缺氧培养组细胞的凋亡率、p38MAPK的磷酸化水平、sFlt-1和sEng的释放较缺氧培养组均下降,体外迁移能力增强(P<0.05);磷酸化p38MAPK的释放水平与sFlt-1及sEng含量呈正相关(r1=0.69,P<0.05;r2=0.71,P<0.05)。结论 SB203580通过特异性阻断p38MAPK信号转导通路,对缺氧培养的HUVECs产生保护作用。     

原子力显微镜技术研究重组人内皮抑素对内皮细胞的作用

目的应用原子力显微镜(atomic force microscope,AFM)技术研究重组人内皮抑素(recombinant human endostatin,rhES)对内皮细胞的作用。方法采用MTT法检测不同浓度的rhES(0.05~2.4μg/ml)对人脐静脉内皮细胞ECV304增殖活力的影响;分别用0.8和2μg/ml的rhES处理ECV304细胞,应用AFM观察内皮细胞整体形貌的变化,SPI 3800 New DFM动力显微镜观察ECV304细胞表面局部形貌的变化。结果 rhES可明显抑制ECV304细胞增殖,且呈剂量效应(P0.001);rhES可降低贴壁的ECV304细胞的厚度,且呈剂量依赖效应,使较光滑的细胞表面变粗糙,产生了一些微小的突起;经rhES处理的ECV304细胞表面结构呈现不规则的变化。结论 AFM技术具有样品制备简便和分辨率较高等优点,适合贴壁培养细胞的原位观察。     

KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用

目的观察KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用。方法将质粒pEGFP-N1/KDRn3扩增并鉴定后,以脂质体介导转染人脐静脉血管内皮细胞,培养一定时间后,在荧光显微镜下观察绿色荧光蛋白的表达,ELISA检测细胞培养上清中KDRn3的含量,MTT法检测KDRn3蛋白对人脐静脉血管内皮细胞增殖的影响。结果转染后48h,在荧光显微镜下可见pEGFP-N1/KDRn3转染组人脐静脉血管内皮细胞发出绿色荧光,72h发出绿色荧光的细胞增多,强度增强;转染后24、48和72h,细胞培养上清中KDRn3含量与转染前比较均提高,且差异有显著意义;转染后48和72h,pEGFP-N1/KDRn3转染组与对照组比较,细胞增殖有明显的抑制,且差异有显著意义。结论KDRn3蛋白可在人脐静脉血管内皮细胞中表达,并能抑制其增殖。     

LHRH-PE40的免疫原性及其抗体对细胞毒作用的影响

目的观察连续静脉注射重组毒素LHRH-PE40所引起的家兔抗LHRH-PE40抗体产生的规律以及该抗体对细胞毒作用的影响。方法将家兔分为3组,第1组隔日连续静脉注射LHRHPE40,第2组用弗氏佐剂乳化的LHRHPE40皮下注射为阳性对照组,第3组静脉注射人血白蛋白为阴性对照组。用ELISA检测血清中的抗体水平,XTT检测LHRH-PE40与血清中和后对Hela细胞的细胞毒作用。结果抗LHRHPE40抗体水平随静脉注射时间的延长而升高,8d可以检测到抗LHRH-PE40抗体,在24d基本达到最高水平。血清中和后LHRHPE40对Hela细胞的半数抑制量较阴性血清提高1～2倍,而阳性血清较阴性血清提高14倍。结论连续静脉注射LHRH-PE40产生较低水平的抗体,这种抗体未造成LHRHPE40对Hela细胞半数抑制量产生严重影响,这为临床连续使用LHRH-PE40提供了依据。     

靶向人Gadd45α基因的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞生物学功能损伤的影响

目的研究靶向人生长阻滞和DNA损伤45α(Growth arrest and DNA damage 45 alpha,Gadd45α)基因和表达绿色荧光蛋白(Green fluorescert protein,GFP)的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞(Human umbilical vein endothelialcells,HUVECs)表达上调的Gadd45α的沉默作用,初步阐明Gadd45α在缺氧应激致HUVECs生物学功能损伤过程中的作用。方法慢病毒包装后感染HUVECs,筛选最适MOI及感染时间。感染72h后,采用Real-time PCR和Western blot检测细胞中Gadd45αmRNA和蛋白的表达;流式细胞术检测细胞的凋亡率;Transwell小室实验检测细胞的迁移率;ELISA检测细胞sFlt-1和sEng的分泌水平。结果包装后慢病毒载体的滴度为1×108TU/ml,最适MOI为20,最适感染时间为72h,对HUVECs的感染效率约为80%。Gadd45α shRNA慢病毒载体对Gadd45α基因的沉默效率可达80%,阴性对照慢病毒载体对Gadd45α的表达无抑制作用。缺氧可致HUVECs中Gadd45α的表达上调,靶向Gadd45α基因的shRNA慢病毒颗粒可有效抑制缺氧致HUVECs的凋亡,减少sFlt-1及sEng的释放,同时增强其体外迁移能力,与缺氧组相比,差异具有统计学意义(P<0.05)。Gadd45α蛋白的表达水平与sFlt-1及sEng的分泌水平呈正相关(r1=0.89,r2=0.77,P均<0.05)。结论沉默Gadd45α基因对缺氧应激条件下的HUVECs生物学功能具有保护作用;Gadd45α可能是一个关键上游位点,参与子痫前期时sFlt-1及sEng的释放。     

Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

Mycosis fungoides (MF) and Sézary syndrome (SS), the most common types of cutaneous T-cell lymphoma (CTCL), are characterized by proliferation of mature CD4+ T-helper cells. Patients with advanced-stage MF and SS have poor prognosis, with 5-year survival rates of 52%. Although a variety of systemic therapies are currently available, there are no curative options for such patients except for stem cell transplantation, and thus the treatment of advanced MF and SS still remains challenging. Therefore, elucidation of the pathophysiology of MF/SS and development of medical treatments are desired. In this study, we focused on a molecule called OX40. We examined OX40 and OX40L expression and function using clinical samples of MF and SS and CTCL cell lines. OX40 and OX40L were co-expressed on tumor cells of MF and SS. OX40 and OX40L expression was increased and correlated with disease severity markers in MF/SS patients. Anti-OX40 antibody and anti-OX40L antibody suppressed the proliferation of CTCL cell lines both in vitro and in vivo. These results suggest that OX40–OX40L interactions could contribute to the proliferation of MF/SS tumor cells and that the disruption of OX40–OX40L interactions could become a new therapeutic strategy for the treatment of MF/SS.     

Involvement of HNP‐1 in different oxidation mechanisms in human endothelial cells

The objective of this study was to elucidate the different mechanisms of low‐density lipoprotein (LDL) oxidation by human endothelial cells. High level of LDL stimulated human alpha‐defensin 1 (HNP‐1) expression strongly, activated myeloperoxidase (MPO), and accumulated malondialdehyde (MDA). In addition, lipopolysaccharides (LPS) had similar effect, however the cell response occurred even earlier. After 12 h of LDL co‐culture, the cells were washed and fresh LDL was added again. MPO activity and MDA generation were increased. Adding fresh LDL after the LPS co‐culture for 3 h, a similar trend, however weaker effect was observed. Culturing cells with LDL and adding various kinds of calcium antagonists at the same time did not make obvious changes to the expression of HNP‐1. In contrast, culturing cells with LPS, calcium antagonists increased HNP‐1 expression. Adding the anti‐radical drug sodium ferulate had no significant effect on oxidative function activated by LDL, but the oxidation activated by LPS was suppressed significantly and the HNP‐1 expression was not changed significantly. The endogenous irritant LDL and the exogenous irritant LPS activated human endothelial cells in different manners and HNP‐1 expression changes were involved.     

辛二酰苯胺氧肟酸对人脐静脉内皮细胞增殖及血管形成的影响

目的探讨组蛋白去乙酰化酶抑制剂(Histone deacetylases inhibitor,HDACi)辛二酰苯胺氧肟酸(Suberoy-lanilide hydroxamic acid,SAHA)对人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)增殖及血管形成能力的影响。方法收集处于对数生长期的HUVEC,以不同浓度的SAHA分别处理24和48 h,另设不加SAHA的对照组,采用CCK-8法检测细胞的增殖活力,并计算增殖抑制率和半数抑制浓度(IC50)。采用流式细胞术检测经15μmol/L SAHA处理48 h的HUVEC凋亡和细胞周期,基质胶体外血管生成试验检测HUVEC的体外成管能力,Western blot法检测HUVEC细胞周期及凋亡相关蛋白的表达水平。结果随着SAHA浓度的增加及作用时间的延长,其对HUVEC增殖的抑制作用增强,SAHA浓度高于80μmol/L时,抑制率增加不明显,24和48 h的IC50值分别为60.53和30.49μmol/L;经15μmol/L SAHA处理48 h,与对照组相比,HUVEC的凋亡率明显增加(P<0.001),S期细胞比例明显升高(P<0.001),G0/G1期比例明显降低(P<0.001),体外成管能力明显下降,P21、caspase-3激活型、caspase-9酶原和激活型蛋白的表达水平均明显升高(P<0.001)。结论 SAHA能够抑制HUVEC增殖及体外血管形成能力,并使P21、caspase-3和caspase-9蛋白水平上调,为肿瘤的治疗提供了一种新的思路。     

Behavior of human umbilical vein endothelial cells on micro-patterned amorphous hydrogenated carbon films produced by plasma immersion ion implantation & deposition and plasma etching

Amorphous hydrogenated carbon films synthesized by plasma immersion ion implantation and deposition (PIII&D) were micro-patterned using argon plasma etching. Wells containing organized arrays of square holes were produced by plasma etching for 40 min on the surface of the a-C:H films covered by a steel stainless mask. The characteristics of the micro-patterned surfaces including the chemical composition, structure, surface morphology, contact angle, and surface energy were investigated and the effects of surface micro-patterning on the behavior of cultured human umbilical vein endothelial (HUVE) cells were investigated. All the micro-patterned samples exhibit a more hydrophobic nature and both cell adhesion and proliferation on the micro-patterned samples are improved compared to the un-patterned hydrogenated carbon film. The micro-patterns on the hydrogenated carbon films are observed to influence the water contact angle, surface energy, and HUVE cell behaviors, and the surface energy and hydrophobic surfaces appear to be the predominant reasons for the better cell adhesion and more rapid proliferation. Our results demonstrate the feasibility of using micro-patterning to modulate HUVE cell behavior.     

Stimulations of strontium-doped calcium polyphosphate for bone tissue engineering to protein secretion and mRNA expression of the angiogenic growth factors from endothelial cells in vitro

The vascularization of tissue-engineered bone is the key problem needed solving before application of tissue-engineered bone in clinical practice. Meanwhile, endothelial cells are the major and important source of seed cells in bone tissue engineering, and significant on promoting vascularization in tissue-engineered bone. Vascularization (namely angiogenesis) is a process mainly controlled by several angiogenic growth factors (VEGF, bFGF and MMP-2) which can be secreted by endothelial cells. Therefore, the research on the stimulations of SCPP to the secretion of the angiogenic growth factors from endothelial cells is very important. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the protein secretion levels and mRNA expression of VEGF, bFGF and MMP-2 from cultured endothelial cells. As a control, we also researched the effect of HA on the mRNA expressions and protein secretion of angiogenic growth factors from cultured endothelial cells. We cultured endothelial cells with SCPP scaffolds containing various concentration of strontium and HA. The results obtained in the MTT and SEM tests indicated that endothelial cells on SCPP scaffold exhibited higher proliferation rate and were easy to get a good spread than them on CPP, the best state of growth and proliferation of cells could be observed on 8%SCPP. The results of ELISA demonstrated that the protein levels of VEGF, bFGF and MMP-2 from cultured endothelial cells increased with the increasing Sr doped in calcium polyphosphate in SCPP groups, the peaks appeared on 8%SCPP. All SCPP groups showed a better ability to stimulate the protein secretion of VEGF, bFGF and MMP-2 from endothelial cells relative to CPP group and HA group. The results of RT-PCR suggested that the 8%SCPP group exhibited a significantly higher mRNA expression of VEGF, bFGF and MMP-2 relative to CPP group and HA group. In conclusion, the results of this study demonstrated that 8%SCPP had obvious promotion for secretion and mRNA expression of angiogenic growth factors from cultured endothelial cells.     

Circulating YKL-40 Level,but not CHI3L1 Gene Variants,Is Associated with Atherosclerosis-Related Quantitative Traits and the Risk of Peripheral Artery Disease

YKL-40, a pleotropic cytokine, is emerging as a risk factor and a prognostic predictor of atherosclerotic cardiovascular disease. We attempted to elucidate the genetic, clinical and biochemical correlates of circulating YKL-40 level and, by combining it with CHI3L1 gene variants, with the risk and long-term mortality of peripheral artery disease (PAD). Plasma YKL-40 concentrations were measured in 612 Taiwanese individuals who had no clinically overt systemic disease. Clinical parameters, CHI3L1 gene promoter variants and 18 biomarker levels were analyzed. Eighty-six PAD patients were further enrolled for analysis. Significant associations were found between CHI3L1 genotypes/haplotypes and YKL-40 levels for the health examination subjects (smallest p = 8.36 × 10⁻⁷ for rs4950928 and smallest p = 1.72 × 10⁻¹⁰ for haplotype TGG) and also for PAD patients. For the health examination subjects, circulating YKL-40 level, but not CHI3L1 gene variants, were positively associated with age, smoking, and circulating levels of triglyceride, lipocalin 2 and multiple inflammatory biomarkers and negatively associated with low-density-lipoprotein cholesterol levels. Circulating YKL-40 level is also significantly associated with the risk of PAD (p = 3.3 × 10⁻²³). Circulating YKL40 level, but not CHI3L1 gene promoter variants, is associated with the risk of PAD in Taiwanese. The association of YKL-40 levels with multiple quantitative traits relating to the risk of PAD may provide a molecular basis linking YKL-40 to atherosclerotic cardiovascular disease.