红花黄色素可促进人脐静脉内皮细胞的增殖

背景：心血管疾病的发生、发展与内皮细胞增生及凋亡有关。 目的：观察红花黄色素对血管内皮细胞模型的保护作用。 方法：体外培养人脐静脉内皮细胞,先采用红花黄色素进行干预,随后采用溶血卵磷脂体外诱导建立人脐静脉内皮细胞损伤模型,并设置对照组。 结果与结论：与对照组比较,溶血卵磷脂干预的人脐静脉内皮细胞增殖减弱(P < 0.05),细胞凋亡增加(P < 0.05),一氧化氮浓度降低(P < 0.01),而经红花黄色素干预后上述现象均可被逆转(P < 0.05)。结果证实,红花黄色素可通过促进内皮细胞增殖,抑制其凋亡并增加一氧化氮浓度对溶血卵磷脂诱导的内皮细胞损伤起保护作用。     

内皮样细胞与脐静脉内皮细胞的比较

BACKGROUND: Stem cells are induced to differentiate into endothelial-like cells that can be used for the treatment of diabetic lower extremity vascular disease. However, it is unclear whether these endothelial-like cells can completely replace endothelial cells to improve vascular disease and what are the differences between endothelial-like cells and endothelial cells. OBJECTIVE: To explore the differences and similarities between endothelial-like cells and human umbilical vein endothelial cells in the aspects of morphology, function, and viability. METHODS: Umbilical cord mesenchymal stem cells and umbilical vein endothelial cells were isolated, cultured and identified using flow cytometry and immunohistochemical method. Isolated umbilical cord mesenchymal stem cells were induced in DMEM-LG/F12 containing 10 µg/L vascular endothelial growth factor, 10 µg/L basic  fibroblast growth factor and 2% fetal bovine serum to differentiate into endothelial-like cells followed by immunohistochemical identification. To compare endothelial-like cells with human umbilical vein endothelial cells, cell migration detection, active substance measurement and three-dimensional angiogenesis test were performed. RESULTS AND CONCLUSION: Isolated umbilical cord mesenchymal stem cells strongly expressed the surface markers of mesenchymal stem cells, and human umbilical vein endothelial cells strongly expressed CD31 and VWF. After induction, the umbilical cord mesenchymal stem cells were identified to highly express CD31 and VWF. Through cell migration, active substance and three-dimensional angiogenesis tests, endothelial-like cells were similar to endothelial cells in the function and activity, and superior to endothelial cells.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

神经肽Y促进3T3L1前脂肪细胞的增殖和分化

目的:观察神经肽Y对3T3L1前脂肪细胞增殖和分化的影响,并对其机制进行初步探讨。方法:3T3-L1细胞由3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine,IB-MX)、胰岛素和地塞米松联合诱导分化,2 d后将细胞分为空白对照组(未加任何诱导剂组)、经典诱导组(胰岛素诱导)和NPY干预组(10-8、10-9、10-10mol/L NPY)。培养的第7、12天用相差显微镜观察各组细胞形态的变化,培养第12天用油红O染色观察脂肪细胞分化程度。MTT检测细胞增殖。Western blot检测脂肪细胞分化相关基因过氧化物体增殖剂活化受体-γ(peroxisome proliferator-activated receptor gamma,PPAR-γ)、CAAT/增强子结合蛋白-α(CCAAT/enhancer-bind-ing protein-α,C/EBP-α)蛋白的表达。结果:10-8mol/L NPY能促进3T3-L1细胞的分化。10-9mol/L NPY,10-8mol/L NPY均能促进3T3-L1细胞增殖。10-8mol/L NPY增加C/EBPα、PPARγ的表达。结论:NPY促进3T3L1前脂肪细胞的增殖和分化,其机制可能与上调PPARγ、C/EBPα的表达有关。     

Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells

After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future.     

Dehydroepiandrosterone and related steroids alter 3T3-L1 preadipocyte proliferation and differentiation.

The purpose of the present study was to determine if the anti-adipogenic effects of dehydroepiandrosterone (DHEA) are mediated solely by DHEA or by one or more of its downstream metabolites. In Experiment 1, preconfluent proliferating cultures of 3T3-L1 preadipocytes were incubated for either 24 or 72 h with 0, 1, 5 or 25 microM DHEA, DHEA sulfate (DHEAS), testosterone, estrone and 17beta-estradiol. Pregnenolone, a precursor of DHEA(S), was also tested at these concentrations. After 24 h of incubation, DHEAS, 17beta-estradiol and estrone at the 1 microM level stimulated preadipocyte proliferation. In contrast, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. After 72 h of incubation, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. In Experiment 2, post-confluent cultures of differentiating 3T3-L1 preadipocytes were incubated for 6 days with 0, 5, 30, or 60 microM levels of these steroids. Preadipocyte differentiation, as assessed by lipid content and glycerol-3-phosphate dehydrogenase activity, decreased markedly when treated with 30 and 60 microM DHEA, 17beta-estradiol, estrone and pregnenolone. In contrast, DHEAS had no impact on preadipocyte proliferation or differentiation. These results suggest that the anti-adipogenic actions of DHEA in adipose tissue may be mediated, in part, by one or more of its distal metabolites, including 17beta-estradiol.     

脂肪酸诱导的3T3-L1脂肪细胞胰岛素抵抗的研究

目的: 观察不同种类、不同浓度脂肪酸对3T3-L1脂肪细胞葡萄糖转运的影响，探讨高脂负荷在胰岛素抵抗形成中的意义，并建立最佳的脂肪酸诱导胰岛素抵抗产生的细胞模型。方法: 以3T3-L1前脂肪细胞和诱导分化成熟的3T3-L1脂肪细胞为研究对象，利用2-脱氧-［³H］-D-葡萄糖掺入法，观察最大葡萄糖摄取率时最佳胰岛素作用浓度和时间；在此基础上研究不同浓度油酸（C18:1）、棕榈酸(C16:0)对前脂肪细胞和脂肪细胞摄取葡萄糖的影响。结果:胰岛素刺激15 min（P<0.05）-1 h（P<0.01），葡萄糖转运呈升高的趋势，至6 h（P>0.05）逐渐下调；胰岛素浓度升高至50 nmol/L时，葡萄糖转运增加336%（P<0.01），100 nmol/L时达最高峰，是基础状态的492%（P<0.01）。0.125 mmol/L油酸或棕榈酸均可明显抑制胰岛素刺激状态下的3T3-L1前脂肪细胞葡萄糖转运（P<0.05），并呈浓度依赖性抑制；油酸及棕榈酸浓度分别达0.5 mmol/L和1.0 mmol/L时，分化成熟脂肪细胞葡萄糖转运显著受抑（P<0.05）。结论: 胰岛素刺激下的3T3-L1脂肪细胞葡萄糖转运有一定的时序性和浓度依赖性，100 nmol/L胰岛素刺激1h，葡萄糖转运率最高。1 mmol/L油酸或棕榈酸作用16-18 h可显著诱导3T3-L1脂肪细胞胰岛素抵抗的发生。     

IKCa1通道阻滞剂TRAM-34对HUVEC增殖作用的影响

目的 体外应用IKCa1通道阻滞剂TRAM-34干预人脐静脉内皮细胞(HUVEC),探讨其抑制HUVEC增殖作用的影响.方法 采用免疫荧光和RT-PCR法观察HUVEC细胞IKCa1的表达,常规MTT法分析不同浓度TRAM-34作用24、48、72 h后HUVEC细胞增殖的变化.结果 免疫荧光和RT-PCR法均证实HU...     

SHP-2 positively regulates adipogenic differentiation in 3T3-L1 cells

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of proliferation and differentiation in various cell types. Here, we investigated the ability of SHP-2 to mediate insulin-induced adipogenic differentiation of mouse 3T3-L1 cells. We found that the expression of SHP-2 was increased along with adipogenic differentiation. Overexpression of wild-type SHP-2 in 3T3-L1 cells resulted in enhanced adipocyte differentiation. Furthermore, insulin-stimulated adipogenic differentiation of 3T3-L1 cells was abolished by down-regulating SHP-2 expression using short interfering RNA. These results suggest that SHP-2 is a positive effector in signal transduction pathways necessary for adipocyte differentiation. In SHP-2 knockdown cells, the expression of peroxisome proliferator-activated receptor gamma, a master regulator of adipogenesis, was entirely suppressed even in the late phase of differentiation, whereas the expression level of C/EBPdelta was unchanged. These results highlight a novel role of SHP-2 in the signal transduction pathways regulating adipocyte differentiation.     

Glyphosate-based herbicides with different adjuvants are more potent inhibitors of 3T3-L1 fibroblast proliferation and differentiation to adipocytes than glyphosate alone

Glyphosate-based herbicides are extensively used for weed control all over the world. Thus, it is important to investigate the putative toxic effects of these commercial formulations which contain not only glyphosate but also different adjuvants. 3T3-L1 fibroblasts are a useful tool in the study of adipocyte differentiation. We have previously reported that a commercial glyphosate formulation inhibits proliferation and differentiation in these cells. In the present investigation, we further evaluated the effect of different commercial glyphosate-based herbicides on 3T3-L1 fibroblast proliferation and differentiation and compared their effect with glyphosate itself. After treatment of 3T3-L1 fibroblasts with different concentrations of three glyphosate formulations or glyphosate itself, the increase in cell number or cytosolic lipid accumulation was determined. To evaluate the presence of polyethoxylated adjuvants, matrix-assisted laser desorption/ionization (MALDI) analysis of the three commercial glyphosate-based herbicides was also performed. We found that the three commercial formulations tested were more potent inhibitors of both physiological processes: proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts than glyphosate. We also found by MALDI analysis that the adjuvants were polyethoxylated in two of the formulations but not in the third one. It is well known the toxic effect of polyethoxylated adjuvants, but according to our results, non-polyethoxylated adjuvants may also contribute to the toxic effects. Our results further support the ability of glyphosate-based herbicides to disturb cellular physiology and highlight the importance of toxicological assessment of commercial formulations rather than glyphosate alone.     

尼古丁对人脐静脉内皮细胞凋亡及坏死的影响

目的不同浓度尼古丁对人脐静脉内皮细胞（HUVECs）凋亡及坏死的影响。方法用CD34以免疫组织化学法鉴定HUVECs。通过3种不同浓度的尼古丁（3、30、300 ng/ml）刺激HUVECs 24 h后,用流式细胞仪检测其凋亡及坏死率。结果低浓度尼古丁促进细胞凋亡最强,而随着尼古丁浓度的增加,细胞凋亡率反而逐渐降低,各组差异具有统计学意义（P〈0.05）;此外,随着尼古丁浓度增加,细胞坏死率也日益增多,各组差异具有统计学意义（P〈0.05）。细胞坏死率与尼古丁浓度呈正相关（r=0.675）。结论尼古丁对HUVECs凋亡的影响具有浓度依赖性,细胞坏死率与尼古丁浓度呈正相关。     

纳米羟基磷灰石薄膜与人脐静脉血管内皮细胞的增殖

背景：合肥大学材料系和中国科学院安徽光学密精机械研究所联合研究应用脉冲激光沉积合成技术制备出一种新型的纳米羟基磷灰石人工心脏机械瓣膜。 目的：观察纳米羟基磷灰石人工心脏机械瓣膜与人脐静脉血管内皮细胞的相容性。 方法：将体外分离培养的传2-4代人脐静脉血管内皮细胞悬液接种于纳米羟基磷灰石人工心脏机械瓣膜上,培养3,7,21 d后,扫描电子显微镜下观察细胞在纳米羟基磷灰石人工心脏机械瓣膜上的生长情况。分别采用纳米羟基磷灰石人工心脏机械瓣膜常温浸提液、纳米羟基磷灰石人工心脏机械瓣膜高温浸提液、高密度聚乙烯浸提液及苯酚溶液培养人脐静脉血管内皮细胞,72 h后采用MTT法检测细胞增殖情况。 结果与结论：扫描电镜观察培养3 d时,人脐静脉血管内皮细胞呈梭形或多边形,伸出突起黏附于纳米羟基磷灰石人工心脏机械瓣膜上;7 d时,可见瓣膜表面细胞伸展充分,连接融合;21 d时,细胞成片融合,牢固覆盖于瓣膜表面,部分区域形成细胞外基质。MTT检测结果显示,纳米羟基磷灰石人工心脏机械瓣膜对人脐静脉血管内皮细胞无细胞毒性,具有良好的细胞相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Enterovirus infection and activation of human umbilical vein endothelial cells

Gastrointestinal tract associated lymphoid tissue is considered to be the main replication site for enteroviruses. In order to invade tissues to reach pancreatic islets, cardiac muscles, and other secondary replication sites, the virus has to survive circulation in the blood and find a way to get through endothelial cells. In the present study, the susceptibility of human endothelial cells to infections caused by human parechovirus 1 and several prototype strains of enteroviruses, representing different species (human poliovirus, human enterovirus B and C), and acting through different receptor families was examined. Primary endothelial cells isolated from human umbilical vein by collagenase perfusion and also an established human endothelial cell line, HUVEC, were used. Primary endothelial cells were highly susceptible to several serotypes of enteroviruses (coxsackievirus A13, echoviruses 6, 7, 11, 30, and poliovirus 1). However, coxsackievirus A 9 and echovirus 1 infected only a few individual cells while human parechovirus 1 and coxsackie B viruses did not show evidence of replication in primary endothelial cells. In general, primary endothelial cells were more sensitive to infection-induced cytolytic effect than HUVEC. Activation of endothelial cells by interleukin-1beta did not change the pattern of enterovirus infection. Immunofluorescence stainings of infected primary endothelial cells showed that expression of activation markers, E-selectin, and intercellular adhesion molecule-1, was clearly increased by several virus infections and the former molecule also by exposing cells to UV-light inactivated coxsackieviruses. In contrast, human leukocyte antigen-DR expression was not increased by virus infection.     

Adhesion of human T lymphocytes to endothelial cells isolated from the umbilical vein: II. Enhanced binding of in vitro activated T lymphocytes

Interactions of the vascular endothelium and cells of the immune system play a major role in the initiation and sustaining of cell mediated immune response in autoimmune diseases, chronic inflammatory processes and graft rejection. In the present investigation, the initial step of T cell-endothelial cell interactions, namely the adhesion of T cells to endothelial cells, was studied with special emphasis on the binding of T cells activated in vitro with lectins and by allogeneic cells as well as on the effect of pretreating endothelial cells with IFN-gamma. Human endothelial cells (EC) were isolated from the umbilical cord vein; human foreskin fibroblasts (HFF) served as control cells. While resting T cells demonstrated an adherence of 53% to EC and 20% to HFF, PHA blasts showed a binding of 90% to EC and 59% to HFF. Similar results were obtained using MLC blasts from mixed leukocyte cultures. Thus, the activation process triggered a striking enhancement of T cell binding not only to EC, but also to HFF that were taken as mesenchymal control cells. Pretreatment of EC and HFF with IFN-gamma, inducing Ia antigens on both cell populations, led to a significant increase of binding of resting T cells to EC. Of special interest, T lymphocytes also exhibited a considerably increased adherence to Ia-positive rheumatoid synovial fibroblasts. Taken together, these findings indicate that both the activation of T cells as well as endothelial cells results in a greatly enhanced binding.     

Overexpression of LYRM1 induces mitochondrial impairment in 3T3-L1 adipocytes

Homo sapiens LYR motif containing 1 (LYRM1) is a recently discovered gene involved in adipose tissue homeostasis and obesity-associated insulin resistance. The exact mechanism by which LYRM1 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of LYRM1 in 3T3-L1 adipocytes resulted in reduced insulin-stimulated glucose uptake, an abnormal mitochondrial morphology, and a decrease in intracellular ATP synthesis and mitochondrial membrane potential. In addition, LYRM1 overexpression led to excessive production of intracellular of reactive oxygen species. Collectively, our results indicated that the overexpression of LYRM1 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of LYRM1-induced insulin resistance.     

天麻素抑制脂多糖诱导的人脐静脉内皮细胞的自噬

目的 探讨天麻素(Gas)在脂多糖 (LPS) 诱导的人脐静脉内皮细胞 ( HUVECs) 自噬中的作用。方法 用不同浓度的Gas(5、10、20、50μmol/L) 预处理 HUVECs 1h,再加入1mg/L LPS共培养 12 h后,分别提取细胞总蛋白及mRNA,采用 Western blotting 及 RT-PCR 检测 Beclin-1、ATG5 及 LC3的表达变化;采用丹酰尸胺( MDC) 染色检测细胞中自噬小体(autophagosome)的表达变化;采用MTT检测 HUVECs 生存率的变化。 结果 LPS 诱导 HUVECs 产生自噬;Gas 预处理后自噬水平明显降低且呈剂量依赖性;与此同时细胞的生存率明显增加。 结论 LPS诱导 HUVECs 产生自噬;Gas 通过抑制由LPS 诱导产生的自噬,提高 HUVECs 的生存率。     

醒脑静抑制重组人肿瘤坏死因子介导的人脐静脉内皮细胞增殖

背景：前期研究将传统名方“安宫牛黄丸”经提取精制而成的新型水溶性静脉注射液醒脑静注射液应用于临床治疗冠心病 并取得很好的疗效,但其具体机制未完全明确。 目的：观察醒脑静对重组人肿瘤坏死因子介导的人脐静脉内皮细胞增殖的影响,探讨醒脑静抑制血管内皮细胞损伤增生的 应用价值。 方法：取3~5代人脐静脉内皮细胞,在培养基中添加10 μg/L重组人肿瘤坏死因子α诱导增殖,醒脑静组加入不同浓度的 醒脑静干预,阳性对照组添加氟伐他汀,并设立单纯细胞培养的空白对照组。 结果与结论：倒置相差显微镜下可见不同浓度的醒脑静均可使人脐静脉内皮细胞呈现胞浆收缩,贴壁细胞减少,坏死细胞 增多。醒脑静组的细胞增殖率低于肿瘤坏死因子α组(P < 0.05),且呈剂量及作用时间依赖关系。证实醒脑静能有效抑制重 组人肿瘤坏死因子α介导的人脐静脉内皮细胞增殖。     

PACAP up-regulates the expression of apolipoprotein D in 3T3-L1 adipocytes. DRG/3T3-L1 co-cultures study

The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation.     

几种外用中药成份对离体人脐静脉内皮细胞增殖的影响

目的:体外观察几种常用外用中药有关成分对血管内皮细胞增殖作用的影响,初步探讨其对创伤愈合的可能作用。方法:采用MTT比色法观察中药人参皂苷Rg1、Rh1、黄芪多糖、鹿茸多肽、川芎嗪、麝香酮、桂皮醛和乳香水提物对人脐静脉内皮细胞(HUVEC)增殖的影响。结果:9.75mg/L-2.5g/L的黄芪多糖对HUVEC未表现出增殖促进作用;1.94mg/L-0.5g/L的人参皂苷Rh1促进HUVEC的增殖(P＜0.05或P＜0.01),31mg/L的人参皂苷Rg1抑制细胞增殖(P＜0.05);1mg/L-0.5g/L的鹿茸多肽明显促进HUVEC的增殖,以10mg/L作用最明显(P＜0.01),桂皮醛2g/L时促进HUVEC的增殖(P＜0.05);1g/L的麝香酮明显抑制HUVEC的增殖(P＜0.01);0.5kg/L-2.5kg/L(生药)乳香水提物明显抑制HUVEC的增殖(P＜0.01),抑制增殖率为35.56%-55.56%。川芎嗪0.125g/L-0.5g/L明显抑制HUVEC的增殖(P＜0.01)。结论:益气温阳药物人参(Rh1)、鹿茸(鹿茸多肽)、肉桂(桂皮醛)促进HUVEC增殖;通络活血药物麝香(麝香酮)、乳香(乳香水提物)、川芎(川芎嗪)抑制HUVEC增殖。