兔骨髓基质干细胞向血管内皮样细胞的诱导分化

目的:研究血管内皮细胞生长因子(VEGF)联合碱性成纤维细胞生长因子(bFGF)促进兔骨髓基质干细胞向血管内皮样细胞的定向诱导分化,为血管化组织工程骨研究提供实验基础.方法:采集2周龄兔后肢长骨骨髓,用全骨骨髓贴壁法进行原代培养,将获得的第2代骨髓基质干细胞以1× 105/mL密度接种于内皮细胞条件培养基(含10 μg/L VEGF,10 μg/L bFGF,10％胎牛血清的DMEM/F12培养液)进行体外诱导培养,对诱导2周的细胞进行细胞形态观察和表型、功能鉴定.结果:经血管内皮细胞条件培养基诱导2周后的细胞呈扁平形,多边形,表达血管内皮细胞特异性标志CD31、VWF因子,细胞具有吞噬DiI-Ac-LDL和摄取FITC-UEA-1的功能,诱导的细胞可在BD基质胶内形成管腔样结构.结论:血管内皮细胞生长因子联合碱性成纤维细胞生长因子可以成功诱导兔骨髓基质干细胞为血管内皮样细胞,有希望作为组织工程骨的血管化的种子细胞.     

Ang-I(2-10)对大鼠急性心肌梗死后VEGF和bFGF表达及血管形成的影响

目的　观察血管紧张素Ⅰ (2 10 )对大鼠急性心肌梗死后 ,心肌细胞血管内皮细胞生长因子 (VEGF)和碱性成纤维细胞生长因子 (bFGF)表达和梗死区血管新生的作用。方法　选雄性大白鼠 45只 ,随机分为实验组、对照组和假手术组。实验组和对照组结扎左冠状动脉 45分钟后再灌注制成非透壁性心肌梗死模型。实验组给予血管紧张素Ⅰ (2 10 ) 5 0 pmol/kg·d ,而对照组和假手术组给予等量的蒸馏水 ,第 15天处死。免疫组织化学染色 ,光镜观察和图像分析处理 ,使用SPSS 10统计分析 3组数据。结果　实验组大鼠心肌细胞内bFGF和VEGF均比对照组表达明显增强 ,图像分析结果其灰度值下降 (为 81 2 4± 0 79比 90 2 9± 0 2 6 ;79 31± 0 72比 89 5 2± 0 5 6 ,P <0 0 0 1) ,微血管密度也明显增多(2 8 2 2± 1 0 1比 2 0 33± 0 83,P <0 0 0 1)。结论　血管紧张素Ⅰ (2 10 )可增强急性心肌梗死后心肌细胞表达VEGF和bFGF ,促进缺血区血管新生 ,发挥保护心肌的缺血性损伤作用。     

骨髓基质细胞在梗塞区与非缺血区心肌中的分化

目的观察心肌梗塞环境对移植骨髓基质细胞(MSC)向心肌样细胞分化的影响.方法采用成年近交系Wistar大鼠,经左冠状动脉前降支结扎建立急性心肌梗塞模型.术后1周,将来自同种供体、经体外扩增和DAPI标记的MSC分别植入梗塞区和非缺血区心肌.移植后1、2、4周取材、连续冰冻切片,观察两种环境中MSC的分布、迁移及心肌特异性肌钙蛋白T(TnT)的表达.结果植入梗塞区中的MSC扩散较快、增殖似更明显.两种环境中都可见移植细胞表达TnT,且有少数细胞呈现心肌样横纹,但梗塞区移植细胞TnT阳性率显著高于非缺血区.结论急性心肌梗塞环境有利于骨髓基质细胞向心肌样细胞分化.     

AP-2α在小鼠血管内皮细胞中上调VEGF的表达

转录因子AP-2α(activating protein 2α)在哺乳动物发育、分化以及肿瘤的发生等生命现象中起重要作用,最近的研究发现.在HaCaT角化细胞中,AP-2α可以影响vaseular endothelial growth factor(VEGF)的表达.对C 57BL/6J小鼠胸主动脉血管内皮细胞进行了原代培养,取2～4代细胞分别以转染质粒pCMV-Myc,pCMV-Myc-AP-2α,pCMV-Myc-VEGFA以及SiRNA-AP-2α,Control-RNAi-AP-2α,SiRNA-VEGFA,Control-RNAi-VEGFA.然后采用实时定量RT-PCR和Western Blot分别检测Ap-2α和VEGF mRNA与蛋白的表达水平.结果发现在小鼠血管内皮细胞中AP-2α的过表达可使VEGF mRNA和蛋白质表达水平上升:培养细胞转染SiRNA-AP-2α后可使VEGF mRNA的表达和蛋白的表达水平下降:而VEGF的过表达及SiRNA-VEGFA对AP-2α mRNA和蛋白的表达水平没有明显影响,因此,在小鼠血管内皮细胞中AP-2α可上调VEGF的表达.     

次声作用后血管内皮细胞超微结构的实验观察

目的: 观察8 Hz、130 dB次声不同时间暴露后大鼠血管内皮细胞的超微结构及血管内皮生长因子(VEGF)的表达.方法: 用8 Hz、130 dB的次声连续作用大鼠1 d、7 d、14 d,每天2 h,观察血管内皮细胞超微结构及VEGF表达的改变.结果: 在次声暴露期间,与对照组比较,7 d时内皮细胞出现线粒体肿胀和内质网扩张,14 d时出现内皮细胞脱落;VEGF表达随时间较对照组增强.结论: 次声可引血管内皮细胞超微结构与VEGF表达的变化,这些变化和次声暴露的时间有关.     

同种异体骨髓干细胞移植联合VEGF干预对心肌梗死后左室功能的影响

目的:探讨同种异体骨髓间质干细胞(BMSCs)移植联合血管内皮生长因子VEGF静脉注射对兔急性心肌梗死心功能的影响.方法:体外分离、纯化、培养兔BMSCs,以BrdU标记细胞.结扎冠状动脉建立急性心肌梗死模型后,随机将其分为4组(n=8),进行心肌内BMSCs移植和/或VEGF静脉注射,组Ⅰ:BMSCs移植+VEGF静脉注射;组Ⅱ:BMSCs移植;组Ⅲ:VEGF静脉注射:组Ⅳ:DMEM注射作为对照组.4周后行免疫组化和超声心动图检查.结果:组Ⅰ和组Ⅱ梗死及缺血心肌处可见大量BrdU标记的移植细胞,EF值组Ⅰ>组Ⅱ>组Ⅲ,组Ⅳ(均为P<0.01).结论:同种异体骨髓间质干细胞移植联合VEGF静脉注射能改善心功能,有利于心肌梗死后的康复.     

犬骨髓分化细胞种植人工血管体内植入实验研究

目的:骨髓分化细胞预种植,探讨增强人工血管内皮化方法.方法:选取选杂种犬8条.每组实验犬2条.实验犬采自体骨髓,定向分化提取培养为内皮细胞,种植人工血管内膜表面.对照犬采用血清和培养基预凝支架.将人工血管重建犬右室流出道,于术后第1、3月观察植入的人工血管通畅情况,采用免疫组织化学方法鉴定细胞,在光镜和电镜下观察人工血管新生内膜表面内皮化情况.结果:骨髓细胞分化后鉴定,第2带细胞CD34染色阳性,第5代细胞CD31、Ⅷ因子染色阳性.细胞种植人工血管7天内皮细胞覆盖.光镜观察植入后1月实验组部分内皮化.对照组无内皮细胞覆盖;术后第3月实验组基本实现内皮化,对照组少量内皮细胞覆盖,存在钙化灶.1月实验组扫描电镜显示细胞排列紧密,对照组细胞少,有少量血栓附着;3月实验组血管表面细胞排列成片,对照组部分覆盖.结论:骨髓细胞定向分化所获取为内皮细胞.内皮细胞种植于去细胞后牛颈静脉血管,可获得较理想的内皮化,减少体内不良反应发生.     

过表达bFGF促进大鼠急性缺血心肌的毛细血管生成

研究过表达人碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）在大鼠急性心肌缺血模型中的表达及其对心肌血管 再生的影响. 将含人bFGF质粒pcDNA/b转染人脐带静脉内皮细胞（HUVEC） ,进行MTT检测. pcDNA/b转染人肾细胞293细胞系,进行G418稳定筛选, Western 印迹检测.建立大鼠急性心肌梗死模型,分别将生理盐水、空质粒 pcDNA3.1(+)和pcDNA/b分3点注射于梗死交界处心肌内4周后取材,做常规 HE 染色和Masson 染色,测量各组微血管数量和梗死面积. 经免疫组织化 学染色鉴定和电镜观察,过表达外源bFGF可以促进HUVEC（细胞）生长; pcDNA/b能在293细胞中高效表达外源bFGF基因. pcDNA/b注射组梗死交界处 可见大量新生血管,毛细血管总数明显大于对照组,梗死面积明显小于对 照组. pcDNA/b组在梗死交界区有bFGF阳性表达.电镜观察显示,在梗死交界 处心肌细胞间有毛细血管增生,分化成2个血管腔.本实验证明,过表达bFGF 具有促进大鼠急性缺血心肌的毛细血管生成的作用,为bFGF基因治疗缺血 性心肌病的研究提供实验基础.     

冻融小鼠卵巢移植后细胞凋亡及血管内皮生长因子表达的变化及意义研究

目的:探讨冻融小鼠卵巢同种异体移植后细胞凋亡及血管内皮生长因子表达的变化及意义。方法:收集C57BL/6j雌鼠和BALB/c雄鼠杂交后F1代4周龄小鼠卵巢,慢冻速融后移植至杂交后F1代8～12周雄鼠的肾被膜下,分别于移植后1d(24h)、2d(48h)和7d回收移植物,将冻融以及移植后不同时间段的卵巢组织进行HE染色、全卵巢卵泡计数、电镜观察、免疫组织化学分析细胞凋亡及RT-PCR检测VEGF基因表达。结果:冻融小鼠卵巢移植后随着时间的推移、各级卵泡数和卵泡存活率逐渐下降;移植后48h内细胞凋亡指数最高;电镜观察发现小鼠卵巢组织移植后损伤主要发生在移植后48h内;移植后VEGF的表达有上升的趋势,至第7d仍维持较高水平;移植后48hVEGF120mRNA和VEGF188mRNA水平明显升高(P0.05),至7d下降恢复至移植前水平,而VEGF164mRNA水平移植后无明显变化(P0.05)。结论:小鼠卵巢组织移植后48h内细胞凋亡最为严重,移植后引起大量卵泡的丢失;在移植后血管化的过程中VEGFmRNA表达量增加,VEGF120mRNA和VEGF188mRNA可能参与卵巢移植后早期血管化过程。     

卵巢癌细胞与腹膜间皮细胞相互作用对VEGF和bFGF表达的影响

目的探讨卵巢癌细胞与腹膜间皮细胞(HPMC)相互作用对血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)表达的影响。方法用Millicell将卵巢癌细胞SKOV3与HPMC进行非接触性共培养,用RT-PCR检测细胞VEGF及bFGF mRNA表达,ELISA检测细胞条件培养液中VEGF及bFGF蛋白水平。结果共培养后,SK-OV3 VEGF及bFGF mRNA表达分别为3.62±0.23及3.41±0.25,条件培养液中VEGF及bFGF蛋白水平分别为(523.5±24.9)pg/ml及(156.4±17.3)pg/ml,与SKOV3单独培养时相比,差别均有显著性(P<0.01)。HPMC共培养后VEGF及bFGF mRNA表达分别为3.96±0.09及3.54±0.21,条件培养液中VEGF及bFGF蛋白水平分别为(1567.62±45.42)pg/ml及(682.9±33.7)pg/ml,均明显高于HPMC单独培养时的水平(P<0.01)。结论卵巢癌细胞与HPMC均可合成VEGF及bFGF;二者共培养时,相互刺激表达更高的VEGF及bFGF。     

Model analysis of difference between EGF pathway and FGF pathway

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

pp42/44MAP Kinase Is a Component of the Neurogenic Pathway Utilized by Nerve Growth Factor in PC12 Cells

Nerve growth factor-stimulated mitogen-activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE-Sephacel, phenyl-Sepharose CL4B, and S-200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr-97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor-stimulated kinase activation was diminished by down-regulation of protein kinase C with 200 nM 12-phorbol 13-myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor-mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutyryl cyclic AMP, do so independently of activating this kinase system.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Inhibition of FGF‐FGFR and VEGF‐VEGFR signalling in cancer treatment

The sites of targeted therapy are limited and need to be expanded. The FGF‐FGFR signalling plays pivotal roles in the oncogenic process, and FGF/FGFR inhibitors are a promising method to treat FGFR‐altered tumours. The VEGF‐VEGFR signalling is the most crucial pathway to induce angiogenesis, and inhibiting this cascade has already got success in treating tumours. While both their efficacy and antitumour spectrum are limited, combining FGF/FGFR inhibitors with VEGF/VEGFR inhibitors are an excellent way to optimize the curative effect and expand the antitumour range because their combination can target both tumour cells and the tumour microenvironment. In addition, biomarkers need to be developed to predict the efficacy, and combination with immune checkpoint inhibitors is a promising direction in the future. The article will discuss the FGF‐FGFR signalling pathway, the VEGF‐VEGFR signalling pathway, the rationale of combining these two signalling pathways and recent small‐molecule FGFR/VEGFR inhibitors based on clinical trials.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.