The Increase of VEGF Secretion From Endothelial Progenitor Cells Post Ultrasonic VEGF Gene Delivery Enhances the Proliferation and Migration of Endothelial Cells

We investigated the feasibility of exogenous gene expression in endothelial progenitor cells (EPCs) through the use of ultrasonic microbubble transfection (UMT). EPCs originating from porcine peripheral blood were cultured in a medium containing constructed vascular endothelial growth factor (VEGF) pDNA followed by UMT. Simultaneously, comprehensive functional evaluations were conducted to investigate the effects of UMT of the VEGF gene on the EPCs. The results showed that UMT yielded significant VEGF protein expression. VEGF-containing supernatant originating from EPCs post UMT led to significantly enhanced activities of proliferation by more than 20% and migration by approximately 30% in human aortic endothelial cells. The duration of additional secretion of VEGF protein attributable to the exogenous VEGF gene in the EPCs post UMT lasted more than 96 hours. In conclusion, UMT successfully delivers the VEGF gene into porcine EPCs, and VEGF-containing supernatant derived from EPCs post UMT enhances the proliferation and migration of human aortic endothelial cells.     

人外周血内皮前体细胞的分离与体外培养

目的研究体外诱导培养外周血来源内皮前体细胞(endothelial progenitor cells,EPCs)的可行性并检测其表型和功能。方法用密度梯度离心法从人外周血中分离单个核细胞。在加有胎牛血清(fetal blood serum,FBS)、血管内皮生长因子(vascular end othelial growthfactor,VEGF)和碱性成纤维生长因子(basic fibroblast growthfactor,bFGF)的M199培养液中培养。用流式细胞仪检测其表面标志CD34和KDR的表达情况。通过荆豆凝集素-1(ulex europaeus agglutinin-1,UEA-1)结合试验和乙酰化低密度脂蛋白(acetylatedlowdensitylipoprotein,acLDL)吞噬试验确定其内皮细胞功能。结果培养过程中可观察到典型的内皮细胞。培养7天后,粘附细胞表达CD34和KDR,阳性率分别为29.5%±9.9%和33.8%±9.2%。粘附细胞的UEA-1结合试验和acLDL吞噬试验均为阳性。结论从成人外周血中可分离到EPCs,并能在体外增殖分化为内皮细胞。体外培养是获得EPCs移植和体外组织工程技术所需要的足够EPCs数量的一条途径。     

血管内皮祖细胞参与人结肠腺癌肺转移灶血管新生的实验研究

目的：初步观察血管内皮祖细胞参与人结肠癌肺转移灶血管生成的情况。方法：体外传代扩增血管内皮祖细胞（EPC）后，采用4’6-二脒基-2-苯基吲哚盐酸盐（DAPI）标记。由尾静脉注入接种人结肠腺癌细胞株LS-174-T细胞7周的荷瘤裸鼠体内，分别于接种1周、2周后处死动物，摘取肺脏冰冻切片，荧光显微镜下观察血管内皮祖细胞参与结肠癌肺转移灶血管形成。结果：荧光显微镜下可以观察，接种血管内皮祖细胞1周后的裸鼠肺部血管中有发出蓝色荧光的血管内皮祖细胞，接种2周后肺部转移灶中有蓝色荧光。结论：血管内皮祖细胞可参与人结肠癌肺转移灶血管形成。     

C.elegans fat-1基因对SGC7901细胞的促凋亡作用

目的通过在人胃癌SGC7901细胞系上转入外源C.elegans fat-1基因观其对细胞增殖或对调亡的影响。方法构建携带有能够编码n-3多聚饱和脂肪酸脱氢酶的小秀丽隐杆线虫fat-1基因的重组质粒,转染人胃癌细胞SGC7901,检测n-6/n-3脂肪酸的比例,观察胃癌细胞的增殖和凋亡情况。结果转染成功后携带有fat-1基因的胃癌细胞,显示了对于n-3脂肪酸饱和酶的高表达。脂类分析表明,n-6PUFAs的比例大幅度降低,n-3PUFAs水平明显提高,n-6/n-3脂肪酸比例,从10.45下降到了0.79,尤其是花生四烯酸和二十碳戊烯酸的比率。相应地,在表达有fat-1基因的胃癌细胞中,来源于n-6PUFAs的类花生酸含量有显著减少。同时,fat-1基因的转移导致大量胃癌细胞的凋亡,抑制胃癌细胞的增殖。结论n-3脂肪酸去饱和酶的基因转移,能够有效调整人肿瘤细胞n-6/n-3脂肪酸的比例,起到抗癌作用,证明在癌症的预防和治疗中,n-6与n-3脂肪酸的比例扮演着一个重要的角色。     

Bioeffects of Ultrasound-Stimulated Microbubbles on Endothelial Cells: Gene Expression Changes Associated with Radiation Enhancement In Vitro

Ultrasound can be used to target endothelial cells in cancer therapy where the destruction of vasculature leads to tumor cell death. Here, we demonstrate ultrasound bioeffects in which the levels of genes in endothelial cells can be significantly altered by ultrasound-stimulated microbubble exposure. These were compared with established effects of radiation on endothelial cells at a gene level. Human-endothelial cells were exposed to ultrasound and microbubbles, radiation or combinations of ultrasound, microbubbles and radiation. Gene expression analyses revealed an up-regulation of genes known to be involved in apoptosis and ceramide-induced apoptotic pathways, including SMPD2, UGT8, COX6B1, Caspase 9 and MAP2K1 with ultrasound-stimulated microbubble exposure but not SMPD1. This was supported by immunohistochemistry and morphologic changes examined with cell microscopy, which showed changes in SMPD1 gene product in cells with microbubble exposure. This supports the hypothesis that ultrasound-stimulated microbubbles can induce significant bioeffect-related changes in gene expression and can affect ceramide signaling pathways in endothelial cells, leading to apoptosis.     

Ultrasound and Microbubble-Targeted Delivery of Small Interfering RNA Into Primary Endothelial Cells Is More Effective Than Delivery of Plasmid DNA

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.     

洛伐他汀对血管内皮细胞与平滑肌细胞增殖调节作用的对比实验研究

目的探讨洛伐他汀对人血管内皮细胞与平滑肌细胞的调节作用及其调节差异性。方法配制含不同浓度洛伐他汀的培养基f0、0．001、O．01、0,1、1、10μmol／L）,分别于96孔板中进行人脐静脉内皮细胞（HUVECs）与血管平滑肌细胞（VSMCs）培养,在培养24、72、120h后以MTT法对两种细胞进行细胞活性及增殖情况评估。结果培养24h,0．1μmol／L的洛伐他汀对HUVECs存在明显的促进增殖作用;培养72h,1斗mol／L的洛伐他汀对HUVECs存在明显的促进增殖作用;培养120h后,O．001、0．01、0．1、1μmol／L的洛伐他汀与对照组相比HUVECs增殖活性无明显差异,10μmol／L的洛伐他汀表现出明显的增殖抑制作用。培养24h和120h,各浓度组洛伐他汀对VSMCs的增殖无显著调节作用;培养72h,0．001、0．01、1、10μmol／L的洛伐他汀对VSMCs的增殖存在部分抑制作用,未发现剂量相关规律性。结论洛伐他汀对人血管内皮细胞增殖存在双向调节作用,与浓度相关。体外浓度0.1、1μmol／L时可以表现出明显的促进增殖作用,浓度达10μmol／L时则表现出抑制作用。相同浓度下,洛伐他汀无促进VSMCs增殖的作用,并且存在部分抑制VSMCs增殖的作用。     

利用基因芯片技术研究造血生长因子在脐静脉内皮细胞中的表达

为探讨利用基因芯片技术研究脐静脉内皮细胞中造血生长因子基因的表达情况，将体外培养的人脐静脉内皮细胞(ECV304)分为血管内皮生长因子(VEGF)处理组(添加50ng／ml的rhVEGF165)和正常对照组；培养24小时后收获细胞，提取总RNA，制备Cy3-dUTP标记的对照组cDNA探针和Cy5-dUTP标记的VEGF组cDNA探针，并与表达谱芯片杂交。杂交信号经荧光共聚焦显微镜扫描后，计算机分析基因表达情况。结果显示。两组细胞均表达多种造血生长因子及受体如Epo/R、GM—CSF／R、G—CSF／R、LIF、IL-3、TPO、Fit-3和SCF；同时也表达多种生长因子(VEGF、IGF2、PDGFA、PDGFB、TGFβ1)和生长因子受体(神经纤维黏蛋白-1，神经纤维黏蛋白-2，TGFβ-R1)；此外，共有24个基因存在差异表达。结论：利用基因芯片技术能够在短时间内迅速而准确地分析脐静脉内皮细胞众多造血生长因子及其受体的表达情况，为进一步研究内皮细胞的生物学特性提供了有效手段。     

骨髓基质细胞对人造血CD_34~+细胞体外扩增及基因转导的作用

目的：探讨造血基质细胞对人外周血干细胞体外培养扩增及基因转导的促进作用。方法：应用基质细胞支持培养扩增体系进行人造血干细胞体外培养扩增及基因转导。结果：骨髓基质细胞和造血生长因子共同支持组的造血ＣＤ＋３４细胞在体外培养３周后,其造血祖克隆形成能力较单纯造血生长因子支持组高３０．７％（Ｐ＜０．０５）。在有基质细胞支持时,逆转录病毒载体上清转导人造血ＣＤ＋３４细胞后,其造血细胞克隆中Ｎｅｏ基因阳性克隆是无基质支持对照组的２倍。结论：基质细胞的支持有维持造血干细胞原始造血活性及促进基因转导的双重好处。     

沉默Caveolin-1基因对膀胱癌细胞增殖和凋亡的影响

目的探讨沉默 Caveolin-1（Cav-1）基因对人膀胱癌细胞株5637增殖和凋亡的影响。方法通过siRNA转染人膀胱癌细胞株5637（沉默组）,以转染 FAM-siRNA为阴性对照组,未转染细胞为正常对照组。Re-al-time PCR法检测Cav-1基因沉默的效率,CCK-8法检测各组细胞的增殖情况,流式细胞术检测各种细胞的凋亡情况。结果 siRNA转染可显著降低人膀胱癌细胞株5637中 Cav-1 mRNA 的表达水平。沉默组细胞的增强能力明显减低,而细胞凋亡率明显升高（均P＜ 0.05）。结论沉默Cav-1基因可抑制人膀胱癌细胞株5637的细胞增殖并促进细胞凋亡。     

人血管内皮生长因子165基因联合间充质干细胞治疗对扩张型心肌病胶原重塑的影响

目的：探讨人血管内皮生长因子165基因（hVEGF165）联合间充质干细胞（mesenchyma1stemce11s,MSCs）治疗对扩张型心肌病胶原重塑的影响。方法：将40只扩张型心肌病（DCM）大鼠模型随机分为4组：PBS组、MSCs心肌移植联合基因治疗组（MSCs—GENE组）、MSCs心肌移植组（MSCs组）以及基因治疗组（GENE组）。心肌局部注射所构建MLC-2v／pIRES2-EG-FP-hVEGF165与MSCs,采用逆转录-聚合酶链反应（RT—PCR）检测I型、Ⅲ型胶原和转化生长因子-β（TGF-β）基因表达,蛋白免疫印迹（Westrenb1ot）检测hVEGF165蛋白表达。结果：PBS组、MSCs＋GENE组、MSCs组以及GENE组的I型胶原PCR产物灰度比分别为（0．359±0．010）、（0．240±0．012）、（0．313±0．058）、（0．230±0．011）;而Ⅲ型胶原分别为（0．831±0．011）、（0．842±0．015）、（0．917±0．058）、（0．688±0．015）;TGF-β1分别为（0．548±0．067）、（0．370±0．012）、（0．561±0．014）、（0．369±0．098）;MSCs＋GENE组TGF-81与GENE组比较差异无统计学意义,但两者显著低于PBS组和MSCs组,提示TGF-β1表达下调;I型／III型胶原灰度比分别为（0．436±0．072）、（0．290±0．023）、（0．337±0．021）、（0．333±0．011）,其中MSCs组与GENE组比较差异无统计学意义;MSCs组与GENE组的I型／III型胶原灰度比减低,2组比较差异无统计学意义,但MSCs—GENE组进-步使I型／III型胶原灰度比减低;TGF-β1表达分析结果显示,hVEGF165基因治疗较MSCs移植使TGF-β1表达下调的作用更显著。结论：MSCs移植与hVEGF165基因治疗有可能通过下调TGF－β1表达,降低I型／III胶原比值,增加心肌顺应性,减轻心肌胶原网络重塑而改善心功能。     

非诺贝特对LPC诱导的脐静脉内皮细胞NO及eNOS基因表达的影响

【目的】探讨非诺贝特对LPC诱导的脐静脉内皮细胞一氧化氮（NO）及内皮型一氧化氮合酶（eNOS）基因表达的影响。【方法】体外培养人脐静脉内皮细胞（HUVECs）,分为：①正常对照组;②溶血卵磷脂（LPC）组;③低浓度非诺贝特组（10μmol／L）;④中浓度非诺贝特组（50μmol／L）;⑤高浓度非诺贝特组（100μmol／L）。采用实时定量PCR（real-time PCR）及流式细胞仪（FCM）分别观测LPC对内皮细胞eNOS mRNA及蛋白表达的影响,采用硝酸还原酶法测定NO含量,并观测非诺贝特干预后的变化。【结果】与正常对照组比较,LPC使HUVEC eNOS mRNA及蛋白表达降低,NO合成减少。非诺贝特可干预LPC对内皮细胞的作用,使eNOS mRNA及蛋白表达升高,NO合成增加,且其作用呈时间-效应、浓度-效应依赖关系。【结论】非诺贝特可改善LPC对HUVECs的影响,使内皮细胞eNOS mRNA及蛋白表达升高,NO合成增加,从而起到抗动脉硬化作用。