H2-B1基因转染小鼠内皮细胞对其免疫原性的影响

目的 转染pEGFP-N1-H2B1质粒载体至小鼠血管内皮细胞(VEC),观察导入H2-B1基因后VEC免疫原性的变化,探讨借鉴母胎免疫耐受模型诱导心脏移植术后免疫耐受的机制.方法 以未处理的VEC为对照,采用流式细胞仪和逆转录-聚合酶链反应(RT-PCR)技术分别于转染H2-B1基因后24、48、72 h检测质粒转染效率以及H2-B1基因的mRNA相对表达量;将同源小鼠外周血单个核细胞(PBMC)和VEC按100:1和10:1两种不同效靶比混合,观察PBMC对靶细胞杀伤活性的变化.结果 H2-B1基因组H2-B1基因在VEC中的mRNA表达量明显高于对照组(0.5mg/L组,P<0.05;1.0 mg/L组,P<0.01).H2-B1基因转染48 h后,当效靶比为100:1时,与对照组细胞毒性14.29%比较,0.5 mg/L基因组细胞毒性为4.28%,提示转染H2-B1基因组PBMC对VEC的毒性作用明显降低(P<0.05).结论 H2-B1基因可能具有免疫调节功能,可降低VEC的免疫原性,抑制PBMC对VEC的杀伤活性,诱导免疫耐受.     

H2-B1基因转染小鼠内皮细胞对其免疫原性的影响

目的 转染pEGFP-N1-H2B1质粒载体至小鼠血管内皮细胞(VEC),观察导入H2-B1基因后VEC免疫原性的变化,探讨借鉴母胎免疫耐受模型诱导心脏移植术后免疫耐受的机制.方法 以未处理的VEC为对照,采用流式细胞仪和逆转录-聚合酶链反应(RT-PCR)技术分别于转染H2-B1基因后24、48、72 h检测质粒转染效率以及H2-B1基因的mRNA相对表达量;将同源小鼠外周血单个核细胞(PBMC)和VEC按100:1和10:1两种不同效靶比混合,观察PBMC对靶细胞杀伤活性的变化.结果 H2-B1基因组H2-B1基因在VEC中的mRNA表达量明显高于对照组(0.5mg/L组,P<0.05;1.0 mg/L组,P<0.01).H2-B1基因转染48 h后,当效靶比为100:1时,与对照组细胞毒性14.29%比较,0.5 mg/L基因组细胞毒性为4.28%,提示转染H2-B1基因组PBMC对VEC的毒性作用明显降低(P<0.05).结论 H2-B1基因可能具有免疫调节功能,可降低VEC的免疫原性,抑制PBMC对VEC的杀伤活性,诱导免疫耐受.     

血管形成素-1基因转染对兔缺血心肌血管形成的影响

血管形成素-l(Angl)可促进缺血区血管新生，对血管内皮细胞具有特异性及独特的拮抗微血管渗出作用而备受瞩目。本研究观察Angl在兔缺血心肌中促血管形成作用，报道如下。     

基因转染内皮细胞衬里人工血管的研究进展

目的：讨论使用外源性抗凝基因修饰内皮细胞后衬里人工血管，使人工血管局部抗凝活性提高以及对内皮细胞其他生物活性的影响等。方法：复习近几年的相关文献，作综述报道。结果：内皮细胞在转染纤溶酶原激活物(t-PA、u-PA、Urokinase)、血栓调节蛋白(TM)和水蛭素(hirudin)等基因后，均使局部血管的抗凝活性增高，但同时也导致内皮细胞粘附性、增殖功能下降。结论：外源性基因能使内皮细胞预衬的人工血管抗凝活性提高，但仍需进行深入研究。     

腺病毒介导入VEGF165和ANG-1双基因转染大鼠BMSCs及对其增殖的影响

[目的]探讨携带人血管内皮细胞生长因子165(VEGF165)和血管生成素-1(ANG-1)双基因的腺病毒表达载体pAd-VIA转染大鼠骨髓基质干细胞(BMSCs)的体外表达及对BMSCs增殖的影响.[方法]将本室构建的编码人VEGF165和ANG-1双基因的腺病毒质粒pAd-VIA在QBI-293A细胞内进行包装和扩增,体外转染大鼠BMSCs,通过绿色荧光蛋白(GFP)表达、Western-blotting、酶联免疫吸附分析(ELISA)方法检测外源基因的表达,利用MTT法检测MOI(multiplicity of infection)=50、100、200、400 pfu/cell不同浓度腺病毒转染后对BMSCs增殖活性的影响.[结果]重组腺病毒质粒pAd-VIA在QBI-293A细胞内成功包装和扩增,体外转染BMSCs后,有大量的GFP 表达,Western-blotting检测显示,转染组与VEGF165和ANG-1抗体结合,在45 KD和14.4 KD左右出现印迹条带;ELISA结果显示:转染组第1、2、3 d,上清中的VEGF165浓度和ANG-1浓度持续增加,未转染组均未检测到外源基因的表达,差异有统计学意义(P<0.01);不同浓度的腺病毒载体转染BMSCs后,促进细胞增殖,细胞生长曲线上移,在1、3、5、7 d转染组的OD值均大于未转染组,差异有统计学意义(P<0.01),第9 d,细胞进入平台期,转染组和未转染组的OD值差异无统计学意义(P>0.05).[结论]重组腺病毒pAd-VIA转染大鼠BMSCs后,外源基因在体外得到有效表达,同时在观察时间内可以促进大鼠BMSCs的增殖.     

同源盒B2基因在内皮细胞增殖中的作用及血管内皮生长因子对内皮细胞放射损伤的保护作用

目的　探讨同源盒B2 (HOXB2)基因在人脐静脉内皮细胞 (HUVEC)增殖中的作用及血管内皮生长因子 (VEGF)对HUVEC放射损伤的保护效应。　方法　分离、培养HUVEC。(1)配制寡脱氧核苷酸 (ODN)或反义ODN(ASODN)浓度为 0 .2 5、0 .5 0、1.0 0、2 .5 0mg/L的脂质体 HOXB2 ASODN、脂质体 ODN,用以刺激HUVEC。以噻唑蓝 (MTT)法和氚标记胸腺嘧啶脱氧核苷 (3 H TdR)掺入法检测刺激后HUVEC增殖活性的变化 ,以流式细胞仪进行细胞周期分析 ,以逆转录聚合酶链反应 (RT PCR)检测HUVEC内HOXB2mRNA的表达水平。 (2 )对HUVEC进行下述处理 :加入 5 0 μg/LVEGF;以 6、12Gy的60 Coγ射线照射 ;12Gy60Coγ射线照射后加入 5 0 μg/LVEGF。2 4、4 8h后进行形态学观察 ,采用MTT法检测细胞增殖活性的变化。　结果　 (1)与脂质体 ODN比较 ,脂质体 HOXB2 ASODN对HUVEC的增殖有明显抑制作用 (P <0.0 5或 0 .0 0 1) ,且ASODN浓度越高变化越明显。 2 .5 0mg/L的ASODN可明显减少HUVEC的S期细胞数量及HOXB2mRNA表达水平 (P<0.0 1或 0 .0 0 1)。 (2 ) 60 Coγ射线照射可造成HUVEC核增大 ,胞浆多空泡 ,多核及核肿胀。当以 6Gy照射 2 4、4 8h时 ,细胞增殖活性分别由未照射时的 0 .36 5± 0 .0 4 7、0 .4 87± 0 .0 2 2升至 0 .5 5 7± 0     

HLA-G1抑制人自然杀伤细胞杀伤猪血管内皮细胞的研究

目的　研究HLA G1基因抑制人自然杀伤细胞 (NK细胞 )对猪血管内皮细胞杀伤的作用。方法　利用脂质体介导的基因转染技术将 pcDNA3 HLA G1转入原代培养的猪血管内皮细胞 ,以间接免疫荧光技术在蛋白质水平上检测HLA G1分子在猪内皮细胞上的表达 ;以NK细胞系(NK92 )和外周血单个核细胞为效应细胞 ,用四甲基偶氮唑盐 (MTT)法检测转染细胞对NK细胞杀伤活性的抑制效应。结果　与未转染HLA G1的对照组相比 ,NK92和外周血单个核细胞对转有HLA G1的猪血管内皮细胞的杀伤效率均有明显降低 (P <0 .0 5 )。结论　HLA G1分子可以明显抑制人NK细胞对猪血管内皮细胞的杀伤。     

腺病毒介导人VEGF_(165)和ANG-1双基因转染大鼠BMSCs及对其增殖的影响

[目的]探讨携带人血管内皮细胞生长因子165(VEGF165)和血管生成素-1(ANG-1)双基因的腺病毒表达载体pAd-VIA转染大鼠骨髓基质干细胞(BMSCs)的体外表达及对BMSCs增殖的影响。[方法]将本室构建的编码人VEGF165和ANG-1双基因的腺病毒质粒pAd-VIA在QBI-293A细胞内进行包装和扩增,体外转染大鼠BMSCs,通过绿色荧光蛋白(GFP)表达、Western-blotting、酶联免疫吸附分析(ELISA)方法检测外源基因的表达,利用MTT法检测MOI(multiplicity of infection)=50、100、200、400 pfu/cell不同浓度腺病毒转染后对BMSCs增殖活性的影响。[结果]重组腺病毒质粒pAd-VIA在QBI-293A细胞内成功包装和扩增,体外转染BMSCs后,有大量的GFP表达,Western-blotting检测显示,转染组与VEGF165和ANG-1抗体结合,在45 KD和14.4 KD左右出现印迹条带;ELISA结果显示:转染组第1、2、3 d,上清中的VEGF165浓度和ANG-1浓度持续增加,未转染组均未检测到外源基因的表达,差异...     

转染人补体调节基因对猪血管内皮细胞的保护作用

供者血管内皮细胞(EC)是猪到人异种移植时引发超急性排斥反应的靶抗原的主要分布部位。利用转基因技术让猪的内皮细胞表达人补体调节基因(hDAF)，可能会增强其血管内皮细胞的防御能力，借以克服超急性排斥反应的发生，联合转染一种以上人补体调节蛋白基因可能效果更好。为此，我们进行了如下实验。     

Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells

BACKGROUND: Cyclophilin A (CypA) is a cytosolic protein which involves many biological functions including immune modulation, cell growth, tumorigenesis, and vascular disease. The objective of this study was to determine the effect of CypA on cell proliferation and several gene expressions in human endothelial cells and vascular smooth muscle cells. METHODS: Human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HMVEC-L), and human aorta smooth muscle cells (HAoSMC) were used in this study. Cells were treated with 10 nM CypA for 24 h. The cell proliferation was determined by [3H]thymidine incorporation. The mRNA levels of 13 genes including CD147 (receptor for CypA), PDGF-BB, endothelin-1 (ET-1), vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1), NRP-2, eNOS, iNOS, nNOS, ICAM-1, and PECAM-1 were semiquantitatively determined by real time RT-PCR as standardized with a house keeping gene beta-actin. RESULTS: CypA significantly increased cell proliferation of HAoSMC and HMVEC-L by 31% and 45%, respectively, as compared to controls, but had no effect on HCAEC. Blocking CD147 did not affect the mitogenic action of CypA. In addition, CypA also significantly increased the mRNA expression of CD147 by 43% and VEGFR-2 by 65% in HAoSMCs (P < 0.05, t test). HAoSMCs expressed much higher CD147 and neuropilin-1 (NRP-1) mRNA than HMVECs-L and HCAECs (P < 0.017, ANOVA). Furthermore, CypA increased ET-1 mRNA by 22% and VEGFR-1 mRNA by 23% in HMVECs-L, but had limited effects on HCAECs. HMVECs-L had much higher expressions of PDGF-BB, ET-1, VEGFR-2, VEGFR-1, VEGFR-3, and NRP-2 than HAoSMCs and HCAECs (P < 0.017, ANOVA). By contrast, HCAECs had much higher ICAM-1 mRNA levels than HMVECs-L and HAoSMCs (P < 0.017, ANOVA). CONCLUSIONS: These data demonstrate that CypA has a mitogenic effect on HAoSMCs and HMVECs-L, but not HCAECs. CD147 may not mediate the action of CypA. In addition, CypA substantially alters the mRNA levels of several key genes in human vascular cells, indicating potential multifunctional roles of CypA in vascular system. Furthermore, this study provides several new aspects of gene expressions in vascular cells.     

不均一性核糖核蛋白A2/B1在脂多糖诱导的血管内皮细胞通透性增加中的作用

目的 探讨不均一性核糖核蛋白A2/B1(heterogeneous ribonucleoprotein A2/B1,hnRNP A2/B1)在脂多糖(lipopolysaccharide,LPS)刺激的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)通透性中的作用.方法以HUVECs为研究对象,筛选出具有hnRNP A2/B1基因沉默作用的小干扰RNA(small interfering RNA,siRNA),按不同处理方法实验分为转染阴性对照siRNA组(NC siRNA组)、 转染hnRNP A2/B1 siRNA组(hnRNP A2/B1 siRNA组)、 转染阴性对照siRNA组并进行LPS干预组(NC siRNA组-LPS组)、转染hnRNP A2/B1siRNA组并进行LPS干预组(hnRNP A2/B1 siRNA组-LPS组),分别给予或不给予LPS(1 mg/L)刺激,检测不同时间点(0、6、12、24 h)各组细胞跨膜电阻抗(transendothelial electrical resistance,TEER)值,检测LPS刺激12 h后细胞表面血管内皮钙黏蛋白(vascular endothelial cadherin,VE-cadherin)及细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)蛋白表达变化.结果在相同剂量LPS诱导下,hnRNP A2/B1 siRNA-LPS组HUVECs的TEER在12 h低于NC siRNA-LPS组(P<0.05);在LPS刺激12 h后,hnRNP A2/B1 siRNA-LPS组HUVECs表面VE-cadherin表达明显低于NC siRNA-LPS组,ICAM-1表达明显高于NC siRNA-LPS组(P<0.05).结论hnRNP A2/B1可能通过调节VE-cadherin及ICAM-1蛋白表达影响血管内皮细胞的通透性,发挥血管内皮屏障保护功能.     

The effect of flow on the neutrophil-mediated Ca2+ responses in human vascular endothelial cells stimulated by endotoxin

2+ levels ([Ca²⁺]_i). Cultured human umbilical vein ECs stimulated by endotoxin were labeled with Fura-2 and exposed to fluid flow with neutrophils. The individual changes in [Ca²⁺]_i were monitored. The application of flow with neutrophils to stimulated ECs led to an increase in [Ca²⁺]_i although either flow without neutrophils or neutrophils without flow rarely induced a rise in [Ca²⁺]_i. Furthermore, flow application with neutrophils to unstimulated ECs also rarely promoted a rise in [Ca²⁺]_i. These findings suggest that the flow might thus induce or enhance the inflammatory process by the induction of Ca²⁺ signaling in endotoxin-stimulated endothelium facing neutrophils in the blood flow. (Received for publication on Sept. 28, 1998; accepted on Mar. 11, 1999)     

Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps

BACKGROUND: Neovascularization occurs through two mechanisms: angiogenesis and vasculogenesis. Therefore, there are two strategies to promote neovascularization: therapeutic angiogenesis and therapeutic vasculogenesis (endothelial progenitor cells therapy). MATERIALS AND METHODS: In this study, we examined whether or not endothelial progenitor cells combined with vascular endothelial growth factor (VEGF) gene therapy is useful for ischemia surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted endothelial progenitor cells (EPCs) transducted with VEGF165 gene and EPCs alone. EPCs were isolated from cord blood of healthy human volunteers, cultured in vitro for 7 days and identified by immunofluorescence. After transduced with VEGF165 gene in vitro, proliferative activity of EPCs was assessed using MTT assay. CM-DiI was used to trace EPCs in vivo 4 days after injection of 5 x 10(5) VEGF-transduced EPCs(VEGF-transduced EPCs group, n = 10), 5 x 10(5) EPCs (non-transduced EPCs group, n = 10) in 500 microL EBM-2 media, or 500 microL EBM-2 media (EBM-2 media group, n = 10) local, a cranially based flap was elevated on the back of nude mice. The percent flap survival, neovasculariztion and blood flow recovery of flaps was detected. RESULTS: EPCs expressed cell markers CD34, KDR, and CD133. A statistically significant increase in percent flap survival was observed in mice of VEGF-transduced EPCs group as compared with that of non-transduced EPCs group: 67.99 +/- 6.64% versus 59.43 +/- 4.69% (P < 0.01), and 41.24 +/- 2.44% in EBM-2 media group (P < 0.01). The capillary density and blood flow recovery of flaps in VEGF-transduced EPCs group were both improved. CM-DiI-labeled VEGF-transduced EPCs were observed in vivo and the numbers of cells increased. CONCLUSION: EPCs from human cord blood can increased neovascularization of ischemic flaps and augmented the survival areas, and VEGF-transduced EPCs have more powerful ability of promoting neovascularization in animal model of ischemic flaps.     

SH2-B对结肠癌HT-29细胞增殖的影响

目的：探讨SH2-B对结肠癌HT-29细胞周期演进和细胞增殖的影响。
方法：免疫荧光筛选SH2-B低表达结肠癌细胞（HT-29）。用LipofectAMINE2000将pcDNA3.1-SH2-B质粒转染至HT-29细胞（转染组），并设空载体转染组和未转染组作为对照。Western-blotting分析SH2-B转染后SH2-B的表达，MTT法分析SH2-B转染后HT-29细胞的增殖，流式细胞术分析SH2-B转染后HT-29细胞周期变化。
结果：将pcDNA3.1-SH2-B质粒转染至HT-29细胞后可获得SH2-B的有效表达。转染SH2-B 后HT-29细胞增殖显著增强（P＜0.05）。流式细胞仪检测结果显示，转染SH2-B组S期细胞显著高于未转染组和空载体组（P＜0.05）。
结论：SH2-B促进结肠癌HT-29细胞增殖和细胞周期演进。

     

Effects of rHuEpo on cellular proliferation and endothelin-1 production in cultured endothelial cells

BACKGROUND.: Although elevation of blood pressure is considered to be themain adverse effect under rHuEpo therapy in haemodialysis patients,the precise mechanism remains obscure. The direct effect ofrHuEpo on endothelial cells (EC) has been suggested as one ofcontributing factors of rHuEpo-induced hypertension. METHODS.: EC were incubated with various concentrations of rHuEpo (0,1000, 5000, 10000 mU/ml) for up to 7 days, and cell numbers,DNA and protein synthesis by EC and supernatant concentrationsof immunoreactive endothelin-1 (ET) were determined by haemocytometer,³H-thymidine and ³H-leucine incorporation, and RIA, respectively.The effect of rHuEpo on EC proliferation was confirmed by anti-rHuEporabbit antiserum. The effect of cycloheximide or actinomycinD was also examined on the increase in ET production by rHuEpo. RESULTS.: rHuEpo dose-dependently stimulated the proliferation of culturedEC, and this proliferative effect was inhibited by anti-rHuEporabbit antiserum. DNA and protein syntheses by EC were alsoincreased by rHuEpo. The supernatant concentrations of ET culturedwith rHuEpo at 5000 mU/ml or more showed significantly greatervalues than those without rHuEpo and the increase in ET in thesupernatants of media containing 5000 mU/ml rHuEpo was inhibitedby incubation with 0.2 µg/ml actinomycin D or 10 µg/mlcycloheximide. Further, rHuEpo increased DNA synthesis by ECwhich had been cultured in E-BM medium containing 0.5 or 2%FBS for 3 h and which were recultured in E-BM medium containing5% FBS for 15 h. CONCLUSIONS.: rHuEpo directly stimulates EC proliferation as a competencefactor, and it also accelerates endothelin-1 production in associationwith stimulation of DNA and protein syntheses by EC.     

DNA损伤修复酶hOGG1降低食管鳞癌细胞对H2O2和顺铂的敏感性

目的 观察食管鳞状上皮癌( ESCC)细胞中,DNA损伤修复酶人8-羟基鸟嘌呤DNA糖苷酶( hOGG1)的高表达,对该细胞H2O2氧化剂和化疗药物顺铂(DDP)敏感性的影响。方法 构建重组腺病毒pAd-CMV5-DEST-hOGG1,并转染食管鳞癌细胞,建立高表达hOGG1的食管鳞癌细胞( EC9706-hOGG1)模型。H2O2和顺铂分别作用于转染重组腺病毒的EC9706细胞、转染空腺病毒EC9706细胞(EC9706-LACZ)以及未转染病毒EC9706( EC9706)细胞。用噻唑蓝(MTT)比色法和锥虫蓝染色法比较3种细胞的存活率,用8-羟基鸟嘌呤(8-oxoG)免疫组织化学比较3种细胞的氧化损伤程度,用TUNEL法和流式细胞仪(FCM)检测法比较3种细胞的凋亡。结果 高表达hOGG1的食管鳞癌细胞较两对照组细胞有更高的存活率、更低的8-oxoG氧化损伤程度和凋亡指数。用1000μmol/L H2O2作用1.5h后,用流式细胞仪检测,发现EC9706-hOGG1细胞较EC9706-LACZ及EC9706细胞凋亡减少。凋亡率为EC9706-hOGG1:5.50％,EC9706-LACZ:12.54％,EC9706:13.48％。用10 mg/L DDP作用1.5h后,发现EC9706-hOGG1细胞较EC9706-LACZ及EC9706细胞凋亡减少。凋亡率分别为EC9706-hOGGl:3.95％,EC9706-LACZ:11.59％,EC9706:11.48％。结论 食管鳞癌细胞中hOGG1蛋白高表达,可能会导致食管鳞癌细胞对氧化剂H2O2和化疗药物顺铂耐受。