沙立度胺抑制缺氧所致胃肠道血管发育不良的机制初探

目的 探讨沙立度胺抗血管生成的作用机制.方法 收集胃肠道血管发育不良患者手术切除的肠段标本,免疫组化检测病变肠段的血管内皮生长因子(VEGF)表达.体外缺氧条件下培养人脐静脉内皮细胞至对数生长期,随机分为6组,同步化后用不同浓度沙立度胺(40、60、80、100μ/ml)刺激72 h.ELISA和实时定量PCR法测定VEGF的表达,Western blot法测定低氧诱导因子1α(HIF-1α)的表达.结果 免疫组化结果显示,病变肠段血管扩张、扭曲,VEGF的表达明显增强.ELISA检测表明,缺氧培养条件下细胞上清中VEGF蛋白表达水平明显高于常氧状态[(1199.3±61.4)ng/L比(864.7±41.2)ng/L,P<0.05];沙立度胺可有效抑制缺氧条件下人脐静脉内皮细胞的VEGF蛋白表达.实时定量PCR结果亦提示沙立度胺能明显抑制缺氧状态下VEGF mRNA的表达(P<0.05).Western blot结果表明,沙立度胺可抑制缺氧状态下VEGF的上游调节因子HIF-1α的表达,且这一抑制作用呈剂量相关性.结论 初步体外研究表明,沙立度胺通过抑制缺氧条件下人脐静脉内皮细胞HIF-1α及VEGF的表达,从而抑制血管生成,是其有效治疗血管发育不良所致消化道出血的可能机制之一.     

沙利度胺对人脐静脉内皮细胞增殖和血管生成的影响

目的 探讨沙利度胺治疗血管发育不良所致消化道出血的机制.方法 体外培养人脐静脉内皮细胞至对数生长期,分为空白对照组、溶剂对照组(二甲基亚砜)和不同浓度(10、20、40、60、80、100μg/ml)沙利度胺组,根据加或不加成纤维细胞生长因子(bFGF,10 ng/ml),共分为16组.刺激72 h后,MTT法检测细胞增殖情况,酶联免疫吸附法和实时定量PCR法测定血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)表达.结果 加或不加bFGF刺激,中、高浓度(≥40/μg/ml)沙利度胺均能抑制人脐静脉内皮细胞增殖.未加bFGF刺激时.20μg/ml沙利度胺能明显抑制VEGF表达.加bFGF刺激时,10 μg/ml沙利度胺即能明显抑制VEGF表达.未检出TNF-α表达.结论 体外实验中,沙利度胺能抑制人脐静脉内皮细胞增殖和VEGF表达,从而抑制血管生成,达到治疗血管发育不良所致消化道出血的目的 .     

Therapeutic and prophylactic thalidomide in TNBS-induced colitis: Synergistic effects on TNF-α, IL-12 and VEGF production

AIM: To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor α (TNF-α), IL-12, and vascular endothelial growth factor (VEGF), hallmarks of intestinal inflammation in Crohn's disease (CD).METHODS: Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4+ T cells, macrophages, and VEGF+ cells were detected by immunohistochemistry. TNF-α and IL-12 were quantified in the supernatant of organ cultures by ELISA.RESULTS: Significant reduction in the inflammatory score and in the percentage of VEGF+ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-α and IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide.TNF-α levels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-α and IL-12 were significantly correlated with the inflammatory score and the number of VEGF+ cells.CONCLUSION: Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-α, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD.     

The role of HIF-1, angiopoietin-2, Dll4 and Notch1 in bleeding gastrointestinal vascular malformations and thalidomide-associated actions: a pilot in vivo study

OBJECTIVE: To investigate plasma levels of hypoxia inducible factor‐1 (HIF‐1), angiopoietin‐2 (Ang‐2), Delta‐like ligand 4 (Dll4) and Notch1 in patients with recurrent gastrointestinal bleeding due to gastrointestinal vascular malformation (GIVM) with or without thalidomide treatment. METHODS: Ten eligible patients with recurrent gastrointestinal bleeding due to GIVM, who received thalidomide 100 mg/d for 4 months, were followed up for 1 year. The effective response was the proportions of patients with yearly bleeding episodes reduced by ≥50% at 1 year after treatment. Plasma levels of HIF‐1, Ang‐2, Dll4 and Notch1 were measured using enzyme‐linked immunosorbent assay in the GIVM thalidomide treatment group before and after treatment (10 patients), the GIVM non‐thalidomide treatment group (25 patients) and the control group (18 participants). RESULTS: In the GIVM thalidomide treatment group, eight patients (8/10) achieved effective response and five (5/10) displayed complete cessation of bleeding. Mean plasma levels of HIF‐1, Ang‐2, Dll4 and Notch1 were all higher in the GIVM thalidomide and non‐thalidomide treatment groups than in the control group (all P < 0.001). However, Ang‐2 decreased more significantly in the effective subgroups (P = 0.003) and no‐bleeding patients (P = 0.008). CONCLUSIONS: HIF‐1, Ang‐2, Dll4 and Notch1 might participate in the formation of GIVM. Thalidomide is an effective and safe treatment agent for GIVM and its associated bleeding. The reduction degree of Ang‐2 after a 4‐month treatment of thalidomide may offer values for evaluating its prognosis and efficacy.     

Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti‐invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia‐mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF‐1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP‐2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia‐induced HIF‐1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF‐1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP‐2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia‐induced glioblastoma cell migration and invasion via inhibition of HIF‐1α. Considering the fact that overexpression of the HIF‐1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.     

缺氧条件下人脐静脉内皮细胞Ang-2、Notch1、Dll4、HIF-1α表达以及沙利度胺对其表达的影响水

背景：沙利度胺治疗胃肠道血管畸形所致的难治性消化道出血安全、有效,而胃肠道血管畸形所致的消化道出血可能与缺氧引起的血管代偿性增生有关。目的：探讨人脐静脉内皮细胞（HUVEC）血管生成素2（Ang-2）、Noteh1、Dll4和缺氧诱导因子（HIF）-1α在缺氧环境中的表达以及沙利度胺的干预机制。方法：HUVEC分别于常氧和缺氧环境下培养。缺氧培养的HUVEC分为溶剂对照组和不同浓度（25、50、100、200μg／mL）沙利度胺组。以Real-time PCR和蛋白质印迹法分别检测Ang-2、Noteh1、Dll4、HIF-1α mRNA和蛋白表达。结果：缺氧条件下Ang-2、Noteh1、Dll4、HIF-1α mRNA和蛋白表达均高于常氧状态,差异有统计学意义（P〈0．05）。沙利度胺可呈浓度依赖性地抑制HUVEC Ang-2、Noteh1、Dll4 mRNA和蛋白表达（P〈0．05）。结论：沙利度胺对Ang-2、Noteh1、Dll4表达的抑制作用可能是其抑制血管生成从而治疗胃肠道血管畸形致消化道出血的机制之一。     

Prognostic value of hypoxia inducible factor 1α in esophageal squamous cell carcinoma

Hypoxia inducible factor 1α (HIF 1α) plays a major role in the pleitropic response observed secondary to hypoxic conditions in tumors. Its expression in the tumor cells has been correlated to tumor aggressiveness and prognosis in squamous cell carcinoma (SCC) of the esophagus in Far Eastern population, but limited information is available on the prognostic role of HIF 1α in SCC of esophagus in European population. This information may help in choosing appropriate therapeutic strategies and possibly developing a monoclonal antibody with therapeutic potential targeting the HIF 1α. Tumor samples from 36 patients diagnosed with SCC of the esophagus were collected. Prepared tissue sections were stained with validated and specific monoclonal antibodies for HIF 1α and the expression was correlated with the disease pattern and survival. Out of 36 patients, 17 patients showed low and 19 high expression of HIF 1α. There was no difference in the disease‐free and overall survival between these two groups (P > 0.05, log rank test). Regression analysis showed that HIF 1α was not an independent prognostic factor for survival (P > 0.05). HIF 1α did not show prognostic value in SCC of the esophagus in our study on European population, in agreement with previous studies. Novel strategies on the therapeutic manipulation of HIF 1α in cancer are to be explored further and may have a role to play in improving treatment outcome.     

沙利度胺治疗血管发育不良所致消化道出血的疗效观察

目的 观察和研究沙利度胺治疗血管发育不良所致消化道出血的临床疗效及其机制.方法 收集2004年11月-2006年12月血管发育不良所致反复发作的消化道出血患者18例,给予沙利度胺片100 mg/d口服,疗程4个月.中位随访时间16.7个月,观察治疗前、后患者临床疗效和血清血管内皮生长因子(vEGF)和肿瘤坏死因子一a(TNF-a)水平变化.结果 患者临床疗效总评分由治疗前的(15.000±3.630)分降至治疗后的(5.330±3.325)分,差异有统计学意义(P＜0.01).随访期间,18例患者平均出血次数为(1.000±1.237)次,输血量为(100.000±240.098)ml,均比治疗前显著减少[(11.220±6.404)次和(1422.220±1556.601)ml,P值均＜0.01];血红蛋白含量为(9.533±2.278)g/ml,比治疗前显著上升[(5.950±1.656)g/ml,P＜0.01].5例患者服药前血清中VEGF浓度为180 pg/ml、TNF-α浓度为58 Pg/ml,治疗后VEGF和TNF-α浓度明显降低(116和34 pg/ml,P值均＜0.01).结论 沙利度胺能明显抑制血管发育不良消化道出血患者的血清VEGF、TNF-α水平,对血管发育不良所致的消化道出血疗效显著.     

缺氧诱导因子⁃1在弓形虫感染孕鼠中的表达

目的　探讨缺氧诱导因子⁃1（hypoxia⁃inducible factor⁃1, HIF⁃1）在小鼠妊娠早期感染弓形虫后母胎界面的表达及可能发挥的作用。方法　将20只妊娠C57BL/6小鼠（体质量为16～20 g）随机分为4组,即12 d对照组、12 d感染组和18 d对照组、18 d感染组。在小鼠孕6 d时,给予12 d感染组、18 d感染组小鼠腹腔注射弓形虫PRU株速殖子150个/只,孕12 d、孕18 d对照组孕鼠则注射等量PBS。于孕12 、18 d分别处死相应对照组和感染组小鼠,取各组孕鼠胎盘和子宫组织观察其组织病理变化;采用实时荧光定量PCR（qPCR）技术检测孕鼠胎盘和子宫中HIF⁃1α、HIF⁃1β及血管内皮生长因子（VEGF）mRNA表达水平,并分析HIF⁃1α和VEGF mRNA表达水平的相关性。此外,设置孕12 d感染组和孕18 d感染组PBS阴性对照组,采用免疫组化染色检测孕鼠胎盘和子宫组织中HIF⁃1α表达水平。结果　与孕12 d、孕18 d对照组相比,孕12 d、孕18 d感染组孕鼠出现畸胎、胎盘发育不良等不良妊娠结局。HE染色结果显示,与相应对照组相比,孕12 d、孕18 d感染组小鼠胎盘组织迷路区细胞分别出现肿胀和瘀血,且均出现血窦减少、炎性细胞浸润;两组小鼠子宫组织均有柱状上皮细胞损伤和炎性细胞浸润。qPCR检测结果显示,与同期对照组相比,孕12 d感染组小鼠胎盘组织中HIF⁃1α、HIF⁃1β mRNA表达水平显著上升（F ＝ 132.6、286.9,P 均＜0.05）,子宫组织中HIF⁃1α、HIF⁃1β mRNA表达水平显著下降（F ＝111.5、55.2,P均＜ 0.05）;孕18 d感染组小鼠胎盘和子宫组织中HIF⁃1α、HIF⁃1β mRNA表达水平均显著下降（F ＝ 215.8、418.9、156.8、200.1,P 均＜ 0.05）。与同期对照组相比,孕12 d感染组小鼠胎盘组织中VEGF⁃A、VEGF⁃B和VEGF⁃C mRNA表达水平均显著下降（F ＝426.2、104.6、566.9,P 均＜0.05）,子宫组织中VEGF⁃A、VEGF⁃B和VEGF⁃C mRNA表达水平显著上升（F ＝ 95.8、502.4、610.0,P均＜0.05）;孕18 d感染组小鼠胎盘组织和子宫组织中VEGF⁃A、VEGF⁃B和VEGF⁃C mRNA表达水平均显著上升（F ＝521.9、100.6、275.9、224.6、108.2、333.4,P均＜ 0.05）。免疫组化染色结果显示,与相应对照组相比,孕12 d、孕18 d感染组小鼠胎盘迷路区HIF⁃1α分别呈强阳性和弱阳性表达,子宫组织中HIF⁃1α均不表达。结论　小鼠孕早期感染弓形虫后,胎盘组织中HIF⁃1α表达呈现孕中期上升、孕晚期下降的趋势,且与VEGF⁃A、VEGF⁃B、VEGF⁃C表达变化趋势相反;推测HIF⁃1α通过抑制VEGF表达而抑制小鼠妊娠期间胎盘血管生成,导致不良妊娠结局。     

Therapeutic and prophylactic thalidomide in TNBS-induced colitis： Synergistic effects on TNF-α, IL-12 and VEGF production

AIM： To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor α （TNF-α）, IL-12, and vascular endothelial growth factor （VEGF）, hallmarks of intestinal inflammation in Crohn＇s disease （CD）.
METHODS： Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4＋ T cells, macrophages, and VEGF＋ cells were detected by immunohistochemistry. TNF-α and IL-12 were quantified in the supernatant of organ cultures by ELISA.
RESULTS： Significant reduction in the inflammatory score and in the percentage of VEGF＋ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-α and IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide. TNF-α levels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-α and IL-12 were significantly correlated with the inflammatory score and the number of VEGF＋ cells.
CONCLUSION： Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-α, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD.     

Hypoxia improves expansion potential of human cord blood-derived hematopoietic stem cells and marrow repopulation efficiency

Objectives: In bone marrow, hematopoietic stem cells (HSCs) reside in the most hypoxic endosteum niche, whereas the proliferating progenitors are located near the relatively oxygen‐rich vascular region. High oxygen tension is potentially detrimental to HSCs. The objective of this investigation was to compare cellular, functional, and molecular responses of human umbilical cord blood (UCB)‐derived hematopoietic stem and progenitor cells in culture under hypoxic and normoxic conditions. Methods: CD133‐enriched UCB cells were cultured in growth factor containing serum‐free and serum‐supplemented medium under 5% O₂ (hypoxia) or 21% O₂ (normoxia) for 10 d. The phenotypes of expanded cells were analyzed by flow cytometry and the engraftability by SCID‐repopulation assay. The expression of hypoxia‐inducible factor (HIF)‐1α and some of its target genes was analyzed by real‐time RT‐PCR. Results: In hypoxic culture, CD34⁺ CD38^? cells were expanded about 27‐fold, which was significantly (P < 0.01) higher than that obtained in normoxic culture. Serum‐free culture did not support the growth of cells in the presence of 21% O₂. Myeloid colony‐forming potential of cells was significantly (P < 0.05) increased in 5% O₂ compared with 21% O₂ culture. SCID‐repopulation efficiency seems to be better preserved in the cells cultured under hypoxic conditions. Hypoxia significantly (P < 0.05) induced the expression of HIF‐1α, vascular endothelial growth factor (VEGF), and ABCG2 genes and also upregulated CXCR4 receptor expression. Conclusions: Low oxygen tension enhanced the proliferation of UCB‐derived HSC/progenitor cells and maintenance of SCID‐repopulating cells than normoxia. These expanded cells are expected to be beneficial in the patients who lack human leukocyte antigen (HLA)‐matched donors.