缺氧诱导因子1和缺氧诱导因子2在人类着床前胚胎的表达

目的研究缺氧诱导因子1(HIF-1)和缺氧诱导因子2(HIF-2)在人类着床前胚胎各个阶段的表达,探讨这两个氧调节基因在人类早期胚胎发育过程中的作用和意义。方法收集不育症患者捐赠的胚胎,采用巢式逆转录聚合酶链反应(RT-PCR)和实时荧光定量PCR分别定性和定量在5%和20%O2条件下体外培养的人胚胎的HIF-1α和HIF-2αmRNA。采用免疫荧光染色检测人胚胎的HIF-1α和HIF-2α蛋白。结果巢式RT-PCR分别检测了5%和20%O2体外培养的2、4、6、8细胞胚胎和囊胚发现,所有34个胚胎均表达HIF-1α和HIF-2αmRNA。实时荧光定量PCR5%O2培养的人囊胚HIF-1α与18SrRNA的Ct比值为(1.22±0.05);20%O2培养的人囊胚,这一比值是(1.02±0.07);两者比较差异显著(P<0.05)。人胚胎在体外常氧培养条件下的HIF-1αmRNA水平显著高于低氧培养。5%O2培养的人囊胚HIF-2α与18SrRNA的Ct的比值为(1.29±0.04);20%O2培养的人囊胚,这一比值是(1.19±0.11);两者比较无显著差异(P>0.05)。人胚胎在体外低氧培养条件下的HIF-2αmRNA水平与常氧培养无显著差异。5%和20%O2体外培养的2、4、6、8细胞,各个发育阶段人胚胎的HIF-1α和HIF-2α免疫荧光染色均阳性。结论研究结果显示人类着床前胚胎在体外常氧和低氧培养时均表达HIF-1α和HIF-2α。二者可能通过广泛的靶基因系统参与早期胚胎的生长发育和着床过程,在早期胚胎的调控可能不在转录水平,而在转录后水平。     

结直肠癌中缺氧诱导因子1-α与FasL 表达相关性的研允

目的 探讨结直肠癌侵袭转移过程中缺氧诱导因子1-α(alpha,HIF-1α)与FasL表达的相关性.方法 采用分子克隆方法,将我室已构建的FasL-pcD-NA3.1(+)质粒与pcDNA3.1(一)质粒进行重组,得到新的FasL-pcDNA3.1(一)质粒并加以鉴定;通过脂质体转染法将空质粒、FasL-pcDNA3.1(+)与FasL-pcDNA3.1(一)质粒分别转染人直肠癌HR-8348细胞,构建侵袭力不同的结直肠癌细胞HR-8348L、HR一8348F和HR一8348As,未转染细胞HR一8348B为空白对照,应用Tran-swell,小室检测各组细胞的侵袭能力;采用化学缺氧法构建四组细胞的缺氧模型,Western blot方法定量检测缺氧0h、6h、12h及24h各组细胞内HIF-1α的表达.结果 FasL-pcDNA3.1(一)质粒符合要求,FasL片段大小约900bp,测序结果正确率99.2%;单层细胞体外侵袭实验见HR一8348F细胞穿透Transwell滤膜的细胞数目为(12.930±2.434),显著多于HR-8348B(8.133±1.959)、HR-8348L(7.670±2.093)和HR-8348As(7.870±1.685)细胞(P<0.05);Western blot检测示HIF-1α蛋白于120kD处显色,缺氧0h与6h,各组样品中HIF-α仅表达微量,HIF-1α水平无显著性差异(P>0.05);缺氧12h与24h,HR一8348F细胞内HIF-1α水平较0h和6h时明显增高(P<0.05),而HR-8348B、HR-8348L及HR-8348As细胞内HIF-1α表达与6h时无明显变化(P>0.05),HR-8348F细胞HIF-1α水平显著高于HR-8348B、HR-8348L及HR-8348As细胞(P<0.01).结论 缺氧环境中结直肠癌细胞FasL表达增强是除低氧分压外另一个诱导HIF-1α表达增高的因素,FasL与 HIF-1α水平呈正相关,高侵袭能力的结直肠癌细胞对缺氧的适应能力加强,促进肿瘤的远处转移.     

胃癌组织缺氧诱导因子-1α与胰岛素样生长因子-Ⅱ表达对肿瘤血管生成的影响及预后评估

目的探讨胃癌组织中缺氧诱导因子-1α（HIF-1α）及胰岛素样生长因子-Ⅱ（IGF—Ⅱ）mRNA的表达水平及其与血管生成和预后的关系。方法采用原位杂交检测118例胃癌组织中HIF-1αmRNA和IGF—ⅡmRNA的表达情况,用免疫组化SP法标记CD34单克隆抗体,计算肿瘤微血管密度（MVD）。结果HIF—1αmRNA和IGF—ⅡmRNA在胃癌组织中的阳性表达率分别为49．2％和47．4％。且与胃癌临床侵袭转移病理指标均相关。HIF-1αmRNA和IGF-ⅡmRNA阳性表达者的MVD值,均高于阴性表达者;HIF-1αmRNA和IGF-ⅡmRNA表达呈正相关,MVD分别与HIF-1αmRNA和IGF—ⅡmRNA的表达水平呈正相关。HIF—1αmRNA和IGF—ⅡmRNA表达阳性及MVD值≥41．5个／0．72mm^2的患者的平均生存时间及5年生存率均低于表达阴性及MVD值〈41．5个／0．72mm^2者。结论HIF-1α与IGF—Ⅱ对胃癌的浸润转移,特别是对肿瘤血管形成具有重要作用,可作为预测患者预后和指导治疗的新途径。     

缺氧诱导因子-1α在前列腺癌中的表达及意义

目的　探讨缺氧诱导因子 1α(HIF 1α)蛋白在前列腺癌 (CaP)中的表达及意义。　方法　应用免疫组化SP法检测 42例CaP和相应癌旁组织 ,12例高分级前列腺上皮内瘤 (PIN )以及 9例正常前列腺组织 (NP)中HIF 1α的表达 ,免疫印迹技术分析不同氧浓度诱导下CaP细胞株PC 3M的HIF 1α蛋白水平。　结果　 3 3例 (78.6% )CaP和 9例PIN组织HIF 1α蛋白表达阳性 ,有远处转移的CaP组织中HIF 1α表达显著高于无转移者 (P <0 .0 5) ,癌旁组织及正常前列腺组织中无阳性表达 ;PC 3M细胞株中HIF 1α表达水平随氧浓度减小而明显升高 ,差异有显著性意义 (P <0 .0 1)。　结论　HIF 1α表达是CaP形成的早期事件 ,其水平升高可能导致肿瘤向恶性发展表型 ,HIF 1α可作为CaP的早期诊断指标和治疗新靶点     

缺氧诱导因子-1α在去势前后大鼠腹叶前列腺中表达的变化

目的探讨与缺氧相关的缺氧诱导因子-1α(HIF-1α)是否参与去势后前列腺萎缩过程.方法24只SD大鼠分为3组,其中A组(n=8)为假手术对照组,B组(n=8)为去势组,C组(n=8)为雄激素替代组(去势后肌注十一酸睾酮50mg/kg);术后3天处死,通过半定量RT-PCR检测与HIF-1α在去势前后前列腺表达变化.结果去势后大鼠前列腺的体积萎缩变小;雄激素替代组出现前列腺增生变大;对照组正常的大鼠前列腺有HIF-1 α mRNA低水平表达,去势组HIF-1α mRNA表达量增加,雄激素替代组HIF-1αmRNA表达量减少,与正常对照组比较,去势组的HIF-1α mRNA的表达量显著增加(P＜0.05),雄激素替代组的HIF-1αmRNA的表达量显著减少(P＜0.01).结论前列腺组织的缺氧参与去势后大鼠前列腺的早期萎缩过程.     

低氧诱导因子-1α在绝经后骨质疏松中调控作用的实验研究

目的探讨在绝经后骨质疏松的发生、发展过程中,低氧诱导因子-1α（HIF-1α）对于成骨细胞功能的调控作用。方法2004年10月至2006年5月,应用Cre—Loxp重组酶技术,建立成骨细胞条件性敲除HIF-1α小鼠,取3个月龄雌性野生型和敲除型小鼠各24只行卵巢切除术,术后0、4、8周取材行HE染色、四环素荧光双标记、Micro-CT、RT—PCR、Western-blotting检测。结果与野生型小鼠相比,敲除型小鼠骨小梁的数目、体积、厚度,骨密度,骨矿沉积速度,血管内皮生长因子（VEGF）、RunX2、ALP、OC基因在mRNA水平的表达,VEGF、RunX2在蛋白水平的表达均明显降低,尤其以术后8周最为明显。结论在绝经后骨质疏松的发生、发展过程中,成骨细胞条件性敲除HIF-1α后成骨功能降低,HIF-1α能够调控成骨细胞的成骨功能。     

gax和HIF-1α基因在原发性肝癌组织中的表达及意义

目的探讨同源盒基因(gax)和缺氧诱导因子(HIF1α)表达产物与原发性肝癌发生、发展的关系。方法采用逆转录聚合酶链反应(RTPCR)和蛋白质印迹技术(Westernblot)分别检测20例原发性肝癌患者癌及癌旁肝组织中gax和HIF1α的mRNA以及蛋白质表达水平。结果肝癌组织中HIF1αmRNA(118.1±33.2)及蛋白表达(10.86±2.76)水平显著高于癌旁组织(分别为42.7±10.5与3.52±1.53,P均<0.05);肝癌组织中gaxmRNA表达(39.5±13.8)及其蛋白表达(4.10±1.26)水平低于癌旁组织(P均<0.05)。结论gax和HIF1α基因是调控肝癌组织血管生长的重要基因之一,与gax表达下调及HIF1α表达下调肝癌发生发展密切相关。     

Erythropoietin protects the kidneys against ischemia reperfusion injury by activating hypoxia inducible factor-1alpha

BACKGROUND: Ischemia/reperfusion (I/R) injury is closely associated with tissue damage in various organs, as well as in kidney transplants. Erythropoietin (EPO) has been shown to have a cytoprotective effect against hypoxia. We examined the effect of EPO against renal I/R injury and the underlying mechanism. METHODS: Human umbilical vein endothelial cells and human renal proximal tubular epithelial cells were cultured under hypoxic conditions with various EPO concentrations at 37 degrees C and examined the mechanism of cell proliferation by EPO. Moreover, to demonstrate the renoprotective effect in vivo, we treated Sprague-Dawley rats with 100 IU/kg EPO every 2 days for 2 weeks (a total of 6 doses). One day after the last injection, the operations to produce renal I/R injury (bilateral renal occlusion for 60 min) were done, and rats were killed at the end of the reperfusion period (24 hr and 72 hr after reperfusion began). RESULTS: First, we demonstrated in vitro that EPO increased hypoxia inducible factor-1alpha (HIF-1alpha) expression and stimulated proliferation of both cells under hypoxic conditions. Next, we demonstrated in vivo that EPO treatment increased the number of HIF-1alpha-positive cells, and markedly induced the expression of vascular endothelial growth factor messenger RNA. Using pimonidazole, a molecular probe that detects hypoxia, we found that EPO markedly attenuated tubular hypoxia, and reduced the number of terminal transferase dUTP nick end labeling-positive apoptotic cells and alpha-smooth muscle actin-positive interstitial cells. CONCLUSIONS: We suggested a novel HIF-1alpha induction pathway by EPO under hypoxic conditions. Thus, EPO may protect the kidneys against ischemia reperfusion injury by activating HIF-1alpha.     

腹主动脉瘤发病中低氧诱导因子-1α介导血管平滑肌细胞凋亡的研究

目的　研究腹主动脉瘤 (AAA)发病不同时期动脉壁中低氧诱导因子 (HIF) 1α的表达及与血管平滑肌细胞 (SMC)凋亡的关系。方法　将 60只Wistar大鼠制成AAA模型 ,分别于术后 1、3、7、14、2 1d切取腹主动脉 ,应用原位DNA片段末端标记 (TUNEL)检测SMC中的凋亡信号 ,用免疫组织化学方法检测HIF 1α及凋亡相关基因p5 3、bcl 2的蛋白表达。结果　HIF 1α的表达于术后 3d达表达高峰 ,为 (18.4± 3 .5 ) % ;术后 7d左右是TUNEL染色及p5 3蛋白表达的高峰时段 ,分别为(19.8± 3 .8) %、(17.6± 3 .3 ) % ;bcl 2蛋白在术后 1d时表达较强烈 ,为 (14 .1± 2 .9) %。结论　HIF 1α参与了腹主动脉缺血缺氧状态下的继发损害过程 ,并可通过调节动脉壁中p5 3、bcl 2的蛋白表达水平促进SMC的凋亡 ,是AAA发病过程中一个重要的中间因子。     

Immunohistochemical expression of hypoxia inducible factor-1alpha and its downstream molecules in sarcomatoid renal cell carcinoma

PURPOSE: Sarcomatoid renal cell carcinomas, highly aggressive variants of renal cell carcinoma subtypes, often present with or develop metastases soon after the primary diagnosis. Most metastatic cases do not respond to immunotherapy or aggressive chemotherapy. Recently targeted therapies, particularly those targeting hypoxia inducible pathway molecules, have been tested clinically on metastatic clear cell renal cell carcinoma with promising initial results. No such studies are available on sarcomatoid renal cell carcinoma. We investigated the hypoxia inducible pathway marker immunohistochemical expression profile, and any potential therapeutic implications that such expression may have, in these tumors. MATERIALS AND METHODS: Immunohistochemical staining for hypoxia inducible factor-1alpha, glucose transporter 1, carbonic anhydrase IX and vascular endothelial growth factor was performed in 22 clear cell and 12 nonclear cell sarcomatoid renal cell carcinomas. The immunoreactivity in the tumors was graded from 0 to 3+ (0-no staining, 1+-1% to 25% cells positive, 2+-26% to 50% cells positive and 3+-greater than 50% cells positive). The results were then compared with various clinical parameters to assess for associations. RESULTS: Most clear cell renal cell carcinomas over expressed (2+ or 3+) hypoxia inducible factor-1alpha (in 59%), carbonic anhydrase IX (95%), glucose transporter 1 (91%) and vascular endothelial growth factor (95%). None of the nonclear cell sarcomatoid renal cell carcinomas showed 2+ or 3+ expression of hypoxia inducible factor-1alpha, carbonic anhydrase IX or glucose transporter 1, but 92% showed diffuse positivity for vascular endothelial growth factor. Over expression of carbonic anhydrase IX showed no association with survival, unlike that reported in (nonsarcomatoid) clear cell renal cell carcinoma. There was significant discordance in the staining grades among hypoxia inducible factor-1alpha, carbonic anhydrase IX and glucose transporter 1 in clear cell renal cell carcinoma, suggesting that mechanisms other than hypoxia inducible pathway may be involved in some sarcomatoid clear cell renal cell carcinoma. CONCLUSIONS: Hypoxia inducible pathway markers continue to be over expressed in sarcomatoid clear cell renal cell carcinoma, and can be of diagnostic usefulness in such high grade tumors. Over expression of vascular endothelial growth factor in the clear and nonclear cell groups raises the possibility that vascular endothelial growth factor targeted therapies may have a role in the management of sarcomatoid renal cell carcinoma, and deserve further investigation.     

门静脉高压症大鼠断流术后胃黏膜缺氧诱导因子-1α和血管内皮生长因子的表达

目的 观察缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）在断流术前后肝前性门静脉高压症（PHPH）大鼠胃黏膜的表达，探讨HIF-1α和VEGF在门静脉高压胃病（PHG）的发生发展的作用。方法 取Wistar大鼠制备PHPH模型80只，设假手术组（SO）60只，在术后3周施断流术。检测HIF-1α、VEGF和CD34在大鼠胃壁组织中的表达。结果 断流术前PHPH组大鼠胃壁中HIF-1α（5．8±1．3）和VEGF（12．0±3．0）的表达均明显高于SO组[HIF-1α（0．03750±0．05175），VEGF（0．7±0．1），P〈0．01]。术后HIF-1α、VEGF和MVD均有升高趋势。结论 HIF-1α和VEGF可能参与了PHG的发生发展，断流术本身能通过某种机制影响HIF-1α和VEGF的表达，加重PHG。     

缺氧诱导因子-1对肾微血管内皮细胞缺氧再复氧损伤免疫调节作用

目的探讨低氧诱导因子1(HIF1)在肾微血管内皮细胞(HGMECs)缺氧再复氧损伤中的免疫调节作用。方法建立HGMECs缺氧再复氧模型,应用特异性脯氨酰羟化酶抑制剂3,4DHB和环氧酶2阻断药物NS398预处理HGMECs,设对照、缺氧复氧、3,4DHB和NS398四组。采用逆转录聚合酶链反应、免疫印记和酶联免疫吸附试验等方法检测各组细胞HIF1α、血红素氧合酶1(HO1)的mRNA和蛋白的表达及上清中可溶性细胞间黏附因子1(sICAM1)的水平;将各组HGMECs与异体淋巴细胞混合培养(MELR),检测淋巴细胞增殖程度和MELR上清中白细胞介素2(IL2)的表达水平。结果与对照组相比,HGMECs经缺氧复氧损伤后HIF1α蛋白和HO1mRNA表达水平显著增高(P<0.01),上清中sICAM1(14.55±1.13vs7.90±1.61)ng、异体淋巴细胞增殖率(0.191±0.053vs0.069±0.032)和MELR上清中IL2(40.0±5.0vs18.0±3.0)pg水平均有显著增高(P<0.01)。与缺氧复氧组比较,3,4DHB干预组HIF1α蛋白和HO1mRNA表达水平显著增高(P<0.05),而淋巴细胞增殖率(0.079±0.038,P<0.01)及IL2(25.7±6.1pg,P<0.01)和sICAM1(8.71±1.32ng,P<0.01)水平则显著下调。与缺氧复氧组比较,NS398干预组HIF1α蛋白和HO1mRNA表达水平显著减低(P<0.01),而淋巴细胞增殖率(0.268±0.079)及IL2(51.0±11.0)pg和sICAM1(20.33±3.05)ng表达水平则显著增高(P<0.05)。结论缺氧复氧会对HGMECs造成免疫学损伤,HIF1α稳定表达可起到保护性作用。     

HIF-1α和P53蛋白的表达对肾透明细胞癌预后的影响

目的本研究旨在探讨缺氧诱导因子1α（HIF-1α）和P53蛋白在人肾透明细胞癌组织中的表达及其对患者预后的影响。方法应用免疫组化方法检测65例肾透明细胞癌组织中HIF-1α和P53蛋白的表达情况,通过生存曲线法比较阳性组和阴性组患者的预后情况。结果 HIF-1α和P53蛋白表达的阳性率分别为40.0%（26/65）和52.3%（34/65）;HIF-1α表达与肾透明细胞癌的组织学分级、临床分期和淋巴结转移有关（P〈0.05）;HIF-1α蛋白阳性组患者的生存期明显较阴性组短（P=0.004）;P53蛋白表达与肾透明细胞癌患者性别、年龄、肿瘤大小、组织学分级、临床分期、淋巴结转移及生存期均无关（P〉0.05）;HIF-1α蛋白P53蛋白的表达之间存在相关性（r=0.402,P=0.001）。结论 HIF-1α的表达与肾透明细胞癌患者的预后相关,其阳性表达提示患者预后不良;检测HIF-1α表达水平有助于肾细胞癌预后评估。     

Experimental varicocele induces hypoxia inducible factor-1alpha, vascular endothelial growth factor expression and angiogenesis in the rat testis

PURPOSE: In this study we investigated hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression, and angiogenesis in an experimental model of varicocele in the rat testis. MATERIALS AND METHODS: A total of 30 adult male Sprague-Dawley rats were investigated in 3 groups, namely varicocele group 1 (13), sham operated group 2 (9) and control group 3 (8). At 30 days after surgery was completed in groups 1 and 2 orchiectomy was performed in all rats. Histological findings in the left testicles of rats from each group were compared. HIF-1alpha and VEGF expression was immunohistochemically studied and CD31 panendothelial antigen was used to identify the number of microvessels, that is microvessel density (MVD), in paraffin embedded sections of testis tissue. Data were analyzed using the chi-square test, Fisher's exact test, 1-way ANOVA and the Tukey HSD test for post hoc comparison. RESULTS: HIF-1alpha expression was detected in 12 specimens (92.3%) in group 1, 4 (44.4%) in group 2 and 2 (25%) in group 3. The frequency of HIF-1alpha positivity in group 1 was significantly higher than the rates in groups 2 (p = 0.023) and 3 (p = 0.003). VEGF expression was detected in 8 specimens (61.5%) in group 1 but none of the group 2 or 3 specimens were VEGF positive. The frequency of VEGF positivity in group 1 was significantly higher than that in groups 2 (p = 0.006) and 3 (p = 0.007). Mean MVD +/- SD in group 1 was 7.53 +/- 1.50 (range 6 to 12), and findings in groups 2 and 3 were 5.88+/-1.45 (range 4 to 8) and 5.12 +/-1.12 (range 4 to 7), respectively. Mean MVD in group 2 was higher than in group 3 but this difference was not significant (p = 0.509). Mean MVD in group 1 was significantly higher than the mean values in groups 2 (p = 0.030) and 3 (p = 0.002). CONCLUSIONS: Previous study of experimental varicocele models in rats documented HIF-1alpha and VEGF expression combined with angiogenesis in the testis. The results of this study show that varicocele can lead to tissue hypoxia and related pathophysiological events, such as angiogenesis.     

Subselective preoperative embolization for meningiomas. A radiological and pathological assessment

Over a 2-year period the authors have studied the effects of preoperative subselective embolization of meningiomas. Thirty-six consecutive patients shown by computerized tomography (CT) to have a meningioma underwent selective internal and external carotid artery angiography, and any significant external carotid artery feeders were embolized (27 cases). It was found that CT and dynamic radioisotope scan findings were unable to predict the degree of vascularity of the tumor or its suitability for embolization. Furthermore, these tests, repeated after embolization, were unreliable in detecting either the extent of necrosis or reduction in blood flow. The effects of embolization upon surgery were recorded, and the excised tumor specimen examined for evidence of thrombosis and infarction. Subselective embolization was determined to be a simple, safe, and effective method of producing tumor necrosis and intraoperative hemostasis in selected patients.