双链小片段干扰RNA抑制缺氧条件下乳鼠心肌细胞缺氧诱导因子1α表达

目的　构建含缺氧诱导因子 1α(HIF 1α)小片段干扰RNA(siRNA)靶序列的U6启动子表达框结构 ,观察其对缺氧条件下乳鼠心肌细胞HIF 1α表达的影响。　方法　分离培养乳鼠心肌细胞 ,分为常规培养液对照组、RNA干扰 (RNAi)组 (转染无效干扰序列Ⅳ )、RNAi抑制组 (转染有效干扰序列并按下游引物不同分为Ⅰ、Ⅱ、Ⅲ组 )。设计、合成 3对 (Ⅰ、Ⅱ、Ⅲ )含HIF 1α编码基因片段(正、反义 )和 1对 (Ⅳ )随机序列 (正、反义 )的PCR下游引物。PCR法构建U6启动子表达框及相应正、反序列表达框 ,同时转染心肌细胞。每组每时相点 5皿细胞。于缺氧 1h后 ,用蛋白免疫印迹法(Western blot)测定其蛋白水平表达 ,免疫组织化学法检验干扰效果。缺氧 6h后采用逆转录 聚合酶链反应 (RT PCR)法检测HIF 1αmRNA的表达。　 结果　筛选出的最佳抑制片段为Ⅱ组序列。缺氧 1h,对照组、RNAi组心肌细胞HIF 1α蛋白水平显著增高 ,Ⅰ、Ⅱ、ⅢRNAi抑制组HIF 1α蛋白水平较对照组明显降低 ,其中Ⅱ组降低最为显著 (P <0.0 1);缺氧 6h,RNAi组心肌细胞HIF 1αmRNA水平较常氧条件下明显增高 (P <0.0 1);RNAi抑制Ⅱ组未见明显增高 (P >0.0 5 )。　 结论　构建的HIF 1αⅡ组表达框能有效地抑制缺氧乳鼠心肌细胞HIF 1α表达     

Distinct signaling pathways are involved in hypoxia- and IL-1-induced VEGF expression in human articular chondrocytes.

Recent data suggest that vascular endothelial growth factor (VEGF) is involved in the pathogenesis of osteoarthritis (OA). Cartilage is an avascular tissue, leading to a low cartilage O2 level. Thus, in a variety of pathologic or physiologic conditions, VEGF is partly regulated by hypoxic stress. The implications of hypoxia for VEGF expression by OA chondrocytes, however, are not known. We investigated the regulatory system of VEGF in OA chondrocytes under hypoxic conditions. Chondrocytes were obtained from articular cartilage of patients with OA. Cells were cultured and then incubated under hypoxic (95% N2, 5% CO2) or normoxic conditions, with or without interleukin (IL)-1 (10 ng/mL) stimulation. The mitogen activated protein kinase (MAPK) inhibitors were also used. VEGF levels in the culture supernatants were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to examine the expression of hypoxia inducible factor (HIF)-1alpha. Hypoxia significantly increased VEGF levels (p<0.05). Hypoxia-induced VEGF secretion was abolished by p38MAPK inhibitor, but not by JNK inhibitor. In contrast, IL-1-induced VEGF secretion was blocked by JNK inhibitor, and not by p38MAPK inhibitor. Both hypoxia and IL-1-induced HIF-1alpha were attenuated by p38 MAPK and JNK inhibitors. We demonstrate that hypoxia and IL-1 induce VEGF production in chondrocytes through distinct MAPK signaling pathways, indicating that VEGF is induced in a HIF-1-dependent or -independent manner in chondrocytes.     

Hypoxia and HIF-1α expression in the epiphyseal cartilage following ischemic injury to the immature femoral head

HIF-1α has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1α activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1α activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process.Materials and methodsFemoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1α western blot, RT-qPCR of HIF-1α, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed.ResultsSevere hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1α protein level and HIF-1α expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p < 0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks.ConclusionsAcute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1α activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.     

Expression of hypoxia inducible factor-1 alpha and correlation with preoperative embolization of meningiomas

OBJECT: Vascular endothelial growth factor (VEGF) has been implicated in meningioma tumorigenesis and growth. The production of VEGF is regulated by hypoxia inducible factor-1alpha (HIF-1alpha), especially under conditions of hypoxia. In this study, the authors examine the expression of HIF-1alpha and VEGF in meningiomas, with a special emphasis on conditions of hypoxia, such as preoperative embolization, and on in vitro studies in cultured cells. METHODS: Meningiomas obtained in 142 patients were studied using immunohistochemical methods to detect HIF-1alpha and the results were correlated with the extent or lack of preoperative embolization and expression of VEGF. Primary meningioma cell cultures were established and cell culture experiments were performed using a hypoxia chamber to stimulate HIF-1alpha and VEGF production. Expression of HIF-1alpha in primary meningioma cell cultures was measured using immunoblot assays. The VEGF secretion was measured using enzyme-linked immunosorbent assay. Half of the meningiomas studied were positive for HIF-1alpha, with a strong correlation between complete embolization and HIF-1alpha expression. Most of the meningiomas studied expressed VEGF protein, and VEGF expression did not correlate with the degree of embolization. A strong correlation was found between VEGF and HIF-1alpha expression in immunohistochemical studies. Secretion of VEGF is increased by hypoxia and growth factor stimulation. In meningiomas, growth factors stimulate HIF-1alpha expression. The role of hypoxia is less clear. CONCLUSIONS: The expression of HIF-1alpha is increased by complete preoperative embolization of meningiomas. The expression of HIF-1alpha also correlates with VEGF secretion in meningiomas. Growth factor and hypoxic stimulation both contribute to VEGF control, but which is most important (or whether both are equally important) will require further studies.     

低氧环境对小鼠未成熟关节透明软骨细胞培养的影响

目的研究低氧和低氧模拟化合物氯化钴对小鼠未成熟关节透明软骨细胞氧感应基因和细胞表型的影响。方法经酶消化分离出小鼠未成熟关节透明软骨细胞，分别在21％氧、2％氧和150μmol/L氯化钴条件下培养一定时间。通过倒置显微镜、透射电镜和扫描电镜观察细胞形态学变化。应用定量PCR和Western Blot检测葡萄糖转运体-1（GLUT-1）、葡萄糖转运体-3（GLUT-3）、磷酸果糖激酶-1（PGK-1）和低氧诱导因子-1α（HIF—1α）的表达。应用定量PCR观察软骨细胞表型改变。应用四甲基偶氮唑盐法（MTT）检测低氧及氯化钴对软骨细胞活性的影响。用葡萄糖检测试剂盒测葡萄糖摄取量。结果不同培养条件下软骨细胞形态无明显差异。2％氧和氯化钴可增加GLUT-1、GLUT-3及PGK-1mRNA表达。2％氧和氯化钴可促进GLUT-1、GLUT-3和HIF—1α蛋白表达。低氧和氯化钴促进细胞活性，增加葡萄糖摄取并促进细胞外基质合成。结论软骨细胞能通过调节氧感应基因适应低氧环境，HIF—1α可能起关键作用。低氧能在一定程度上增加软骨细胞活性和细胞外基质合成。模拟体内氧环境培养细胞能更好地了解软骨细胞特性。     

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1alpha-dependent mechanism.     

Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension

OBJECTIVE: Adult articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from <10% oxygen at the cartilage surface to <1% in the deepest layers. This gradient may be disturbed during diseases of the joint, for example in rheumatoid arthritis when synovial fluid pO(2)falls. We investigated whether changes in oxygen tension modulate gene expression in articular chondrocytes. DESIGN: Bovine articular chondrocytes were cultured in alginate beads in medium maintained at <0.1, 5, 10 or 20% oxygen. A modified RNA arbitrarily primed polymerase chain reaction (RAP-PCR) technique was used to identify several genes whose mRNA abundance in articular chondrocytes was dependent upon oxygen tension. Northern hybridization slot blots were used to quantify changes in mRNA level relative to a housekeeping gene, beta-actin. RESULTS: Genes found by RAP-PCR to undergo up-regulation in hypoxia included TIMP-1 and integrin-linked kinase. Collagen V mRNA levels were down-regulated in hypoxic chondrocytes. This led us to examine mRNA levels for various cytokines, matrix structural molecules and beta1 integrin. Interleukin 1beta, transforming growth factor beta and connective tissue growth factor were all up-regulated by low oxygen tensions, as was beta1 integrin. Collagen II (COL2A1) was down-regulated by hypoxia but aggrecan mRNA levels remained unchanged. The mRNA levels for GAPDH, the archetypal hypoxia responsive gene, were not modulated in articular chondrocytes by changes in oxygen tension. CONCLUSIONS: Oxygen tension modulates the abundance of mRNAs encoding structural molecules, several cytokines, beta1 integrin and integrin-linked kinase in articular chondrocytes. This may be important during disease progression. Chondrocytes are unusual in their response to hypoxia, presumably because they exist physiologically in a low oxygen environment.