ObjectiveTo investigate the effects of hypoxic three-dimensional culture microenvironment on the proliferation of bone marrow mesenchymal stem cells and its mechanism. MethodsP5 generation mouse bone marrow mesenchymal stem cells and P (3HB-co-4HB) were co-cultured under normoxic three-dimensional (20%) and hypoxic three-dimensional microenvironment (4%) respectively. After 24 hours, the proliferation of the two groups was determined by CCK-8 method. The expression of HIF-1α gene was detected by real-time quantitative PCR after 12 hours. Western blotting was used to detect the expression of HIF-1α protein after 24 hours. ResultsAfter 24 hours, the CCK-8 method showed that the OD value of the hypoxia group was significantly higher than that of the normoxia group (0.455±0.027 vs. 0.352±0.090, n=12, P<0.05). After 12 hours of hypoxic culture, the expression level of HIF-1α mRNA in the hypoxia group was significantly higher than that in the normoxia group (P<0.05). Compared with the normoxia group (0.47± 0.05), the relative expression level of HIF-1α protein in the hypoxia group (0.63±0.06) significantly increased in the Western blotting after 24 hours (n=3, P<0.05). ConclusionThe hypoxic three-dimensional microenvironment can promote the proliferation of bone marrow mesenchymal stem cells, which may be related to the activation of HIF-1α signaling pathway.
【摘要翻译】 一 氧化氮合酶( NOS) -2( NOS-2) 的诱导和一氧化氮产物增加是过敏性气道疾病的共同特征。严重哮喘与气道S-亚硝基硫醇减少相关。S-亚硝基硫醇是NO的生化产物, 可通过促炎症转录因子NF-κB 的S-亚硝基化抑制炎症反应。因此, 重建气道S-亚硝基硫醇对治疗可能有益。我们对此假设在以卵清蛋白诱导的过敏性炎症大鼠模型中进行验证。未使用或使用卵清蛋白致敏的动物均在卵清蛋白激发前于气管内灌注S-亚硝基谷胱甘肽( GSNO;50 μl, 10 mM) , 并在48 h 以后进行分析。GSNO 给药增加了肺组织S-亚硝基硫醇水平。与对照组比, GSNO 降低了卵清蛋白致敏动物NF-κB 的活性, 但对支气管肺泡灌洗细胞总数、分类计数及杯状细胞化生标记物均无显著影响。GSNO给药也改变了HIF-1 的活性, 导致未致敏大鼠HIF-1 活化,但抑制卵清蛋白致敏大鼠的HIF-1 活性。我们使用NOS-2基因敲除小鼠来评价内源性一氧化氮合成酶-2 在调节NF-κB和( 或) HIF-1 活性及气道过敏性炎症的作用。尽管在NOS-2 基因敲除小鼠中卵清蛋白诱导的NF-κB 活力轻度增高, 这与支气管肺泡灌洗中性粒细胞轻度增加有关, 其他的过敏性炎症指标和HIF-1 活性在NOS-2 基因敲除及野生型小鼠之间却无明显相差。总体来说, 我们的研究表明GSNO灌注能抑制气道过敏性炎症中NF-κB 活性, 但是并不能显著地影响大部分炎症及杯状细胞化生指标, 这样可能因为对其他信号通道( 比如HIF-1) 的影响而限制了它的治疗价值。【述评】 GSNO 是近年哮喘治疗研究的热点。既往的研究发现GSNO 在哮喘治疗中有一定前景。本研究却发现GSNO 气管内滴注虽能抑制过敏性气道炎症中NF-κB 活性,但并不能显著抑制气道炎症反应及杯状细胞化生这两个哮喘关键病理改变, 可能与GSNO 同时影响了HIF-1 等其他信号通路有关。该研究表明GSNO 对哮喘气道炎症治疗效果有限, 同时表明哮喘气道炎症调控机制较为复杂, 治疗药物的设计需考虑多种信号通路对哮喘气道炎症的影响。
Objective To evaluate the effects of Shengji Yuhong collagen on promoting angiogenesis of the ischemia tissues and probe the possible mechanisms. Methods Forty-eight Wistar rats were divided by random method of paired into blank group， control group （collagen），and experimental group （Shengjiyuhong collagen）. After made the rats hind limb ischemia model， collagens with or without the extracts of Shengji Yuhong Gao were randomly paired implanted locally in hind limb ischemia tissues of rats in experimental group or control group. The samples of collagens and tissues about 0.5 cm large surrounding the collagen were explanted respectively on day 3，7， 14， and 28 for detected the hemog-lobin contents in colagen， microvascular counting by using CD34 immunohistochemical markers， and the expressions of HIF-1α mRNA and VEGF mRNA by using real-time fluorescent quantitative RT-PCR. The blood perfusion of the ischemic tissues at each time were determined by using laser speckle imaging system of Moor-FLPI. Results The results of Moor-FLPI showed that the obvious ischemia condition after model made， the blood perfusion was significantly lower than that before operation （P＜0.01）. On day 3 after operation it showed obvious congestion in the ischemic tissues， and from day 7 to day 14， it showed the ischemia state locally till day 28 after operation which showed improved situation of ischemic. Except for the day 3， the blood perfusion of experimental group were higher than those of blank group （P＜0.05）. There was no statistical significance between the blank group and control group （P＞0.05）. The blood perfusion on day 7 and day 14 after operation of experimental group were higher than those of control group （P＜0.05）. The hemoglobin contentsof each time point in the experimental group were higher than those in the control group （P＜0.01）. The microvascular counting on day 7 and day 14 in experimental group were higher than those of control group （P＜0.05）. The expressions of HIF-1α mRNA and VEGF mRNA at each time point of experimental group were higher than those of control group and blank group （P＜0.05）， and there was no significant differences between the control group and blank group （P＞0.05）. Conclusion The effects on promoting angiogenesis of rat hind limb ischemia tissues with Shengji Yuhong collagen may though inducing the expressions of HIF-1 α mRNA and VEGF mRNA locally.
目的 探讨HIF-1α和BAK蛋白在胃癌中的表达情况，以及二者在胃癌中的相互关系及作用。方法 应用免疫组化SABC染色法检测80例胃癌组织和20例正常胃组织中的HIF-1α和BAK蛋白的表达情况。结果 胃癌中HIF-lα和BAK蛋白的表达阳性率分别为56.3%（45/80）和67.5%（54/80），而在胃正常组织中分别为5.0%（1/20）和20.0%（4/20），二者在胃癌中的表达显著高于胃正常组织，其差异有统计学意义（P＜0.05）。HIF-1α蛋白表达与胃癌组织的浸润范围、分化程度及淋巴结转移有关（P＜0.05），与临床分期、年龄及性别无关（P＞0.05）；BAK蛋白表达与胃癌浸润及分化程度相关（P＜0.05），与淋巴结转移、临床分期、年龄及性别无关（P＞0.05）。胃癌组织中HIF-1α与BAK蛋白的阳性表达之间呈正相关（列联系数r=0.056，P＜0.05）。结论 HIF-1α与BAK蛋白在胃癌的临床分期及浸润转移中存在关系，这对于研究胃癌的发生和发展，以及对于探索以二者为靶点的抗肿瘤治疗有重要意义。
ObjectiveTo systematically review the correlation between the expression of hypoxia inducible factor-1α (HIF-1α) protein and different clinical pathological features of renal cell cancer. MethodsWe electronically searched databases including The Cochrane Library, PubMed, EMbase, CNKI, VIP, CBM and WanFang Data from inception to June 2015 to collect case-control studies investigating the correlation between HIF-1α protein expression and different clinical pathological features of renal cell cancer. Two reviewers independently screened literature, extracted data, and assessed the risk of bias of included studies. Then meta-analysis was performed using RevMan 5.3 software. ResultsA total of 8 case-control studies involving 429 cases of renal cell cancer and 130 cases of normal renal tissue were included. The results of meta-analysis showed that:HIF-1α protein expression was higher in the renal cell cancer group than that in the normal renal tissue group (OR=16.76, 95%CI 8.53 to 32.92, P<0.000 01); HIF-1α protein expression was higher in the lymph node metastasis group than that in the non-lymphnode metastasis group (OR=4.33, 95%CI 2.53 to 7.39, P<0.000 01); HIF-1α protein expression was higher in the TNM Ⅲ-IV group than that in the TNM I-Ⅱ group (OR=0.30, 95%CI 0.18 to 0.51, P<0.000 1); HIF-1α protein expression was higher in the Fuhrman pathology classification G3+G4 group than that in the G1+G2 group (OR=0.54, 95%CI 0.29 to 0.98, P=0.04). However, there were no significant differences in HIF-1α protein expression between the age≥50 group and the age <50 group (OR=1.09, 95%CI 0.54 to 2.19, P=0.82), and between the male group and the female group (OR=0.77, 95%CI 0.48 to 1.25, P=0.29). ConclusionHIF-1α protein expression is significantly correlated to the clinical stage and pathological grading of renal cell cancer. It is possibly involved in the initiation and development of renal cell cancer. Due to the limited quantity and quality of included studies, the above conclusion needs to be further verified by more high quality studies.
ObjectiveTo investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.MethodsThe nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO2, 20%O2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO2, 1%O2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.ResultsHE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).ConclusionHypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.