Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1alpha;) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1alpha; anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1alpha; sense oligonucleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5% glucose solution caudalis veins injection, group C and group D rats received HIF-1alpha; ASODN and HIF-1alpha; SODN caudalis veins injection, respectively(025 mg/kg).The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD18 positive neutrophil cell was（44.93plusmn;3.60）% in group B，（18.66plusmn;1.52）% in group A，（31.66plusmn;4.72）% in group C，（51.00plusmn;5.66）% in group D. Compared with each other groups，the differences are statistically significant （F=42.46, Plt;0.001）. The number of positive staining cells of retinal leukocyte was （46.16plusmn;10.68）in group A，（133.83plusmn;20.43）in group B,（99.83plusmn;9.28）in group C，（121.33plusmn;10.23） in group C. Compared group B with group C，the number of positive staining cells raised about 2.89 times；compared group B with group C and D，the differences are statistically significant （P=0.12，95% confidence interval -3.69~28.69）. Conclusions In vivo, HIF-1alpha; can decreased the expression of CD18 on neutrophils from diabetic ratsprime; peripheral blood and the collection of retinal leukostasis in the diabetic animals. HIF-1alpha; may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy. （Chin J Ocul Fundus Dis，2008，24：268-271）
ObjectiveTo investigate the effect of emodin on the expression of hypoxia inducible factor (HIF)-1α protein in rats with severe acute pancreatitis-associated renal injury and explore the possible mechanisms. MethodsA total of 72 rats were randomly divided into sham-operated group (n=24), severe acute pancreatitis with renal injury group (injury group, n=24), and treatment group (n=24). The sham-operated and injury groups were given 1.5 mL saline through intragastric administration before operation while the treatment group was fed with the same amount of 50 mg/kg emodin diluent. The pancreas and pancreatic tail-segment was dissociated and the head of pancreas was occluded in rats to form the model, and blood vessel forceps were loosed after three hours. All the rats were sacrificed 12, 24 and 36 hours after modeling. The level of ascites, serum amylase, creatinine, blood urea nitrogen were detected. Hematoxylin-eosin staining was used to observe the pancreatic and renal pathological changes, and immunohistochemical method was used to detect the expression of HIF-1α protein level in the kidney. ResultsCompared with the sham-operated group, the level of ascites, serum amylase, creatinine, blood urea nitrogen and the expression of HIF-1α protein level increased significantly. The tissue damage of pancreas and the kidney became more serious. Compared with the injury group, the kidney and pancreas function of the treatment group had a better performance. HIF-1α protein level significantly increased in the treatment group, and the difference had a statistical significance (P<0.05). ConclusionEmodin has a good protective effect on severe acute pancreatitis-associated renal injury. It may function through up-regulation expression of HIF-1α protein level to improve the ability of the kidney to tolerate hypoxia, and then reduce the cell apoptosis and necrosis of the kidney.
Objective To observe the inhibition of LipofectamineTM2000 (LF2000)mediated pSUPER recombinant plasmid expressing small interference RNA targeting hypoxia-induced factor (HIF)-1alpha;(pSUPERsiHIF-1alpha;) on retinal neovascularization in mice. Methods pSUPERsiHIF-1alpha; recombinant plasmid was created. Forty-eight (seven-day-old) C57BL/6J mice were randomly divided into a normal group, the control group, empty vector group and gene therapy group with 12 mice in each group. Mice in the normal group were kept in normal room air, while the other three groups retinal neovascularization was induced by hypoxia. The mice in control group were not treated. The mice in the vector group received intravitreous injection of pSUPER and LF2000 (1 mu;l), and the gene therapy group received pSUPERsiHIF-1alpha; and LF2000 (1 mu;l)one day before being returned to normal room air．Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections．HIF-lalpha;and vascular endothelial growth factor (VEGF) levels in retinas were measured by immune histochemical staining method and reverse transeriptase-polymerase chain reaction (RT-PCR). Results Fluorescent angiography showed radial branching pattern vessels in the normal group and distorted large vessels, obstructed capillaries, many neovascular tuffs, fluorescence leakage in the peripheral retina in the control group and vector group. The gene therapy group demonstrated a significant reduction in neovascular tufts and fluorescence leakage compared with the control group and the vector group. The number of vascular cell nuclei extending breaking through the internal limiting membrane(ILM) of control group and vector group increased significantly compared with normal group (F=5850.016,P＜0.05), while obviously decreasing in the gene therapy group compared with control group (F=3012.469,P＜0.05). Immunohistochemical staining showed the expression of HIF-1alpha; protein in nucleus and VEGF protein in cytoplasm. The expression of HIF-1alpha; protein in retina was negative, while VEGF protein was weakly positive in normal group. The expression of HIF-1alpha; and VEGF protein were both positive in control group and vector group, while weakly positive in gene therapy group. The Results of RT-PCR showed that the expression of HIF-1alpha; mRNA in retina was increased significantly in control group and vector group as compared with normal group (F=3102.326,P＜0.05), while decreasing significantly in gene therapy group as compared with control group (F=3336.425,P＜0.05). Conclusion Retinal neovascularization in the mice is significantly inhibited by intravitreal injection of LF2000-mediated recombinant plasmid pSUPERsiHIF-1alpha;.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Objective To explore the change tendency of hypoxia-inducible factor-1α (HIF-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2) in fetal rat cerebral cortex neurons cultured in vitro after hypoxia-ischemia reperfusion andto investigate their mutual relationship. Methods Cortical neurons obtained from cerebral cortex of 15 pregnant SD rats at16-18 days of gestation underwent primary culture. The primary neurons 5 days after culture were adopted to establ ish model of oxygen and glucose deprivation (OGD). The experiment was divided into 4 groups: the experimental group 1, culture medium was changed to neuron complete medium containing glucose after the preparation of OGD model to form reperfusion, and the neurons were observed 0, 2, 4, 8, 12 and 24 hours after reperfusion; the control group 1, the neurons were treated with normal medium; the experimental group 2, the neurons were pretreated with U0126 followed by the preparation of OGD model, and the neurons were observed 4 and 8 hours after reperfusion; the control group 2, the neurons were pretreated with DMSO, and other treatments were the same as the experimental group 2. Expressions of HIF-1α, VEGF protein, ERK1/2 and p-ERK1/2 were detected by Western blot. Expression and distribution of p-ERK1/2 and HIF-1α protein were detected by SABC immunocytochemistry method. Results Compl icated synaptic connections between cortical neurons processes were observed 5 days after culture. The expression of HIF-1α and VEGF were increased gradually, peaked at 8 hours, and decreased gradually after 12 hours in the experimental group 1, and there were significant differences between the experimental group 1 and the control group 1 (P lt; 0.05). There was no significant difference between the experimental group 1 and the control group 1 in terms of ERK1/2 protein expression (P gt; 0.05). The p-ERK1/2 protein expression in the experimental group 1 started to increase at 2 hours peaked at 4 hours, and started to decrease at 8 hours, showing significant differences compared with the control group 1 (P lt; 0.01). In the experimental group 2, the p-ERK1/2 protein decreased, and HIF-1αand VEGF protein expression subsequentlydecreased, showing significant differences compared with the control group 2 (P lt; 0.05). There was no significant difference between the experimental group 2 and the control group 2 in terms of ERK1/2 protein expression at each time point (P gt; 0.05). Immunocytochemistry staining showed that p-ERK1/2 and HIF-1α expression decreased, and the yellow-brown staining of the neurons was reduced. Conclusion Expressions of HIF-1α and its target-gene VEGF protein in the cortex neurons after OGD reperfusion are time-dependent. Their expressions decrease when ERK1/2 signal ing pathway is inhibited, indicating the pathway plays an important role in the regulation of HIF-1α and VEGF induced by OGD of cortical neurons
ObjectiveTo investigate the effects of hypoxia-inducible factor 1α (HIF-1α) small interfere RNA construct pSUPERH1-siHIF1α on the expression of CD18 and ninjurin-1 by K562 (human chronic myelogenous leukemia cell line) cells cultured with serums from patients with early stage of diabetic retinopathy. MethodsK562 cells were cultured in 4 groups as control group (group A), diabetic group (group B), diabetes and pSUPERH1-siHIF1α transfect group (group C) and diabetes and pSUPER-retro transfect group (group D). The cells in group A were cultured in human serum from age-matched healthy control, and in group B, C and D, the cells were cultured in serum from the subjects of early stage of diabetic retinopathy. Twenty-four hours before the cells were cultured by the serum from the subjects of early stage of diabetic retinopathy, the HIF-1α specific siRNA expression vector pSUPERH1-siHIF1α and empty vector pSUPER-retro were transfected into the cells of group C and D, respectively. The percentages of CD18 and ninjurin-1 positive cell on the surface of K562 cells were measured by Flow Cytometry. The adherent rate between K562 and RF/6A was measured by the rose Bengal staining test. ResultsThe percentages of CD18 positive cell in the group A, B, C and D were significantly different (F=14.33, P=0.01). The percentage of group B was significantly higher than that in group A (P=0.001); the percentage of group C was significantly lower than that in group B (P=0.001) and group D (P=0.02); the difference between group C and A was not significant (95%CI=-14.89-2.13, P=0.12). The differences of the percentage of ninjurin-1 positive cell among the group A, B, C and D were significant (F=39.38, P=0.001). The percentage of group B was significantly higher than that in group A (P=0.00); the difference of the percentage between group C and B was not significant (P=0.06), that was also not significant between group C and D (P=0.49). The differences of the adherent rate between K562 and RF/6A (rhesus monkey retinal choroid blood vessel endothelial cell line) among the group A, B, C and D were significant (F=20.62, P=0.00). The adherent rate of group B was significantly higher than that in group A (P=0.00), the adherent rate in group C was significantly lower than that in group B (P=0.01), but it was still significantly higher than that in group A (P=0.002), the difference of adherent rate between group B and D was not significant (P=0.68). ConclusionUnder the early stage of diabetic retinopathy, HIF-1α small interfere RNA pSUPERH1-siHIF1α may significantly suppress the expression of CD18 on the surface of K562 cells, but it may not significantly influence the expression of ninjurin-1 on the surface of K562 cells.
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。
Objective To investigate the expressions of hypoxia-inducible factor-1α (HIF-1α) and caudal homeobox gene 2 (CDX2) in colorectal adenocarcinoma, and the relationships between them and the clinicopathologic factor of colorectal adenocarcinoma. Methods The expressions of HIF-1α and CDX2 were detected by immunohistochemistry in 62 specimens of colorectal adenocarcinoma and 20 specimens of normal colorectal mucosa tissue. The correlation between the expressions of HIF-1α and CDX2 was analyzed by Spearman rank correlation analysis. Results The positive rates of HIF-1α expression in normal colorectal mucosa tissue and colorectal adenocarcinoma were 5.0% （1/20） and 62.9% （39/62）, CDX2 were 95.0% （19/20） and 69.4% （43/62）, the differences of positive rate between different tissues were significant （Plt;0.05）. In colorectal adenocarcinoma, the expression of HIF-1α or CDX2 was related to tumor differentiation, lymph node metastasis, and Dukes staging (Plt;0.05). There was a negative correlation between HIF-1α and CDX2 expressions in colorectal adenocarcinoma （r=－0.293 2，Plt;0.05）. Conclusions The up-regulation of HIF-1α and down-regulation of CDX2 may be involved in the genesis of colorectal adenocarcinoma， and there is a negative correlation between the two kinds of protein. HIF-1α may participate in modulation of CDX2 expression and lead to accelerate the progression of colorectal carcinoma.