Objective To investigate the effect of small interfering RNA(siRNA) targeting hypoxia inducible factor1alpha; (HIF1alpha;) and vascular endothelial growth factor (VEGF) on expression of VEGF in human vascular endothelial cells. Methods HIF-1alpha; siRNA recombinant plasmid was constructed. Human vascular ndothelial cells were cultured in vitro and divided into normoxia group (20% O2) and hypoxia group (1% O2). Hypoxia group was then divided into control group, vector group, HIF-1alpha; group (HIF-1alpha; siRNA), VEGF group ( VEGF165 siRNA) and cotransfection group (HIF-1alpha; siRNA+VEGF165 siRNA). LipofectamineTM 2000 (LF2000) mediated vector plasmid was transfected to cells in each group except the control group. The expression of HIF-1alpha; siRNA and VEGF165 siRNA recombinant plasmid were identified by reverse transcriptasepolymerase chain reaction (RT-PCR). The expression of VEGF mRNA and protein were detected by RTPCR and immunocytochemical method. Results The expression of HIF-1alpha; siRNA and VEGF165 si RNA recombinant plasmid were detected 24 hours after transfected. The expression of VEGF mRNA and protein was faint in the normoxia group, but increased obviously in hypoxia group. The expression of VEGF mRNA and protein in the HIF1alpha;, VEGF and cotransfection groups were lower than which in the control group. Cotransfection group showed the highest inhibitory effect. Conclusion HIF-1alpha; and VEGF165 siRNA can effectively inhibit the expression of VEGF in human vascular endothelial cells.
ObjectiveTo observe the expression and mechanism of hypoxia-inducible factor-1α (HIF-1α) and p53 protein at the altitude of 5000 meter plateau hypoxia environment in rats, as well as the effect of Astragalus injection. MethodsSixty Sprague Dawley rats were randomly divided into the Astragalus injection intervention group and normal saline control group, 30 rats in each group. Astragalus injection group rats were intraperitoneal injected of Astragalus injection (15 ml/kg) before 30 minutes into the plateau environment simulation cabin, normal saline group rats were intraperitoneal injected with the same volume of saline. 30 minutes after injection, rats in each group were reared in the plateau experiment cabin which simulated altitude of 5000 m (oxygen partial pressure 11.3 kPa) for 2, 6, 8, 12, 24 hours, each time period of 6 rats. When get out, the rats were executed immediately and eyes were harvested. Retinal sections were studied by hematoxylin eosin stain, and immunohistochemical method for HIF-1α and p53 expression. ResultsFor control rats, after 2 hours in the cabin, there was edema in retinal layers. HIF-1α and p53 were expressed mainly in the cytoplasm of retinal layers. When the periods in cabin extended, there was atrophy of retinal nerve fiber layer, swelling and degeneration of ganglion cells. The expression of HIF-1α and p53 was increased. Compared with the control group, the intervention group rat had similar but less severe retinal changes, and the expression of HIF-1α and p53 was significantly decreased (P<0.05). ConclusionAstragalus injection can reduce pathological retinal damage in rats at high altitude environment, and its mechanism may be associated with reduced HIF-1α, p53 expression.
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;.
Objective To investigate the expression of hypoxia inducible factor 1(HIF1alpha;) in ratsprime; retinae during the embryonic and earlier postnatal period. Methods The retinal expression patterns of HIF-1alpha; protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcriptionpolymerase chain reaction (RT-PCR). Results HIF-1alpha; protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when re tina became more mature. Embryonic rat retina had higher expression of HIF-1alpha; protein and mRNA than postnatal retina, the difference was significant (P＜0.01）. Conclusion The expression of HIF1alpha; in ratsprime;retina e differs from embryonic to earlier postnatal stages.
Objective To observe the effect of hypoxia inducible factor-1alpha;（HIF-1alpha;）to the expression of cell surface adhesion molecules CD18 and the adhesion ability of leukocytes and vascular endothelial cells under early stage of diabetic retinopathy condition.Methods The human promyelocytic leukemia cell line HL60 and the rhesus choroid-retina vascular endothelial cell line RF/6A were cultured in RPMI 1640 medium-10% human serum, which was collected from the subjects of early stage of diabetic retinopathy and age-matched healthy control. The cells were cultured in 4 groups as control group (group A), diabetic group (group B), HIF-1 anti-sense oligonucleotides (ASODN) group (group C) and HIF-1 sense oligonucleotides (SODN) group (group D). The percentages of CD18 positive cell in the HL60 cell were measured by flow cytometry and mRNA in the HL60 cell by realtime reverse transcriptionpolymerase chain reaction（RT-PCR）. Results The percentage of CD18 positive cell in the group A, B, C and D was 17.06plusmn;6.01, 42.23plusmn;2.60, 25.33plusmn;3.05 and 32.40plusmn;10.57, respectively, the differences among them were significant (F=36.47,P＜0.001). Compared to the group A,the expression of CD18 mRNA in the group B,C and D was increased about 21.05plusmn;2.07、2.23plusmn;0.96 and 25.07plusmn;2.27 times,respectively, the differences among them were significant (F=180.34, Plt;0.001). The adherent rates of HL60 to RF/6A in group A, B, C and D was 0.06plusmn;0.00,0.09plusmn;0.10,0.05plusmn;0.00 and 0.07plusmn;0.01, respectively,the differences among them were significant（F=13.06，P=0.002）.Conclusion In vitro, HIF-1 could regulate the expression of CD18 by HL60, and the adhesion of HL60 to RF/6A when the cells were exposed to diabetic serum. The effects of human serum weaken with the inhibition of HIF-1 expression.HIF-1 play regulatory role in the expression of CD18 and adhesion of leukocytes and vascular endothelial cells under early stage of diabetic retinopathy condition.
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.
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）
ObjectiveThis study aims to study the effects and mechanism of resveratrol on hepatocellular carcinoma (HCC) cells through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) axis.MethodsHepG2 cells at logarithmic growth stage were treated with different concentrations (0, 12.5, 25.0, and 50.0 μmol/L) of resveratrol, respectively. Then the proliferation of HepG2 cells was detected by the CCK8 method and real time cell anaIysis (RTCA) system, the expressions of signal molecules associated with PI3K/Akt axis was detected by the Western blot method, including PI3K p58, phosphorylation protein kinase B (p-Akt), total protein kinase B (t-Akt), and CyclinA2 protein.ResultsResveratrol had a significant inhibitory effect on the growth of HepG2 cells in a time and dosage dependent manner. After 48 h treatment of resveratrol to HepG2 cells, 50.0 μmol/L resveratrol inhibited the growth of HepG2 cells most significantly. Further, the RTCA system studies also found that resveratrol had a time and concentration dependent effect on the reduction of normalized cell index (NCI) in HepG2 cells. Flow cytometry results showed that, apoptosis rates of 12.5, 25.0, and 50.0 μmol/L group were higher than that of 0 μmol/L group. Compard with 0 μmol/L group, the expressions of PI3K p85, p-Akt, and CyclinA2 protein in HepG2 cells of 12.5, 25.0, and 50.0 μmol/L resveratrol group was significantly higher (P<0.05), although there was no significant effect of resveratrol on the expression of t-Akt in HepG2 cells (P>0.05).ConclusionsResveratrol might have anti-proliferation effects on HepG2 cells through PI3K p85/Akt signaling axis. This study could provide a novel idea for the treatment to HCC.
The aim of this study is to determine the regulatory mechanism of PTEN-induced putative kinase protein 1 short isoform (PINK1S) in cytoplasm. By co-immunoprecipitation (Co-IP) assay, we identified that PINK1S interacted with the beta regulatory subunit of Casein Kinase 2 (CK2β), but not with the catalytic subunits CK2α1 and CK2α2. Furthermore, cells were transfected with PINK1S and CK2β, and then PINK1S was purified by immunoprecipitation. After detecting the phosphorylated proteins by Phos-tagTM Biotin, we found that CK2β overexpression increased auto-phosphorylation of PINK1S. Finally, we generated CK2β knockdown cell lines by RNA interference. Purified PINK1S from CK2β knockdown cells significantly reduced its auto-phosphorylation compared with control cells. These results suggested that CK2β functions as a regulatory subunit of PINK1S kinase complex promoted its activation by self-phosphorylation.