ObjectiveTo observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice. MethodsCREB1 siRNA construct was created, screened and packaged to produce CREB1 RNAi-lentivirus. One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group, oxygen induced retinopathy (OIR) group, empty vector group and CREB1 therapy group with 35 mice in each group. Mice in the normal group were kept in normal room air, while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7). The mice in the OIR group were not treated. The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP, 1 μl), and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography. The areas of RNV and non-perfusion region were calculated. The expression of CREB1, phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels, Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. ResultsThe number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P<0.05), while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P<0.05). The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group, while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group. The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220, 110.090; P<0.05). The mRNA expression of CREB1 and protein expression of P-CREB1, the mRNA and protein expression of VEGF-A, Akt, PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group, while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group. The difference of mRNA expression of CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=6.087, 5.464, 6.191, 8.627; P<0.05). The protein expression of P-CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=162.944, 13.861, 19.710, 22.827; P<0.05). ConclusionRNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation.
ObjectiveTo observe the effect of adenovirus-mediated Tum5 (rAd-Tum5) inhibiting retinal neovascularization (RNV) of oxygen-induced retinopathy (OIR) mouse model. MethodsThe OIR model was induced in 96 C57BL/6J mice aged of 7 days according to the literature. These mice were divided randomly into control group, OIR group, OIR rAd-green fluorescent grotein (GFP) group and OIR rAd-Tum5 group, each group had 24 mice. The rAd-GFP and rAd-Tum5 were injected into the vitreous cavity of mice aged of 12 days in OIR rAd-GFP group and OIR rAd-Tum5 group, respectively. Meanwhile, OIR group and the control group received the injection of physiological saline solution of same volume. The relatively non-perfusion area was evaluated by fluorescence angiography, and the number of pre-retinal nucleus breaking through internal limiting membranes was observed by hematoxylin-eosin staining. The expression of vascular endothelial growth factor (VEGF) was estimated by immunofluorescent (IF) and Western blot. ResultsThe retinal avascular areas of all groups were significantly different (F=61.224, P<0.01). The retinal avascular area of the rAd-Tum5 group was decreased significantly comparing with that in the OIR group and rAd-GFP group (P<0.01). However, there are no significant differences between the OIR group and rAd-GFP group (P=0.827). The number of pre-retinal nucleus breaking through ILM of all groups was significantly different (F=635.738, P<0.01), but no significantly difference was observed in OIR group and rAd-GFP group (P=0.261). Significant differences could also been seen between OIR rAd-Tum5 group and OIR group as well as OIR rAd-Tum5 group and OIR rAd-GFP group (P<0.01). The results of IF and Western blot indicated that expression of VEGF in the OIR group and rAd-GFP group was obviously up-regulated, compared with that in the control group. But the expression was declined in the rAd-Tum5 group compared with that in the OIR group and rAd-GFP group. ConclusionTum-5 peptide can efficiently prevent RNV probably by down-regulating expression of VEGF.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
ObjectiveTo observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. MethodsTo construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. ResultsThe adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). ConclusionThe adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.
Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adenoassociated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo.Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A,B,C,D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 mu;l phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 mu;l recombinant adenoassociated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5 mu;l rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects.Results Green fluorescence can be observed exactly in labeled RGC in B，C，and D groups. The AOD and transfection rate in group D was （95.02plusmn;7.25）% and（20.10plusmn;0.74）% , respectively; which were higher than those in group B and C (F=25.970，25.799；P＜0.01). The difference of the number of RGC among the four groups was not significant(F=0.877,P＞0.05). Conclusion Under the condition of low frequency and with certain energy, ultrasoundmediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.
Objective To compare the transfection effects on soluble fms-like tyrosine kinase receptor-1 (sFlt-1) gene (2-4 transcellular region) mediated by carboxymethylated dextran coated nanoparticle and lipofectamineTM2000.Methods The plasmid pcDNA3.1-EGFP/sFlt-1(2-4) was constructed and assessed by enzyme cut, electrophoresis, and genetic sequencing. Three groups were divided: nanoparticle group, lipofectamine group, and non-transfected group. Twenty-four and 48 hours after the transfection, the distribution of cellular green fluorescence was oberved under the inverted phase contrast fluorescence microscope; the expression rate of green fluorescence was measured by flow cytometry; the expression of sFlt-1(2-4)mRNA and the protein was detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot; the growth of the cells was observed by methyl thiazolyl tetrazolium (MTT) colorimetry and the relative growth rate (RGR) of the cells in each group was calculated; the cellular apoptosis in each group was detected by Hoechst staining.Results The sequence of sFlt-1(2-4) gene was equal to 915 base pair (bp).The transfection rate was 45% in nanoparticle group and 21% in lipofectamine group; the difference between the two groups was significant (t=2.541，Plt;0.05). Forty-eight hours after the transfection, the expression of sFlt-1（2-4）mRNA and protein was obviously higher in nanoparticle group than that in lipofectamine group (t=2.454，2.398；Plt;0.05) . Twenty-four and 48 hours after the transfection,the difference of RGR of the cells between nanoparticle and non-transfected group was not significant（t=1.436，Pgt;0.05）; the RGR in lipofectamine group differed much from that in non-transfected and nanoparticle group (t=2.412，2.545; Plt;0.05) ; the difference of cellular apoptosis was not significant between nanoparticle and nontransfected group (t=1.436，Pgt;0.05), but significant between nanoparticle and lipofectamine group (t=2.236，Plt;0.05). Conclusion The transfection rate of sFlt-1(2-4) mediated by carboxymethylated dextran coated nanoparticle was higher than that mediated by lipofectamineTM2000.
Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group,oxygen-induced retinopathy (OIR) group, empty vector group and treated group,14 mices in each group. Except for the control group,the mice in the other groups were exposed to (75plusmn;2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid.The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane（ILM） was observed and counted by optical microscope.Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group.The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups（F=39.006，Plt;0.001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinalneovascularization in OIR.
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 investigate the expression of miR-195 and the underlying molecular mechanisms of miR-195 regulating HMGB1 in diabetic retinopathy (DR). MethodsExtract 5 ml venous blood from DR patients, diabetes mellitus (DM) patients and normal subjects, then extract and perificate plasma total RNA. MicroRNA array and real time polymerase chain reaction (RT-PCR) was used to screen out miRNAs which were expressed with significant differences in the serum of patients with DR. Bioinformatics was employed to predict the miR-195 related to high mobility group box 1 (HMGB1) regulation. Next, miR-195 was down-regulated or up-regulated in umbilical vein endothelial cells through transfection of miR-195 inhibitor and miR-29b mimics respectively.Then we analyzed expression of HMGB1 mRNA and protein by RT-PCR and Western blot. ResultsMicroRNA array results showed the expression of miR-195 in DR group is decreased by 8.34 times and 11.47 times compared with DM group and the normal group. RT-PCR verification results conforms to the microRNA array results. Compared with the DM group (F=0.034, t=8.057) and the normal group (F=0.370, t=9.522), the expression of miR-195 in DR group were significantly reduced, the differences were statistically significant (P < 0.05). RT-PCR showed that the expression of HMGB1 mRNA was significantly decreased in up-regulation group, compared with blank (F=0.023, t=11.287) and negative control group (F=0.365, t=7.471), the difference was statistically significant (P < 0.05). The expression of HMGB1 mRNA was significantly increased in down-regulation group, compared with blank (F=0.053, t=10.871) and negative control group (F=0.492, t=6.883), the difference was statistically significant (P < 0.05). Western blot showed that the expression of HMGB1 protein was significantly decreased in up-regulation group, compared with blank (F=0.021, t=8.820) and negative control group (F=0.039, t=7.401), the difference was statistically significant (P < 0.05); and significantly increased in down-regulation group, compared with blank (F=0.186, t=10.092) and negative control group (F=0.017, t=12.923), the difference was statistically significant (P < 0.05). ConclusionMiR-195 can inhibit the expression of HMGB1, reduce the inflammation and angiogenesis, thereby delaying or inhibiting the occurrence and development of DR.
Objective To investigate the inhibitory effects of 15-lipoxygenase-1 (15-LOX-1) gene transfer on oxygen-induced retinal neovascularization in mice. Methods Ninety-six 7-day-old C57BL/6J mice were randomly divided into normal control group, oxygeninduced retinopathy (OIR) model group, gene treated group and empty vector group. The mice with their mothers were kept in (75plusmn;2) % 02 environment for 5 days and then returned to normoxia for 5 days to establish the OIR model. At postnatal day 12, the gene treated group received intravitreous injection of recombinant adenovirus (Ad) vector containing both enhanced green fluorescent protein (EGFP) and mouse 15-LOX-1 genes (Ad-15-LOX-1-EGFP) at 1 l, while the empty vector group received the same volume of recombinant Ad vector containing EGFP (Ad-EGFP). The expression of EGFP was observed on flat-mounted retina by fluorescence microscopy 2 days after intravitreous injection of Ad-15-LOX-1-EGFP. At postnatal day 17, the efficacy of 15-LOX-1 gene transfer on retinal tissue was detected by immunofluorescence staining, real-time polymerase chain reaction and Western blot. The changes of retinal vessels, relative retinal non-perfusion and neovascularization areas were evaluated by fluorescein isothiocyanate-dextran fluorescein angiography on flatmounted retina. The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. Results Two days after intravitreous injection of Ad-15-LOX-1-EGFP, the expression of EGFP had been seen by fluorescence microscopy on Flat-mounted retina. Immunofluorescence staining of retinal section revealed that 15-LOX-1 expression was primarily in the outer plexiform layer, inner nuclear layer and ganglion cell layer of retina. The 15-LOX-1 protein and mRNA expression levels were higher in gene treated group than those in OIR model group and empty vector group (tprotein=22.74 and 24.13 respectively.tmRNA=12.51 and 13.40 respectively; P＜0.01). The relative retinal non-perfusion and neovascularization areas were significantly smaller in gene treated group than those in OIR model group and empty vector group (tnon-perfusion=16.22 and 14.31 respectively.tneovascularization=9.97 and 9.07 respectively; P＜0.01), and the number of endothelium cell nuclei breaking through the ILM in gene treated group was obviously lower than the other two groups (t=14.25 and 11.62 respectively，P＜0.01). Conclusion 15-LOX-1 gene transfer can decrease the oxygen-induced retinal non-perfusion areas and inhibit the retinal neovascularization in mice.