ObjectiveTo investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.MethodsThirty patients with DR (DR group), 30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study. Three groups of subjects were taken 5 ml of venous blood, and total plasma RNA was extracted and purified. The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip, and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR). Bioinformatics was used to predict potential target genes for miRNA regulation, and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened. Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L). hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model, which was divided into blank control group, high expression group and negative control group. The expression of miR-1470 was detected by RT-PCR. The expression of EGFR protein was detected by Western blot. The measurement data of the two groups were compared using the independent sample t test. The comparison of the measurement data between the two groups was analyzed by ANOVA. The comparison between the measurement data of the groups was compared by multiple comparisons.ResultsThe results of RT-PCR were consistent with those of the gene chip. The expression of miR-1470 in the plasma of the DR group, the DM group and the normal group was statistically significant (F=63.486, P=0.049). Compared with the DM group and the normal group, the expression of miR-1470 in the DR group was significantly decreased, and the difference was statistically significant (q=111.2, 73.9; P＜0.05). The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082, P=0.015). The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=−39.939, P=0.016). The expression of miR-1470 (F=637.069, P=0.000) and EGFR (F=122.908, P=0.000) protein expression in hREC of blank control group, negative control group and high expression group were statistically significant . Compared with the blank control group and the negative control group, the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7, 328.8; P＜0.05), and the expression of EGFR protein was significantly decreased (q=242.5, 234.6; P＜0.05). There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5, 7.9; P＞0.05).ConclusionThe expression of miR-1470 in the plasma of patients with DR is significantly down-regulated, and the increase of EGFR expression may be related to it.
Objective To investigate the effects of recombinant adeno-associated virus type-2 (rAAV2) mediated delivery of pigment epitheliumderived factor (PEDF) on oxygen-induced retinal neovascularization (OIRNV) in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 mu;l rAAV2-PEDF and rAAV2EGFP into the left eyes (experimental group) and the right eyes (control group). All mice were put into the oxygen box right after the injection to induce the OIRNV model.4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 (P13). Twelve mice underwent retinal angiography with high molecular weight fluoresceindextran, and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative nonperfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software. Results The expression level of PEDF protein was higher in the experimental group than that in the control group.The absolute nonperfusion area was (0.96plusmn;0.22) mm2 in the experimental group and (1.96plusmn;0.34) mm2 in the control group; the difference between the two groups was significant（t=-8.554, P＜0.01）. The relative nonperfusion area was (8.64plusmn;1.52)% in the experimental group and (17.27plusmn;2.98)% in the control group with a significant difference between the two groups (t=-8.97, P＜0.01).The absolute area of retinal neovascularization was (0.37plusmn;0.11) mm2 in the experimental group which was obviously higher than (1.26plusmn;0.38) mm2 in the control group (t=-7.8, P＜0.01); the relative areas in experimental and control groups was (3.96plusmn;0.66)% and (11.45plusmn;2.06)%, respectively, whose difference is apparently(t=-8.51, P＜0.01).The areas of retina neovascularization were (0.11plusmn;0.003) mm2 and (0.41plusmn;0.02) mm2 in the experimental and control groups, respectively, and the difference between the two groups was significant（t=-5.14, P＜0.01）.Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion. rAAV2-PEDF can decrease the retinal non-perfusion areas and inhibit the retinal neovascularization in OIRNV mice.
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.
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.
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 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 construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene.Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Nontransfected NPE cells were set as control group.The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively.Results The opticin mRNA expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were decreased compared with the control group (F=10.239，P＝0.000)；the inhibitory rate were 85.7%，62.87%，54.87% and 48.77% respectively.The opticin protein expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were also decreased compared with the control group (F=17.870，P=0.000)；the inhibitory rate were 78.7%，34.6%，31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
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.