Objective To explore and evaluate the protective effects of soluble fms-like tyrosine kinase recptor-1(sFlt-1) gene 2-3 and 2-4 transcellular region on retinal vascular leakage and phosphatidyl inositol-3 kinase (PI3K)/Akt pathway under hypoxia and/or hyperglycemia. Methods The plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) were constructed.. Two of these plasmids with carboxy methylation glucan (CMD) magnetic particles were transfected into human umbilical vein endothelial cells (HUVEC), which were cultured under hypoxia and/or hyperglycemia. The blood retinal barrier was evaluated by Evans blue permeation (EBP). Then the expression of p-Akt mRNA and protein were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot separately. Results In the diabetic rabbits, The blood retinal barrier breakdown was detected by the retinal vascular leakage of EBP. The expression of p-Akt mRNA and protein in hypoxia and hyperglycemia groups were also obviously increased. These changes were largely prevented by transfection the plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) (P＜0.01 in both groups). Conclusion Both sFlt-1(2-3) and sFlt-1(2-4) can make the retinal blood vessels less leaky and may be beneficial in preventing vision loss from diabetic retinopathy.
Objective
To establish a rapid in vitro culture method of human choroidal endothelial cells (HCEC) and the cellular Characteristics to provide an in vitro model for researches of choroiretinal diseases which involved the HCEC.
Methods
The human choroidal tissues were digested in two steps by trypsin and collagenase, and the HCEC were obtained and cultured after the digested cell suspension was sorted and purified with magnetic beads of CD31 Dynabeads. The characteristics of HCMEC were observed by the morphologic observation method, transmission electron microscopy, and immunohistochemical staining with FⅧ factor, CD31, and CD34.
Results
The cultured HCEC were polygonal and oval, and after amalgamation, the cells had slabstone-like appearance. After the subculture, the configuration of HCEC remained the same, and represented cobblestone appearance with less magnetic beads attached on the cellular surface after HCEC converged into a single layer. The Weibel-Palade body which is the characteristic marker of endothelial cells was found. The staining of FⅧ fatcor, CD31, CD34 were positive.
Conclusion
HCEC can be cultured in vitro successfully with our method, which is easy to get sufficient number of highly purified HCEC.
(Chin J Ocul Fundus Dis, 2007, 23: 126-129)
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 investigate the inhibitory effects of fms-like typrosine kinase receptor sFlt-1 on retinal neovascularization (RNV).Methods Recombinant lentivirus sFlt-1（2-3）and sFlt-1（2-4）expressing the sFlt-1 (2-3) and (2-4) immunoglobulinlike regions of sFlt-1 were constructed. 96 seven-day-old C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1: normal control; group 2: experimental control; group 3: sFlt-1（2-3）; group 4: sFlt-1（2-4）.The mice in group 2-4 were exposed to hyperoxia with （75plusmn;2）% O2 for 5 days and then returned to normoxia with 21% O2;the mice received an intravitreal injection with 1 mu;l virus of empty vector, sFlt-1（2-3）,or sFlt-1（2-4）,respectively. Five days later, all mice underwent perfusion fluorecein angiography and retinal wholemont was made to observe the changes of retinal vessels; retinal sections were stained by hematoxylin and eosin and RNV endothelium cell nucleus were counted; vascular endothelial growth factor(VEGF) and VEGF receptor-2 (KDR/Flk-1) protein were measured by Western blot.Results Seventeen days after birth, the retinal area of fluorescein leakage and RNV, RNV nucleus which breaking through inner limiting membrane in group 3 and 4 were smaller or less than that in group 2（P＜0.01）; while VEGF protein didnprime;t changed much （P＞0.05）the expression of KDR/Flk-1 decreased significantly (P＜0.01). Conclusion sFlt-1（2-3）and sFlt-1（2-4）can inhibit the formation of oxygen-induced RNV,the former virus has a better effect.
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.
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.
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 analyze the consistency of diagnostic results using simple and comprehensive reading methods on stereoscopic color fundus photographs of diabetic retinopathy (DR) with diabetic macular edema (DME).
Methods450 sets of 7-field stereoscopic color fundus photographs of DR DME were compared to standard fundus photographs of early treatment and DR study group. The pictures were read by two groups of reader with similar experience. Two strategies were used to make the judgments, including simple reading which based on the color fundus photographs only, and comprehensive reading which based on color fundus photographs, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). 15 parameters were scored, including micro-aneurysms (MA), intra-retinal hemorrhage (IRH), hard exudates (HE), cotton wood spot (CW), intra-retinal microvascular abnormalities (IRMA), neovascularization on optic disc (NVD), neovascularization elsewhere (NVE), optic fiber proliferation (FPD), fiber proliferation elsewhere (FPE), pre-retinal hemorrhage (PRH), vitreous hemorrhage (VH), retinal elevation (RE), retinal detachment of central macular (RDC), venous beading (VB), Venous leak (VL). The reliability was evaluated using weighted κ(κw) statistic values. According to Fleiss statistical theory, κw≥0.75, consistency is excellent; 0.60≤κw < 0.75, consistency is good; 0.40≤κw < 0.60, consistency is general; κw < 0.40, consistency is poor.
ResultsThe κw values of these 15 parameters were 0.22-1.00, 0.28-1.00 for the simple reading and comprehensive reading respectively. For simple reading, the consistency was poor for 8 parameters (MA, NVD, NVE, FPE, PRH, IRMA, VB, VL), general for 3 parameters (CW, FPD, VH), good for 2 parameters (IRH, HE) and excellent for 2 parameters (RE, RDC). For comprehensive reading, the consistency was poor for 2 parameters (NVE, VB), general for 6 parameters (MA, IRH, CW, FPE, IRMA, VL), good for 2 parameters (NVD, HE), excellent for 5 parameters (FPE, PRH, VH, RE, RDC).
ConclusionThe comprehensive reading has higher consistency to judge the abnormality parameters of the fundus photographs of DR with DME.
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 elucidate the new development, structural features and appl ication of the lumbar interspinous process non-fusion techniques. Methods With the review of the development course and important research works in the field of the lumbar inter-spinous process non-fusion techniques, the regularity summary, science induction, and prospect were carried out. Results The lumbar inter-spinous process non-fusion technique was a part of non-fusion insertof spinal division posterior surface. According to the design, it could be divided into two major categories: dynamic and static systems. The dynamic system included Coflex and device for intervertebral assisted motion; the static system included X-STOP, ExtenSure and Wall is. The lumbar inter-spinous process non-fusion technique was a new technique of spinal division, it could reserve the integrated function of intervertebral disc and zygapophysial joint, maintain or recover the segmental movement to a normal level, and have no adverse effect on the neighboring segments. A lot of basic and cl inical researches indicated that lumbar inter-spinous process insert had extensive appl ication to curatio retrogression lumbar spinal stenosis, discogenic low back pain, articular process syndrome, lumbar intervertebral disc protrusion and lumbar instabil ity and so on. Conclusion With the matures of lumbar inter-spinous process non-fusion techniques and the increased study of various types of internal fixation devices, it will greatly facil itate the development of treatment of lumbar degenerative disease. But long-term follow-up is needed to investigating the long-term efficacy and perfect operation indication.
