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 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 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.