Effects Of Netrin-1 On Placental Angiogenesis In Vitro And In Vivo

Part one Silence of Netrin-1 expression in HUVECs by RNA interference in vitroObjectiveTo inhibit the expression of Netrin-1 in HUVECs by RNA interference in vitro.Methods1. Primary HUVECs were isolated and cultured, then identified by immunofluorescence;2. Plasmid was transfected transiently into HUVECs by RNA interference, and selected by G418;3. Expression of Netrin-1 mRNA of transfected HUVECs was detected by real time quantitative PCR;4. Expression of Netrin-1 protein of transfected HUVECs was detected by Western blotting.Results 1. FactorⅧand CD31 were positive through immunofluorescence;2. Expression of GFP was stable in transfected HUVECs by RNA interference technology;3. Expression of Netrin-1 mRNA of transfected HUVECs was decreased by (86.54±3.10)%;4. Expression of Netrin-1 protein of transfected HUVECs was decreased by (77.62±6.92)%.ConclusionExpression of Netrin-1 mRNA and protein in HUVECs can be inhibited efficiently and stablely through RNA interference technology.Part twoEffects of Netrin-1 on HUVECs ability related to angiogenesis in vitroObjectiveTo study the Effects of Netrin-1 on HUVECs biological function related to angiogenesis in vitro.Methods1.HUVECs were divided into 3 groups:control group (cells were transfected with shVetcor and cultured in normal medium), Netrin-1 group (cells were transfected with shVetcor and cultured in medium with 100 ng/ml recombinant Netrin-1), and shNetrin-1 group (cells were transfected with shNetrin-1 and cultured in normal medium);2. Viability of HUVECs was detected by 3-(4,5-dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide salt (MTT) assay; 3. Proliferation of HUVECs was detected by colony formation assay;4. Migration of HUVECs was detected by using transwell;5. Angiogenic ability of HUVECs was detected by tube formation assay.Results1. After stimulated with Netrin-1 for 24 hours, viability of HUVECs was significantly improved; after inhibited the expression of Netrin-1 for 24 hours, viability of HUVECs was significantly decreased;2. Colony forming rate of HUVECs in Netrin-1 group was increased from (60±6.67)% to(92.22±5.09)%, and which in shNetrin-1 group was reduced from (60±6.67)% to(30±3.33)% (p<0.05);3. The number of cell migration of Netrin-1 group was increased from (81.8±7.95) to (124.2±15.9); which in shNetrin-1 group was decreased to (40.4±8.56) (p<0.05);4. The number of branch points of Netrin-1 group was increased from (32.6±3.13) to (57.4±6.88); which in shNetrin-1 group was decreased to (19.6±4.45) (p<0.05).ConclusionExogenous Netrin-1 can promote the cell viability, proliferation, migration and tube formation of HUVECs in vitro; which can be inhibited by the silence of Netrin-1 gene.Part threeSilence of Netrin-1 expression in placenta by RNA interference in vivoObjectiveTo inhibit the expression of Netrin-1 in placenta by RNA interference in vivo.Methods1. Plasmid was transfected into the placenta of pregnant rats by RNA interference; 2. Transfection effect of placenta was observed through frozen sections under fluorescence microscope;3. Expression of Netrin-1 mRNA of transfected placenta was detected by real time quantitative PCR;4. Expression of Netrin-1 protein of transfected placenta was detected by Western blotting and immunohistochemistry.Results1. Expression of GFP was expected in transfected placenta by RNA interference technology;2. Expression of Netrin-1 mRNA of transfected Placenta was decreased by (63.49±9.58)%;3. Expression of Netrin-1 protein of transfected HUVECs was decreased by (57.93±11.76)%, and Netrin-1 was mainly expressed in placental vascular endothelial cells.ConclusionExpression of Netrin-1 mRNA and protein in placenta can be inhibited efficiently through RNA interference technology.Part fourEffects of Netrin-1 on placenta angiogenesis and fetus development in vivoObjectiveTo study the Effects of Netrin-1 on placenta angiogenesis and fetus development in vivo.Methods1. The pregnant rats were randomly divided into 4 goups, and injected with the corresponding medicine at day 14 of gestation:control group (rats were injected with 20μl saline), Netrin-1 group (rats were injected with 400 ng Netrin-1 dissolved in 20μl saline), shVector group (rats were injected with 20μl transfection compounds containing 10 ug sh Vector), shNetrin-1 group(rats were injected with 20μl transfection compounds containing 10μg shNetrin-1);2. Placenta, fetal size and weight were measured at day 17 of gestation;3. Placental vascular density was detected by using immunohistochemical assay against CD34 at day 17 of gestation.Results1. Compared with control group, placental weight in Netrin-1 group was increased from (1.57±0.1) g to(1.83±0.12) g; placental weight in shNetrin-1 group (1.35±0.09) g was significantly decreased from (1.53±0.11) g in shVector group (p<0.05);2. Compared with control group, placenta microvessels in Netrin-1 group was increased from (80.2±5.89) to (98.4±9.07); placenta microvessels in shNetrin-1 group (54.0±5.34) was significantly decreased from (75.6±7.3) in shVector group (p<0.05);3. Compared with control group, fetal weight in Netrin-1 group was increased from (2.46±0.18) g to(2.67±0.10) g; fetal weight in shNetrin-1 group(2.10±0.20)g was significantly decreased from (2.40±0.14) g in shVector group (p<0.05).ConclusionExogenous Netrin-1 can promote angiogenesis in the placenta of pregnant rat and fetal growth in vivo; which can be inhibited by the silence of Netrin-1 gene.