Objective To study the effect of serum rich in growth factors (SRGF) derived from plateletrich plasma (PRP) on the biological function of human and rat osteoblast.Methods PRP and platelet-poor plasma (PPP) obtained from healthy human and SD rat were activated by thrombin toget SRGF and serum poor in growth factors (SPGF). The level of TGFβ1 and PDGF-AB in human-SRGF and SPGF were assayed by enzyme-linked immunoassay(ELISA). Rat and human osteoblast were cultured and identified. Rat osteoblasts were treated with 5% rat-SRGF, 5% rat-SPGF and serumfree F12 medium, respectively. And human osteoblast were treated with 5% human-SRGF, 5% human-SPGF and serumfree DMEM. Cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay at 24, 48, 72 and 96hours.Results The level of TGF-β1 in human-SRGF was 307.67±35.57 ng/ml, and that of PDGF-AB was 52.76±7.89 ng/ml. The proliferation of rat and human osteoblast were promoted after treated with rat-SRGFand human-SRGF, respectively. In rat osteoblast groups, there were significant differences in absorbency between ratSPGF group and rat-SRGF group at 48 and 96 hours(Plt;0.05). In human osteoblast groups, the differences between human-SPGF group and human-SRGF group were significant at 48, 72 and 96 hours(Plt;0.05). The proliferation of these two kinds of osteoblasts almost stopped in serum-free medium, and the differences in absorbency , compared with othergroups，were significant (Plt;0.05). Conclusion High quality of PRP can be achieved by the improved method and SRGF is capable of up-regulating the proliferation of rat osteoblast and human osteoblast.
Objective To observe the effect of ginsenoside Rg3 on the proliferation, migration, and tube formation of human retinal capillary endothelial cell (HRCEC) cultured in normal and hypoxia condition. Methods HRCEC was cultured in normal condition and treated with 0.0 mmol/L (group A), 0.1 mmol/L (group B) and 0.5 mmol/L (group C) ginsenoside Rg3. HRCEC was also cultured in hypoxia condition and treated with 0.0 mmol/L (group D), 0.1 mmol/L (group E) and 0.5 mmol/L (group F) ginsenoside Rg3. The effects of ginsenoside Rg3 on HRCEC proliferation were measured by methylthiazoletrazolium assay in 24, 48 and 72 hours after culture. In 24 hours after culture, the effect of cell migration was evaluated by transwell chamber; the effect of tube formation was evaluated by Matrigel; the expression of vascular endothelial growth factor (VEGF) protein and mRNA were detected by Western blot and real-time quantitative reverse transcription-polymerase chain reaction. Results Ginsenoside Rg3 could inhibit proliferation of HRCEC, depending on the concentration (F=30.331 and 33.402 in normal and hypoxia condition, respectively; P＜0.05) and time (F=85.462 and 136.045 in normal and hypoxia condition, respectively; P＜0.05). The number of cell migration was 103.33plusmn;3.54, 92..25plusmn;3.68, 78.64plusmn;4.66 in group A, B and C, the difference among three groups was statistically significant (F=28.801, P＜0.05). The number of cell migration was 125.76plusmn;3.11, 90.27plusmn;3.55, 77.81plusmn;5.01 in group D, E and F, the difference among three groups was statistically significant (F=117.594, P＜0.05). The number of tube formed in Matrigel was 24.3plusmn;2.2, 15.7plusmn;1.7, 10.1plusmn;2.3 in group A, B and C, the difference among three groups was statistically significant (F=35.364, P＜0.05). The number of tube formed in Matrigel was 26.2plusmn;1.9, 15.1plusmn;2.6, 8.6plusmn;1.9 in group D, E and F, the difference among three groups was statistically significant (F=50.989, P＜0.05). The expression of VEGF mRNA was 1.00plusmn;0.06, 0.79plusmn;0.06, 0.68plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=31.303, P＜0.05). The expression of VEGF mRNA was 3.88plusmn;0.12, 2.83plusmn;0.09, 1.15plusmn;0.05 in group D, E and F, the difference among three groups was statistically significant (F=682.668, P＜0.05). The expression of VEGF protein was 0.62plusmn;0.03, 0.41plusmn;0.02, 0.32plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=125.471, P＜0.05). The expression of VEGF protein was 0.91plusmn;0.03, 0.82plusmn;0.03, 0.71plusmn;0.02 in group D, E and F, the difference among three groups was statistically significant (F=41.045, P＜0.05). Conclusion Ginsenoside Rg3 can inhibit the proliferation, migration, and tube formation of HRCEC through the inhibition of VEGF expression.
ObjectiveTo observe the serum vascular endothelial growth factor (VEGF), apelin and heme oxygenase-1 (HO-1) levels in patients with type 2 diabetes mellitus (T2DM) and to explore their their relationship with diabetic retinopathy (DR).MethodsA total of 208 patients with T2DM and 50 healthy subjects (control group) from the Central Hospital of Western Hainan during January 2014 and December 2017 were selected in this study. Vision, slit lamp microscope, indirect ophthalmoscope and FFA examinations were performed on all the subjects. According to the results of the examinations combined with the DR clinical staging criteria, the patients were divided into non-DR (NDR) group, non-proliferative DR (NPDR) group, and proliferative DR (PDR) group, with 72, 76 and 60 patients in each, respectively. The clinical data of each group were recorded, and the levels of fasting blood glucose (FPG), HbA1c, total cholesterol (TC), three acylglycerol (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), VEGF, apelin and HO-1 were detected in each group. The receiver operating characteristic curve (ROC) were used to analyze the value of VEGF, apelin and HO-1 in predicting the occurrence of PDR. Correlation analysis of serum VEGF, Apelin and HO-1 with clinical parameters in PDR patients by Pearson correlation analysis.ResultsThe level of VEGF (56.82±10.16 vs 91.74±22.83, 140.15±36.40, 195.28±42.26 pg/ml) and apelin (2.95±0.53 vs 4.68±0.74, 7.25±1.13, 10.16±1.35 ng/ml) in PDR group were significantly higher than those in NPDR, NDR and control groups (F=17.306, 21.814; P＜0.05). The level of HO-1 (50.37±10.14 vs 43.58±8.16, 30.25±6.28, 22.60±4.72 mmol/L) in PDR group was significantly lower than those in NPDR, NDR and control groups (F=15.827, P＜0.05). The ROC curve analysis showed that the best cut-off values of serum VEGF, apelin and HO-1 were 162.50 pg/ml, 8.30 ng/ml, 27.13 mmol/L, and the three combined to predict PDR of AUC (95%CI) was 0.906 (0.849−0.962), and their sensitivity (90.3%) and specificity (83%) were better. The correlation analysis showed that the VEGF, apelin and HO-1 of PDR patients were correlated with the course of diabetes (r=0.382, 0.416, −0.36; P＜0.05), FPG (r=0.438, 0.460, −0.397; P＜0.05) and HbAlc (r=0.375, 0.478, −0.405; P＜0.05), and the serum VEGF were correlated with apelin and HO-1 (r=0.793, −0.594; P＜0.01).ConclusionElevated serum VEGF and apelin levels and reduced HO-1 levels are associated with the progression of DR, and the three combination helps predict the occurrence of PDR.
Objective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina. Methods Forty-eight Sprague -Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group). The rats of DM group were induced with streptozotocin injection creating a diabetic model. Retinas were obtained at eight, 10, 12 weeks after DM induction from both groups. CTGF and VEGF mRNA levels were examined by realtime reverse transcriptionpolymerase chain reaction (RT-PCR). Based on the results of above experiments, 60 rats with same conditions were selected. Fifty rats were induced with streptozotocin injection creating a diabetic model, and 10 rats comprised the control group (CON2 group). Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group, ranibizumab singletarget intervention group, CTGF shRNA singletarget intervention group and nonintervention group. Retinas were obtained at one week after intervention from all the groups. CTGF and VEGF mRNA levels were examined by RT-PCR. Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction, and this upregulation was maintained through the 12th week (t=-2.49, -2.67, -2.42;P＜0.05). There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction(t=-0.443,P=0.669). VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week, and remained to be higher at the 12th week (t=-2.35, -2.57;P＜0.05). The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014), which was similar to CON2 group (t=-1.37,P＞0.05); however, the CTGF mRNA level was significantly increased as compared to the nonintervention group (t=2.48,P＜0.05). In the CTGF shRNA single-target intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=0.23, -2.92;P＜0.05). In the ranibizumab and CTGF shRNA dual targets intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=-6.09, -5.11;P＜0.001), which was similar to CON2 group (t=-1.16, 1.139; P＞0.05). Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina, and the level of CTGF increased earlier than VEGF. Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina.
Objective To observe the influence of prolyl hydroxylase 2 (PHD2) expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial cells (HRECs). Methods The HRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 5 mmol/L glucose +25 mmol/L mannitol, 30 mmol/L glucose) as normal control group, mannitol control group and high glucose group, respectively. After the cells cultured for 24 and 48 hours, the protein levels of PHD2, hypoxia-inducible factor-1alpha; (HIF-1alpha;) and occludin was detected by Western blot; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by enzymelinked immuno sorbent assay (ELISA); the transcription levels of PHD2, HIF-1alpha;, VEGF and occludin were determined by the reversetranscription polymerase chain reaction (RT-PCR); the paracellular permeability between endotheliums was detected by 7times;104 molecular weight FITCdextran. Results Compared with normal control group, the protein level of PHD2 in mannitol control group and high glucose group firstly decreased and then increased, the protein level of HIF-1alpha; increased while that of occludin decreased; the secretion of VEGF increased in high glucose group but not in mannitol control group (PHD2：F=7.618, 8.627;P＜0.05. HIF-1alpha;：chi;2=7.692, 7.652;P＜0.05. occludin：F=23.23, 7.317;P＜0.05. VEGF：F=10.768, 4.562; P＜0.05). Compared with normal control group, the mRNA levels of PHD2, HIF-1alpha;, VEGF and occludin in mannitol control group and high glucose group increased (PHD2：F=5.69, 14.27;P＜0.05. HIF-1alpha;：F=6.07, 10.47;P＜0.05. VEGF：F=12.31, 9.14;P＜0.05. occludin：F=8.77, 8.00;P＜0.05). Compared with normal control group, the paracellular permeability of mannitol control group and high glucose group increased (chi;2=20.57,F=56.09;P＜0.05). Conclusions High glucose induced altered expression of PHD2 which might play an important role in endothelial barrier dysfunction. The mechanism might be associated with HIF-1alpha; and VEGF.
ObjectiveTo explore the effect and mechanism of netrin-1 on blood-retinal barrier permeability in diabetes mellitus (DM) rats. MethodsEighty Sprague-Dawley rats were randomly divided into the normal control group, DM+balanced salt solution (BSS) group, DM+netrin-1 low dose group and DM+netrin-1 high dose group, with 20 rats in each group. DM rats were induced by intraperitoneal injection of streptozocin (STZ). These rats were feed with high sugar and fat for 3 months after STZ injection. All rats were sacrificed at 1 month after intravitreal injection. Retinal vascular permeability was measured by Evans blue. The expression level of occludin was determined by immunohistochemistry. Hematoxylin-eosin (HE) staining of retina was used to observe the pathological change of DM and the level of occludin mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Five rats of each group. ResultsHE staining of retina showed that the degree of edema and vascularization in DM+netrin-1 high dose group was better than DM+BSS group. Staining of occludin in retina was limited to nerve fiber layer, ganglion cells, inner plexiform layer and inner nuclear layer in normal rats, but in DM+BSS group, the color of staining positive of occludin was lighter and more reduced. However, DM+ netrin-1 group occludin staining was deepen and enlarged. The result of RT-PCR showed that the expression of occludin mRNA in other three groups was less than normal control group (P < 0.05). The significant difference during DM+BSS group, low dose group and DM+netrin-1 high dose group (F=177.13, P=0.00), and the more concentrate of netrin-1 the higher expression of occluding. Compared the DM+netrin-1 low dose group with DM+BSS group, there was significant difference expression of occludin (t=-13.98, P=0.00). There was significant difference between the DM+netrin-1 high dose group and normal control group (t=12.87, P=0.00). There was statistically significant difference in DM+BSS group, DM+netrin-1 low dose group and DM+netrin-1 high dose group (F=179.69, P=0.00). Compared the two group of different concentration netrin-1, the quantification of vascular permeability in DM+netrin-1 high dose group reduced more (t=12.73, P=0.00). ConclusionsNetrin-1 can protect the blood-retinal barrier in DM rats. Netrin-1 may decrease BRB leakage in DM rats by protecting the expression of occludin.
Objective To observe the expression of the pigment epithelium derived growth factor (PEDF) in retina and the effects of PEDF for vascular endothelial growth factor (VEGF) and retinal microvascular after recombinant adeno-associated virus mediated pigment epithelium derived factor（rAAV-mediated-PEDF）transferred retina of diabetic rats. Methods Male Wister rats were induced diabetes with intraperitoneal injection of streptozocin, then divided into 1 month group (DM1), 3month group(DM3) and 6 month group randomly. In diabetic rats, right eyes were injected rAAV2CMVhPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1times;1011 v.g/ml) as self-control group. In normal rats, right eyes were punctured but not injected (CONf), left eyes let it be without any disposal. The levels of VEGFmRNA and hPEDFmRNA in retina were evaluated using RT-PCR in different period. The protein levels of VEGF, PEDF within the retina were determined using western blot. The change of retinal capillary were observed through retinal vascular flattening. Result The expression of hPEDFmRNA in retina was enhanced persistence after rAAV2-CMV-hPEDF injected, achieved climax until 6 months. The levels of PEDF were also incerased consecutive, the differences were statistically significant in treatment group compared with own control group （Plt;0.01）. Levels of mRNA and protein of VEGF at different time-points among therapy group were not statistically significant, all obviously higher than normal （Plt;0.05），but all lower obviously than respectively own control group at the same timepoint （Plt;0.01）. The morphology of retinal capillary was not different significant with normal rats in 1 month diabetic rats. Morphology changes of therapy groups were less than those of respective own control group in DM3 and DM6. Conclusion Intravitreous injection rAAV2-CMV-hPEDF can increase expression of mRNA and protein of PEDF，alleviate lesion of retinal microvascular in early period of diabetic rats and supress expression of VEGF in retina of diabetic rats.The regulation occur on mRNA level. （Chin J Ocul Fundus Dis，2008，24：259-264）
Objective To investigate if insulin can affect the expression of vascular endothelial growth factor (VEGF) in the retina of streptozotocin-induced diabetic rats. Methods A total of 60 male SpragueDawley rats were randomly divided into sodium citrate buffer control group (CIT-CON, n=30) and STZ-induced diabetic group (STZ-DM, n=30). At the 16th week, 24 rats from CIT-CON group at random were randomly divided to group A (sodium citrate buffer control group, n=12) and group B (sodium citrate buffer plus insulin group, n=12). The remaining 6 rats from as CIT-CON group served as negative control. At the same time, 24 rats from STZDM group at random were randomly divided to group C (STZinduced diabetic group, n=12) and group D (STZ-induced diabetic plus insulin group, n=12). The remaining 6 rats from STZ-DM group also served as negative control. 4 IU of insulin was injected subcutaneously to rats of group B and D. Immunohistochemistry, Western blot and Real-time polymerase chain reaction (RT-PCR) were used to measure the expression level of VEGF protein and mRNA respectively. RESULTS Insulin significantly increased the VEGF mRNA (7.71plusmn;0.25 vs 5.36plusmn;0.37, t test Plt;0.05) and protein expression (0.4925plusmn;0.0122 vs 0.4272plusmn;0.0110, t test Plt;0.05) in the retina of CITCON rats. However, in retina of STZDM rats, insulin had no effect on VEGF mRNA (8.92plusmn;0.27 vs 9.05plusmn;0.28, t test, Pgt;0.05) and protein expression (0.5152plusmn;0.0109 vs 0.5099plusmn;0.0100, t test Pgt;0.05). Conclusions Insulin had no effect on VEGF expression in the retina of STZ-DM rats.
Objective:To study the effects of growth factor on the proliferation of the cultured huamn retinal glial cells. Methods:EGF(0.5～100.0ng/ml) and NGF (0.5～10.0ng/ml) were added to cultures of human retinal glial cells and the proliferation rates of the cells were measured by MTT method. Results:EGF at a dosage ranging from 0.5ng/ml to 100.0ng/ml and NGF (0.05～10.0ng/ml) stimulated the cellular proliferation effectively with their EC 50 of 17ng/ml and 0.7 ng/ml respectively. Conclusion:Both EGF and NGF NGF had an effective stimulation on human retinal glial cell proliferation.They may play a role in the formation of PVR. (Chin J Ocul Fundus Dis,1998,14:33-34)
ObjectiveTo analyze the expression of VEGF, IL-33 and NO concentration after laser photocoagulation and subthreshold micropulse laser photocoagulation conventional in proliferative diabetic retinopathy (PDR) patients.MethodsA case control study. The clinical data of 39 patients of PDR and 11 patients of idiopathic macular pucker (IMP) from Department of Ophthalmology, Central Theater General Hospital during November 2015 were collected in this study. PDR patients were assigned randomly into three groups. Fifteen PDR patients with 15 eyes were treated with conventional laser as group A. Thirteen PDR patients with 13 eyes were treated with subthreshold micropulse laser as group B. Eleven PDR patients with 11 eyes without any laser therapy were grouped as C. Eleven IMP patients were grouped as D. There was no difference of age (F=0.53, P=0.23), gender ratio (χ2=0.55, P=0.91), body mass index (F=2.62, P=0.07), duration diabetes (F=0.29, P=0.75), glycoslated hemglobin (F=1.72, P=0.19) in four groups. All PDR patients were examined with FFA. Total protein was quantified by a bicinchoninic acid assay kit. Levels of VEGF, IL-33, NO were determined using enzyme-linked immunosorbent assay kits.ResultsThere was no difference of total protein in four groups (F=1.78, P=0.17). Group C had a higher VEGF level than group A and B (F=7.84, P=0.002). Group A had a higher IL-33 level than group C (t=4.15, P=0.02). There was no difference of IL-33 level in group B and C (t=1.34, P=0.20). Group D had a lower NO level than group A, B, C (F=38.42, P＜0.001). There was no difference of NO level in group A, B and C (F=3.29, P=0.06).ConclusionsBoth conventional laser photocoagulation and subthreshold micropulse laser photocoagulation can decrease vitreous VEGF level and subthreshold micropulse laser photocoagulation can induce less IL-33 level.