Objective To observe the degradation of the polyactic glycolate acid (PLGA) microparticles with releasing-slowly vascular endothelial growth factor(VEGF) synthesized by the method of emulsification-diffusion. Methods The method of emulsification-diffusion is to incorporate VEGF into microparticles composed of biodegradable PLGA. The controlled release of microparticles are acquired. The content of the VEGF released slowly from PLGA microparticles in vitro was detected with ELISA at different time. Results We synthesized 100 releasing-slowly VEGF PLGA microparticles with the size of 0.20-0.33 μm by 5 times. The contents were 62±11 ng/L, 89±14 ng/L, and 127±19 ng/L in the 1st, the 2nd and the 3rd months after degradation, respectively. Conclusion The PLGAmicroparticles with releasing-slowly VEGF can be synthesized by the method of emulsification-diffusion.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective To examine an effect of the locally-used platelet derived growth factor-BB (PDGF-BB) on the healing of the medial collateral ligament (MCL) in the knee joints of rats. Methods Forty-eight rats were equally randomly divided into 2 groups: the experimental group (group A) and the control group(group B). MCL of all the rats were ruptured to establish the wound models. In group A, 5 μg of PDGF-BB was locally injected in the wound of each rat and then the wound was sutured; but in group B, the wound was only sutured. After 2 weeks, histological evaluations were performed to determine whether PDGF-BB could promote the healing of MCL. Results There were significantly more fibroblasts formed during the ligament healing process in group A than in group B (213.44±15.32 vs. 180.42±12.78, Plt;0.01). The fibroblasts were more mature andmore regularlyarranged in group A than in group B. The type, content, and crosslink of the collagen were improved to a greater extent in group A than in group B (Plt;0.01). Conclusion PDGF can promote the healing of the injured ligament.
Objective To investigate the influence of vascular endothelial growth factor (VEGF) antagonist bevacizumab on the growth of human choroidal melanoma (CM) OCM-1 cell xenografts in nude mice, and to explore the probable mechanism.Methods OCM-1 cells were subcutaneously implanted on 18 nude mice to establish ectopic model of human CM. The nude mice with the tumor of 5 mm in diameter were randomly divided into three groups: untreated group (group A), normal saline （NS） group (group B), drug treated group (group C). Bevacizumab was intraperitoneally injected for 14 consecutive days in group C, and the same volume of NS was used at a same way in group B. The volume and weight of implanted tumor as well as inhibitory rates of drug on tumor were calculated, ki67 and survivin proteins were measured with immunohistochemistry, and the mRNA expression of VEGF and survivin were assessed by RT-PCR.Results The volume and weight of tumor was (598.86plusmn;321.81) mm3, (0.66plusmn;0.15) g; (1 715.15plusmn;278.16) mm3, (1.54plusmn;0.39) g and (1 750.23plusmn;206.36) mm3, (1.54plusmn;0.31) g in groups C, A and B, respectively. There were significant differences between group C and A (F=34.53, P=0.00) and group C and group B (F=8.69, P=0.01). The inhibitory rate of these three groups were 57.14%, 5.31%, 6.25%, respectively, and the proliferation index (PI) of ki67 in these three groups were (51.85plusmn;1.32)%, (46.30plusmn;1.39)%, (27.90plusmn;0.90)%, respectively, there were significant differences in ki67 PI between C group and A or B group (H=15.17, P=0.00). The expression of survivin mRNA was (0.49plusmn;0.02), (0.82plusmn;0.05) and (0.61plusmn;0.05) in groupss C, A and B, respectively, there were significant differences between C group and A or B group (F=15.17, P＜0.05) . The expression of VEGF mRNA was (0.32plusmn;0.08), (0.73plusmn;0.07), (0.80plusmn;0.04) in groups C, A and B, significant difference was found between group C and A or B group (F=12.05，P＜0.05). Conclusion Bevacizumab can inhibit the growth of human CM in nude mice probably by inhibiting the activity of VEGF and downregulating survivin expression of the tumor as well as inhibiting the growth of the tumor.
Abstract To observe the effect of fibroblast growth factor (FGF) on wound healing, 50 mice were divided into 5 groups. On the back of every mouse, 2 wounds were made by operative cuts, one for experiment and the other for control. The wounds of the experimental group were covered with 0.5ml FGF solution (contented FGF 300 μg/ml, heparin 100 μg/ml), whereas the wounds of the control group were covered with 0.5ml 0.9% NaCl solution. All of the wounds were dressed by sterilized gauze, and received the same treatment once a day. After 1,3,5,7,10 days, the mice in every group were sacrificed and the tissues of the wounds were collected and prepared for microscopic examination. The results showed that the capillaries and fibroblasts in the experimental group were markedly increased and reached the peak 2～3 days earlier than those in the control group. It was suggested that FGF promoted the formation of granulation tissue and the wound healing.
Abstract In order to understand the expression change of epidermal growth factor (EGF) gene and its distribution in tissue duringwound healing, 12 Winstar rats were divided into 4 groups. In each side of the back of every rat, 4 wounds of 1.5cm×1.5cm in size were made. After 4,8,12,16 days, one group of rats was sacrificed and tissues from wound were collected. By DIG-labelled probe hybridization in situ technique, EGF gene mRNA was detected. It was shown that EGF gene expression was evident during the whole stage of woundhealing, and the peak was in 8th day. It suggested that the promotion of EGF gene expression may lead to wound healing earlier.
Objective To investigate the different influence of the expression levels of the epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) after the unilateral phrenectomy in piglets. Methods Thirty-six piglets were divided into 3 groups according to their ages during the operation (10 d,30 d,50 d). In each group, 6 piglets underwent the left cervical phrenectomy and 6 piglets were used as the shamoperation controls. The expression levels of EGF and KGF were determined by the real time quantitative RT-PCR at 2 weeks after operation.Results The melting curves of RTPCR showed that there was a single peark at the temperature of 80.0, 84.5 and 89.0℃ of EGF,KGF and GAPDH, respectively. In the experimental group, the expression levels of EGF were 3.53±0.36 and 1.73±0.29, and the expression levels of KGF were 4.71±0.42 and 2.77±0.29 in thepiglets undergiong the operation at their ages of 10 d and 30 d.Compared with the control group,the expression levels of EGF (4.60±0.41，2.18±0.24) and KGF(6.05±0.42，3.58±0.31) showed that there was a significant decrease postoperatively in the piglets undergoing the operation at their ages of 10 d and 30 d（P＜0.05）. However, there was no significant change in the piglets undergoing the operation at their ages of 50 d（P＞0.05）. The expression levels of EGF and KGF were significantly decreased with the lung development of the piglets（P＜0.05）. Conclusion The unilateral phrenectomy performed in the piglets younger than 30 d may cause abnormity of the EGF and KGF expression levels. The piglets older than 50 d may not cause a significant influence.
Objective To investigate the effects of matrine on cell proliferation and expression of connective tissue growth factor( CTGF) and hypoxia inducible factor-1α( HIF-1α) of human lung fibroblast ( WRC-5) in normoxia ( 21% O2, 74% N2 , 5% CO2 ) and hypoxia ( 1% O2, 94% N2 , 5% CO2 )conditions. Methods MRC-5 cells were cultured and divided into differrent groups interfered with different dose of Matrine ( final concentration of 0 ～3. 2 mmol / L) in normoxia or hypoxia for 24 h. Cells were dividedinto 8 groups according to culture conditions, ie. normoxiagroup( N0 group) , normoxia + matrine 0. 2 mmol / L group( N0. 2 group) , normoxia + matrine 0. 4 mmol / L group( N0. 4 group) , normoxia + matrine 0. 8 mmol / L group( N0. 8 group) , hypoxia group( H0 group) , hypoxia + matrine 0. 2 mmol /L group( H0. 2 group) , hypoxia +matrine 0. 4 mmol /L group( H0. 4 group) , and hypoxia + matrine 0. 8 mmol / L group( H0. 8 group) . The MTT assay was used to measure the cell proliferation activity. Western-blot assay was used to examine the expression of CTGF and HIF-1α. Results Hypoxia promoted the cell proliferation in all groups( P lt;0. 05) .Matrine inhibited the proliferation of WRC-5 cells in a concentration-dependent manner in hypoxia or normoxia conditions( P lt;0. 05) . The expression of CTGF andHIF-1αwas lower in normoxia and higher in hypoxia( P lt;0. 01) . Matrine inhibited the expression of CTGF and HIF-1αin a concentration-dependent manner in hypoxiaand normoxia( P lt;0. 05) . Conclusion Matrine can inhibit the cell proliferation and the expression of CTGF and HIF-1αof WRC-5 cells in normoxia and hypoxia in a concentration-dependent manner.
ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
Objective To determine the association between the geneti c polymorp hisms of vascular endothelial growth factor (VEGF) gene and the prognosis for retinopathy of prematurity (ROP) in Chinese. Methods Twenty infants with threshold ROP who had undergone retinal photocoagulation were in the treated group and 20 infants with self-regressed ROP without any treatment were in the control grou p . In the two groups, all the infants had oxygen-breathing history and the sex a n d gestational age were all suitable to be compared, except birth weight. Polymer ase chains reaction-restriction fragment length polymorphism was used to determine the frequencies of VEGF genes in the two groups. Results The frequencies of +405C allele were higher in the treated group than those in the control group (P＜0.05). The frequencies of the VEGF-460T/C and +936C/T ploymorphisms were similar in both groups (P＞0.05). Conclusions The +4 05C/G ge netic polymorphisms of VEGF may correlate to the prognosis of ROP. The carriers of +405CC allele are more susceptible to ROP.