Objective To explore the effect and mechanism of ultrashort wave (USW) for prevention and treatment of vascular crisis after rat tail replantation. Methods Eighty 3-month old female Sprague Dawley rats (weighing 232.8-289.6 g) were randomly divided into 5 groups. In each group, based on the caudal vein and the coccyx was retained, the tail was cut off. The tail artery was ligated in group A; the tail artery was anastomosed in groups B, C, D, and E to establish the tail replantation model. After surgery, the rats of group B were given normal management; the rats of group C were immediately given intraperitoneal injection (3.125 mL/kg) of diluted papaverine hydrochloride injection (1 mg/mL); the rats of groups D and E were immediately given the local USW treatment (once a day) at anastomotic site for 5 days at the dosage of 3 files and 50 mA for 20 minutes (group D) and 2 files and 28 mA for 20 minutes (group E). The survival rate of the rat tails was observed for 10 days after the tail replantation. The tail skin temperature difference between proximal and distal anastomosis was measured at pre- and post-operation; the change between postoperative and preoperative temperature difference was calculated. The blood plasma specimens were collected from the inner canthus before operation and from the tip of the tail at 8 hours after operation to measure the content of nitric oxide (NO). Results The survival rates of the rat tails were 0 (0/14), 36.4% (8/22), 57.1% (8/14), 22.2% (4/18), and 75.0% (9/12) in groups A, B, C, D, and E, respectively, showing significant overall differences among 5 groups (χ2=19.935, P=0.001); the survival rate of group E was significantly higher than that of group B at 7 days (P lt; 0.05), but no significant difference was found between the other groups by pairwise comparison (P gt; 0.05). At preoperation, there was no significant difference in tail skin temperature difference among 5 groups (P gt; 0.05); at 8 hours, 5 days, 6 days, and 7 days after operation, significant overall difference was found in the change of the skin temperature difference among groups (P lt; 0.05); pairwise comparison showed significant differences after operation (P lt; 0.05): group B gt; group D at 8 hours, group C gt; group D at 5 days, groups A, B, and C gt; group D at 6 days, groups B and C gt; groups A and E, and group B gt; group D at 7 days; but no significant difference was found between the other groups at the other time points (P gt; 0.05). Preoperative plasma NO content between each group had no significant difference (P gt; 0.05). The overall differences had significance in the NO content at postopoerative 8 hours and in the change of the NO content at pre- and post-operation among groups (P lt; 0.05). Significant differences were found by pairwise comparison (P lt; 0.05): group D gt; groups A, B, and C in the plasma NO content, group D gt; groups A and B in the change of the NO content at pre- and post-operation; but no significant difference was found between the other groups by pairwise comparison (P gt; 0.05). Conclusion Rat tail replantation model in this experiment is feasible. USW therapy can increase the survival rate of replanted rat tails, reduce skin temperature at 7 days, improve blood supply, increase the content of nitric oxide at the early period and prevent vascular crisis.
Objective To explore morphological recellularization level of bioprosthetic valve scaffold (BVS) and to provide researching means for fabricating tissue engineered heart valve in vitro.Methods The homograft bioprosthetic aortic tube valve was selected as BVS, which was conserved by liquid nitrogen, and its endothelial cells (ECs) were removed by 0.1% sodium dodecylsulphate (SDS). As implantation cells, the endothelial cells (ECs) differentiating from human bone marrow mesenchymal stem cells (MSCs) in vitro were implanted with high-density seeding (gt;10 5 cells/cm2) on the BVS, which was covered by fibronectin (80 μg/ml) in advance. The complex structure was statically cultured in DMEM (high glucose) with 20% FBS and VEGF (10 ng/ml) for about 20 days in vitro and stained by 0.5% AgNO3. The morphological structure was observed and photographed by stereomicroscope to detect the recellularization level. Results The ECs of the bioprosthetic valve were notonly removed completely, but also the collagen fiber and elastic fibers were reserved. The ECs differentiating from MSCs were successfully implanted on the HBS, whose recellularization levels on 7th, 14th and 20th day were 73%, 85%, and 92% respectively. Conclusion AgNO3 staining technique is effective, convenient, and economic in evaluating the recellularization level of BVS. It is an effective method in morphological observation for fabricating tissueengineered heart valve in vitro.
Objective To investigate the effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
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.
Objective The observe the effects of interferon-inducible protein-10 (IP-10) on proliferation, migration and capillary tube formation of human retinal vascular endothelial cells (HREC) and human umbilical vein endothelial cells (HUVEC). Methods The chemokine receptor (CXCR3) mRNA of HREC and HUVEC were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). In the presence of the different concentrations of IP-10, the difference in proliferation capacity of HREC and HUVEC were analyzed by cell counting kit-8 (CCK-8) methods. Wound scratch assay and threedimensional in vitro matrigel assay were used for measuring migration and capillary tube formation of HREC and HUVEC, respectively. Results RT-PCR revealed both HREC and HUVEC expressed CXCR3. The proliferation of HREC in the presence of IP-10 was inhibited in a dosagedependent manner (F=6.202,P＜0.05), while IP-10 showed no effect on the inhibitory rate of proliferation of HUVEC (F=1.183,P＞0.05). Wound scratch assay showed a significant reduction in the migrated distance of HREC and HUVEC under 10 ng/ml or 100 ng/ml IP-10 stimulation (F=25.373, 23.858; P＜0.05). There was no effect on the number of intact tubules formed by HREC in the presence of 10 ng/ml or 100 ng/ml IP-10. The number of intact tubules formed by HREC in the presence of 1000 ng/ml IP-10 was remarkably smaller. The difference of number of intact tubules formed by HREC among 10, 100, 1000 ng/ml IP-10 and nonintervention group was statistically significant (F=5.359,P＜0.05). Conclusion IP-10 can inhibit the proliferation, migration and capillary tube formation ability of HREC and the migration of HUVEC.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs). Methods The second to fifth passage cultured HUVECs were divided into five groups: control group, CoCl2induced hypoxic group and 50, 100, 200 mu;mol/L TMP treatment groups. HUVECs in control group were not treated. HUVECs inCoCl2induced hypoxic group were treated with 150 mu;mol/LCoCl2for four hours. HUVECs in 50, 100, 200 mu;mol/L TMP treated groups were pretreated with 150 mu;mol/LCoCl2 for four hours, followed by treatment with 50, 100, 200 mu;mol/L TMP for eight hours. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2), hypoxia-induced factor-1alpha;(HIF-1alpha;) and vascular endothelial growth factor (VEGF). Protein levels of PHD2, HIF-1alpha;, and VEGF were detected using Western blot. Results Compared with the control group, theCoCl2 induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734, 3.122;P＜0.05), while those of HIF-1alpha; and VEGF increased (HIF-1alpha; mRNA:t=4.589,P＜0.05; HIF-1alpha; protein:t=3.778,P＜0.05. VEGF mRNA:t=3.926,P＜0.05; VEGF protein:t=3.257,P＜0.05). Compared with theCoCl2 induced hypoxic group, 50, 100, 200 mu;mol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA: t=3.286, 3.617, 3.886;P＜0.05. PHD2 protein: t=2.813, 3.026, 3.078; P＜0.05); while those of VEGF decreased (VEGF mRNA: 50 mu;mol/L TMP: t=1.696,P＞0.05; 100 mu;mol/L TMP:t=2.974,P＜0.05; 200 mu;mol/L TMP: t=3.492,P＜0.05; VEGF protein: 50 mu;mol/L TMP: t=1.986,P＞0.05; 100 mu;mol/L TMP: t=2.976,P＜0.05; 200 mu;mol/L TMP:t=3.136,P＜0.05); although changes in HIF-1alpha;mRNA levels were not statistically significant (t=1.025, 0.726, -1.386;P＞0.05), showed a decrease in HIF-1alpha;protein levels (50 mu;mol/L TMP: t=2.056,P＞0.05; 100 mu;mol/L TMP:t=3.058,P＜0.05; 200 mu;mol/L TMP:t=3.828,P＜0.05). ConclusionIn HUVECs, TMP can upregulate the mRNA and protein expression of PHD2, while down regulating HIF-1alpha; protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.
Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2 induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model. Methods Human eyes were selected to establish CoCl2induced HRECs hypoxia model in this study. Four experimental groups were studied: normal control group, hypoxia group (CoCl2 100 μmol/L, 48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate, PI-88,5 μg/ml, combined with CoCl2 100 μmol/L, 48 hours) and PBS control group. Heparanase, VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence. Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal, hypoxia, PI88 and PBS control groups. ResultsImmunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs, but faint in normal group. Heparanase was also observed in the nucleus of hypoxia HRECs. Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa：F=-4。005, P＜0.05；VEGF：F=-4.063, P＜0.05), and VEGF was decreased in HRECs treated with PI-88(F=5。963, P＜0.05). ConclusionsHeparanase is upregulated that resulted in increase of VEGF expression, therefore enhanced angiogenesis in CoCl2 induced hypoxia HRECs.
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 investigate the effect of small interfering RNA(siRNA) targeting hypoxia inducible factor1alpha; (HIF1alpha;) and vascular endothelial growth factor (VEGF) on expression of VEGF in human vascular endothelial cells. Methods HIF-1alpha; siRNA recombinant plasmid was constructed. Human vascular ndothelial cells were cultured in vitro and divided into normoxia group (20% O2) and hypoxia group (1% O2). Hypoxia group was then divided into control group, vector group, HIF-1alpha; group (HIF-1alpha; siRNA), VEGF group ( VEGF165 siRNA) and cotransfection group (HIF-1alpha; siRNA+VEGF165 siRNA). LipofectamineTM 2000 (LF2000) mediated vector plasmid was transfected to cells in each group except the control group. The expression of HIF-1alpha; siRNA and VEGF165 siRNA recombinant plasmid were identified by reverse transcriptasepolymerase chain reaction (RT-PCR). The expression of VEGF mRNA and protein were detected by RTPCR and immunocytochemical method. Results The expression of HIF-1alpha; siRNA and VEGF165 si RNA recombinant plasmid were detected 24 hours after transfected. The expression of VEGF mRNA and protein was faint in the normoxia group, but increased obviously in hypoxia group. The expression of VEGF mRNA and protein in the HIF1alpha;, VEGF and cotransfection groups were lower than which in the control group. Cotransfection group showed the highest inhibitory effect. Conclusion HIF-1alpha; and VEGF165 siRNA can effectively inhibit the expression of VEGF in human vascular endothelial cells.