Objective To review the research status of the neovascularization of adi pose tissue engineering in the past decade so as to provide theoretical references for the development of the rapid revascularization of tissue engineered adi pose. Methods The l iterature about the revascularization of adi pose tissue engineering was extensively reviewed andanalyzed, centering on 5 elements: specificity of histological structures and blood supply, revascularization mechanism, coculture of different seed cells, modification of scaffold, and microenvironment. Results Adi pose tissue engineering offers a new solution for soft tissue defects. However, there is still the unfulfilled need in the size of engineered adipose tissue (less than 1 mL), which was determined by the degree of neovascularization in engineered tissue. Overall, rapid neovascularization in engineering tissue is a key l ink of experimental study changing into cl inical appl ication. Conclusion Providing a sufficient supply with nutrients and oxygen by means of a sufficient and rapid neovascularization will be at the heart of any attempts to obtain bigger tissue engineered adipose to meet the demand of repairing large soft tissue defect.
Objective To compare two kinds of myofascial flap encapsulating adi pose-derived stromal cells (ADSCs) in adi pogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adi pose tissue. Methods ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3-4 months old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 μg/mL) was appl ied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to thosecells 2 weeks after being induced towards adi pocyte, al izarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 × 107 ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm × 10 mm × 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was appl ied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothel ium. Results The nucleus of ADSCs positive for BrdU label ing showed green fluorescence under fluorescence microscope, with the positive label ing ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red l ipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibreencapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 ± 0.017 3) g and (0.095 3 ± 0.012 7) g, respectively, indicating there was a significant difference between two groups (P lt; 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 ± 4.5 and 19.3 ± 2.6, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adi pocytes and partial capillary endothel ium in groups A and B presented green fluorescence. Conclusion ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adi pogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.
Objective To observe the therapeutic effect of ultrasonic microbubble combined with bevacizumab (Avastin) on choroidal neovascularization induced by photocoagulation in rabbits.Methods CNV was induced by photocoagulation with argon laser in 30 rabbits (60 eyes).All of the rabbits underwent fundus fluorecein angiography (FFA) 21 days after photocoagulation; 6-8 hours later, 3 rabbits were randomly chosen to be executed to having the immunohistochemical examination.Twenty one days after photocoagulation, 27 rabbits were divided randomly into 3 groups: bevacizumb, ultrasonic microbubble + bevacizumb,and control group; each group has 9 rabbits (18 eyes).The rabbits in control group had no interference treatment; while the rats in bevacizumb and ultrasonic microbubble + bevacizumb group underwent injection with bevacizumb or ultrasonic microbubble + bevacizumb respectively.FFA was performed on all of the rabbits 7,14,and 28 days after photocoagulation to observe the inhibition of CNV; immunofluorecence and Western blot were used to detect the expression of VEGF in retina and choroid.Twentyeight days is the time point to determine the therapeutic efficacy. The expression of VEGF and the results of FFA were the sdandards of the judgement of therapeutic efficacy.Results Proliferaion of CNV to the retinal inner layer and the obvious leakage of fluoresein in the photocoagulation area indicated that the model of CNV was set up successfully. Twenty eight days after injection,obvious fluorescent leakage was found in the control group, and the average fluorescent leakage in bevacizumab group differed much from the control group（t=16.2952，Plt;0.05）; while the difference between ultrasonic microbubble + bevacizumb group and bevacizumab group was also significant (t=4.7955，Plt;0.05) . At the same time point, the expression of VEGF in bevacizumab group detected by immunofluorecent assay and Western blot differed much from the control group (t=7.0327，9.2596；Plt;0.05),and the difference of VEGF between ultrasonic microbubble + bevacizumb group and bevacizumab group was significant（t=2.9724,17.1937；Plt;0.05）. this experiment show that ultrasound combined bevacizumab intravitreal injection of the therapeutic effect of CNV superior to other groups(Plt;0.01).Conclusion Ultrasound microbubble combined with bevacizumab injection may improve the therapeutic effect on CNV by inhibiting the expression of VEGF.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
Objective To evaluate the diagnostic value of Heidelberg retinal angiography(HRA) combined with optical coherence tomography (OCT) to detect neovascularization (CNV) in exudative agerelated macular degeneration (AMD) patients. Methods This is a cross-sectional study of a series of clinical cases. AMD diagnosis was established by international standard vision chart, Slit lamp microscope, direct or indirect ophthalmoscope examination. A total of 50 eyes (42 cases) of exudative AMD received HRA and frequency domain OCT scan. All 50 eyes received fundus fluorescein angiography (FFA) and frequency domain OCT simultaneously, and among them 15 eyes also received indocyanine green angiography (ICGA) at the same time. FFA and ICGA were carried out by conventional methods, CNV was localized by real-time localization technology of frequency domain OCT. In the radial and grid-like section from the areas with b fluorescence, image acquisition settings are 7 mu;m fault for each frame, 30 deg; intervals for radial section, 10 vertical and 10 horizontal scan lines for gridlike section. CNV can be divided into 4 types (typical CNV, partial typical CNV, occult CNV, CNV scarring) according to their boundaries demonstrated in FFA. Based on the features of the OCT images, there were 3 types of integrated image (sub-RPE type, sub-retinal type and mixed type). Results CNV was detected in all 50 eyes. There were 4 eyes (8%) of typical CNV, 11 eyes (22%) of partial typical CNV, 32 eyes (64%, including 27 eyes of RPE detachment and 5 eyes of passive late leakage) of occult CNV and 3 eyes (6%) of CNV scarring. There were 4 eyes (8%) of subRPE type (CNV under the RPE light band) , 16 eyes (32%) of subretinal type(interrupted light band of RPE and choroid capillary layer) and 30 eyes (60%) of mixed type of integrated image. Conclusion The image integration technology of the HRA and frequency domain OCT system provide a valuable tool to classify and measure CNV, which will benefit the clinical treatment of AMD patients.
Objective To investigate auto-cortex of crystalline lens-induced neovascular epiretinal membrane（NVERM）by micro-injuring posterior c apsule of crystalline lens. Methods twenty four C57BL/6 mouse between 4-6 weeks were selected, and divided into two groups randomly: auto-cortex of crystalline group and the control group. The auto-cortex of crystalline group was treated by penetrating the posterior capsule of lens and washing out the lens cortex into the mouse vitreous using PBS (phosphate buffered solution), while the control group were injected PBS into vitreous merely. Clinical change s were followed by slit-lamp examination and photograph. The eye balls were enu cleated at the day of 3, 7, 14 and 28 after operation. Both HE and immunohistoch emistry were used to detect the pathological changes. Results postoperative one to three days, 11 of 12 mouse in autocortex of crystalline g roup, lens appear to alba turbid at different levels one after another, and then develop into highdensity chinaware white. Postoperative (po) three days, HE s taining shows cortex of lens debris transmigrated in vitreous cavity, and some o f which approached to internal limiting membrane and lead it to rough and discon tinue; Po7-14 days, the capillary in retina expanded, migrated and broke though t internal limiting membrane which got to the pro retina and became the new ves sels. And typical NVERM were observed. Po28 days, some vascularslike structure formed in vitreous cavity. None of mouse in control group developed NVERM. Conclusion Auto-cortex of crystalline lens can induced neovascular epiretinal membrane in C57BL/6 mouse. (Chin J Ocul Fundus Dis,2008,24:118-121)
ObjectiveTo analyze the regulative rule of mRNA of vascular endothelial growth factor (VEGF) in mice with oxygen-induced retinopathy, and to elucidate the possible mechanism of occurrence of neovascularization in retinopathy of prematurity (ROP).MethodsSixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, 36 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days; in control group, 24 mice were raised in room air. Vascular perfusion of fluorescein and retinal stretched preparation were used to observe the morphologic changes of retinal vessels. Reversal transcriptionpolymerase chain reaction (RT-PCR) was used to observe changes of VEGF mRNA in each group. ResultsIn oxygen-induced retinopathy group, the morphologic characteristics of retinal vessels were the unperfused area at the center of superficial and deepseated vessels, and the neovascularization appeared at mid-peripheral retina after 2 days in relative hypoxia condition. The results of RT-PCR showed space-time corresponding relation between expression of VEGF and neovascularization, which meant that the transcription of VEGF mRNA decreased in hyperxia conditionand increased in relative hypoxia condition. ConclusionHypoxia is the main reason of occurrence of retinal neovascularization; increased expression of VEGF caused by relative hypoxia after hyperxia might be effective in reducing the occurrence of neovascularization in ROP.(Chin J Ocul Fundus Dis, 2005,21:292-295)
Objective To determine the expression of the growth factors and the receptors related to angiogenesis in the intraocular tissues incarcerating in the sclerotomy sites. Methods Ten specimens from prolapsing intraocular tissues in sclerotomy sites during vitrectomy were obtained and serially sectioned in cryostate and were stained with a group of polyclonal antibodies against vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-A(PDGF-A) and transforming growth factor-β1(TGF-β1) as well as their receptors by using a streptavidin peroxidase system. Results The tissues prolapsed from the sclerotomy sites were identified as retina(3 cases), vitreous tissues(3 cases), degenerated red blood cell components(2 cases), ciliary body(one case) and fibrous tissue(one case). All specimens expressed VEGF and bFGF as well as their receptors. PDGF-A, TGF-β1 and their receptors expressed in the most of specimens. The positive cells included retinal cells, ciliary non-pigmented epithelial cells and pigmented epithelial cells, fibrous cells and the cells in vitreous. Conclusions The intraocular tissues incarcerated in the sclerotomy entries express the growth factors and receptors related to angiogenesis. This might be one of the potential factors of developing anterior proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis, 2002, 18: 34-37)
Purpose To estabalish a quantifying model of retinal neovascularization suitable for the study of pathogenesis and therapeutic intervention for the retinal neovascularization． Methods Sixteen one-week-old C57BL/6 mice were exposed to 75% oxygen for 5 days and then to room air and 16 mice of the same age kept in room air as controls.Ink-perfused retinal flatmount was examined to assess the oxygen-induced changes of retinal vessels.The proliferated neovascular response was quantitated by counting the nuclei of endothelial cells of new vessels extending from the retina into the vitreous in 6 mu;m sagittal cross sections． VEGF and bFGF were determined on the cross-sections after immunohistochemcal stain. Results Constriction and closure of the blood vessels were found under the hyperoxia condition,and dilation and proliferation were found under the relatively hypoxia status.There was a mean of 24 neovascular nuclei per cross-section in the oxygen-treated retina and less than 1 nucleus in the control group (P＜0.001).VEGF stain was found ber in the inner retinal layer of oxygen-treated mouse than in that of the controls. Conclusion The quantifying model of retinal neovascularization may fascilitate the further researches of medical intervention and pathogenesis of retinal neovacularization. (Chin J Ocul Fundus Dis，2000，16：213-284)
Purpose To investigate the roles of vascular endothelial growth factor (VEGF),glutamate and gamma;- aminobutyric acid (GABA) in neovascularization of proliferative diabetic retinopathy (PDR). Methods Vitreous samples were collected from 25 patients (27 eyes)with PDR and 14 patients (14 eyes) with idiopathic macular hole.VEGF levels were determined by ELISA.Amino acids analyses were performed by high-performance liquid chromatography. Results Patients with PDR had significantly higher concentrations of VEGF (median 0.41 ng/ml,quartile 0.54 ng/ml) than controls (median 0.017 ng/ml,quartile 0.01 ng/ml)(Plt;0.001).The levels of glutamate [(11.7plusmn;3.0) mu;mol/L] and GABA [(7.2plusmn;3.9mu;mol/L)] were also higher in patients with PDR than glutamate [(5.8plusmn;0.7) mu;mol/L] and GABA [(3.3plusmn;2.9) mu;mol/L) in controls (Plt;0.05).The glutamate level and GABA level had a significantly positive correlation with VEGF level. Conclusions The increased levels of glutamate and GABA indicate retinal ischemia.The correlations of glutamate and GABA levels with an elevated VEGF level provide biochemical support for ischemi induced neovascularization in patients with PDR.The findings present novel modalities in treatment of PDR. (Chin J Ocul Fundus Dis,2000,16:162-165)