To make a rabbit model of Perthes disease and to explore the change and its significance of VEGF expression in the femoral head. Methods Twenty-four 3-month-old New Zealand rabbits (weighing 1.6-1.8 kg) were randomly divided into experimental group (n=16) and control group (n=8). A rabbit model of Perthes disease was made by excision of left l igamentum teres and retinacular blood suppl ies of femoral head. The gross appearance, X-ray film and histological observations were made and the immunohistochemistry and VEGF mRNA in situ hybridization were carried out1, 2, 4, 8 weeks after operation. Results The rabbit model of Perthes disease was made successfully; only 1 was infected5 days after operation and was made quit. The gross appearance: The femoral heads had no necrosis changes in control group at every time. The femoral heads became coarse, tarnish and smaller, and even collapsed in experimental group. The HE staining observation: The femoral heads had no necrosis changes in control group at every time after operations. New vessels and granulation tissues grew into the necrosis part in the experimental group 4 weeks and 8 weeks after operations. New bone could be seen in repaired bone. Immunohistochemistry staining: In the epiphyseal cartilage of the femoral heads in control group, an intensive VEGF immunoreactivity (VEGF-IR) was found in the hypertrophic zone with a low level of VEGF-IR in the prol iferative zone. At 1 week after operation, the percentage of VEGF+ cells in the prol iferative zone of the femoral heads in experimental group was increased compared with that of the femoral heads in control group. The percentage of VEGF+ cells in the hypertrophic zone of the femoral heads in experimental group was significantly decreased compared with that of the femoral heads in control group. At 8 weeks after operation, VEGF-IR was observed throughout the epiphyseal cartilage surrounding the bony epiphysis in the femoral heads in experimental group. The percentage of VEGF-positive cells in the prol iferative zone of the femoral heads in experimental group was significantly increased compared with that of the normal heads. The hypertrophiczone of the femoral heads in experimental group had a similar percentage of the VEGF+ cells to the femoral heads in control group when endochondral ossification was restored at 8 weeks. There were statistically significant differences in the ratios of VEGF+ cells in the prol iferative zone of femoral head 1, 2, 4, 8 weeks after operations (P lt; 0.01); in the ratios of VEGF+ cells in the hypotrophic zone of femoral head 1, 2, 4 weeks after operations (P lt; 0.01) between experimental group and control group. In situ hybridization results: The results were similar to that of histology. VEGF mRNA expression in the hypertrophic zone of epiphyseal catilage after necrosis were lower. VEGF mRNA expression in the prol iferative zone of epiphyseal catilage after necrosis increased. VEGF mRNA expression in the hypertrophic zone of epiphyseal cartilage in experimental group could be seen again after endochondral ossification was repaired. Conclusion It is possible that VEGF may act as a key regulator that couples angiogenesis, cartilage remodel ing, and ossification after ischemic damage to restore endochondral ossification in the epiphyseal cartilage.
ObjectiveTo investigate the expression of tumor metastasis associated genes-1 (MTA1) and vascular endothelial growth factor-C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) and the relationship between them and lymphangiogenesis. MethodA total of 107 patients who received excision for ESCC in the Cardiothoracic Surgery Department of Suining Central Hospital from March 2013 through January 2014 were enrolled. And the paraffinembedded esophageal tissues in 56 healthy persons were collected. The expression of MTA1 and VEGF-C in ESCC was detected using the immunohistochemical method. And D2-40 was used to label the micro-lymphatic endothelial cells of the tumor tissues while the micro-lymphatic vessel density (LVD) was counted. Meanwhile, a statistical analysis was performed for the relationship between MTA1 with VEGF-C and clinical pathological parameters. ResultsThe expression rates of MTA1 protein and VEGF-C protein in ESCC (50.4% and 58.8%, respectively) were higher than those in normal esophageal tissues with a statistical difference (P<0.05). Besides, their high expression rates in stage T3/T4 ESCC and lymph node metastasis group were significantly higher than those in stage T1/T2 ESCC and metastasisfree group, with statistical differences (P<0.05). The high expression rates of MTA1 and VEGF-C protein in ESCC with different TNM stages were compared using Kruskal-Wallis test with statistical differences (P<0.05). Moreover, a positive correlation existed in the expression level between MTA1 protein and VEGF-C protein of ESCC (Spearman coefficient r=0.512, P=0.000). And LVD of the high expression group for MTA1 protein and VEGF-C protein was statistically different from that of the low expression group (P<0.05). ConclusionThe expression of MTA1 is positively correlated with the expression of VEGF-C in ESCC. And they may co-promote lymphangiogenesis and lymphatic metastasis in ESCC. Therefore, both can be used as the laboratory indicators to determine the prognosis of ESCC.
Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the l ipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, weretransfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 ± 0.03, 0.90 ± 0.02, 0.64 ± 0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P lt; 0.05); there was no significant difference among groups C, D and E (P gt; 0.05). Conclusion The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation l iposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abil ities of BMSCs.
To construct the recombinant adeno-associated virus (rAAV) vector co-expressinghVEGF165 and hBMP-7 depending on internal ribosome entry site (IRES) sequence, to measure the virus titer and to ver ify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid pIRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were ampl ified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein (GFP) labeled rAAVIRES GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packagingwas monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells.The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAVhVEGF165- IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of 5.5 × 1011vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement
Objective To systematically review the efficacy of intravitreal injection of anti-vascular endothelial growth factors (anti-VEGF) on macular edema (ME) secondary to retinal vein occlusion (RVO). Methods Databases including PubMed, EMbase, Web of Science, The Cochrane Library, CNKI, WanFang Data and VIP were electronically searched to identify randomized controlled trials on different anti-VEGF drugs in the treatment of RVO-ME from inception to September 17th 2021. Two reviewers independently screened literature, extracted data, and assessed the risk bias of the included studies. Meta-analysis was then performed using RevMan 5.3 software. Results A total of 11 RCTs were included. Data from these studies included 2 436 eyes, of which 1 682 involved central retinal vein occlusion and 754 involved branch retinal vein occlusion. The results of meta-analysis showed that at 6 months of follow-up, anti-VEGF drug treatment of RVO-ME improved corrected visual acuity (MD=14.97, 95%CI 10.09 to 19.86, P<0.000 01) and reduced central retinal thickness (MD= ?218.21, 95%CI ?295.56 to ?140.86, P<0.000 01) compared with control groups. At 12 months, anti-VEGF treatment of RVO-ME showed better improvement in corrected visual acuity compared with control group (MD=5.70, 95%CI 3.90 to 7.50, P<0.000 01). No statistically differences were observed in the improvements corrected visual acuity with different anti-VEGF drugs. However, for central retinal vein occlusion, different anti-VEGF drugs improved the central retinal thickness including aflibercept vs. bevacizumab (MD=?46.79, 95%CI ?83.12 to ?10.46, P=0.01), and bevacizumab vs. ranibizumab (MD=76.03, 95%CI 30.76 to 121.30, P=0.001) had significant differences. Conclusions The current evidence shows that anti-VEGF drugs can improve vision and reduce macular edema in the treatment of RVO-ME. Bevacizumab may be an effective alternative to ranibizumab or aflibercept. Existing evidence cannot determine differences between the improvement of best-corrected vision and the reduction of central retinal thickness during the long-term treatment of RVO, which requires to be verified by further research.
Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
Objective To observe the effect of BMSCs transplantation on gene and protein expression of VEGF receptor fetal l iver kinase 1 (Flk-1) after spinal cord injury (SCI), and to investigate the mechanism of repairing the SCI by BMSCs transplantation. Methods BMSCs were isolated and cultured from five 4-week-old male Wistar rats weighing100-120 g. The SCI model was made by using the modified Allen’s impactor device. Eighty-one adult female Wistar rats weighing 220-250 g were randomly divided into 3 groups: sham-operated group (group A, n=21), in which spinous process and vertebral plate of thorax 8-10 spinal cord segment were removed; DMEM group (group B, n=30), in which rats received four injections of DMEM in the peri-lesion area; and BMSCs group (group C, n=30), in which rats received four injections of BMSCs in the peri-lesion area. The changes of Flk-1 mRNA expression in rats’ spinal cord tissues were detected with RT-PCR method 1, 3 and 5 days after transplantation. The expression of Flk-1 protein was observed by using immunohistochemical technology in spinal cord 3, 7 and 14 days after transplantation. Results Morphology of the primary cultured BMSCs was various. Cell morphology tended to be uniform with the accumulation of passages, which appeared flat and spindle-shaped. RT-PCR results showed that there was no significant differences (P gt; 0.05) in Flk-1 mRNA expression between group C and group B at different time points after transplantation. But Flk-1 mRNA levels of group B and group C significantly increased and peaked 1 day after transplantation (P lt; 0.01), and then decreased 3 days after transplantation (P lt; 0.01) compared with that of group A, and were still higher than that of group A 5 days after transplantation (P lt; 0.05). Immunohistochemical staining results revealed that the expression of Flk-1 in group B was enhanced 3 and 7 days after transplantation compared with group A, which was significantly different (P lt; 0.01). There was no significant difference in the expression of Flk-1 between group B and groupp A 14 days after transplantation (P gt; 0.05). There was no significant difference in Flk-1 protein expression between group C and group B 3 days after transplantation (P gt; 0.05). The expression of Flk-1 protein in group C was significantly higher than that in group B 7 and14 days after transplantation (P lt; 0.01). Conclusion BMSCs transplantation after SCI does not have regulatary effect onthe expression of Flk-1 mRNA, but it does upregulate the Flk-1 protein expression, which may be one of the mechanisms of repairing SCI.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer. MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method. ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05). ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
ObjectiveTo observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. MethodsThe BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined, and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. ResultsThe isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant (P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B (P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B (P>0.05). ConclusionVEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.
Objective To study the expression changes of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), and bone morphogenetic protein 2 (BMP-2) in femoral neck fracture, traumatic, and non-traumatic avascular necrosis of femoral head (ANFH), and to study the relationshi p between the expressions of VEGF, bFGF, BMP-2mRNA and bone mass so as to explore the pathogenesis of ANFH and provide the exprimental basis for individual treatment of ANFH. Methods Femoral head specimens were obtained from 59 donors undergoing total hip replacement, including 22 cases of traumatic ANFH (group A, 13 cases of Ficat stage III and 9 cases of Ficat stage IV), 19 cases of non-traumatic ANFH (group B, 11 cases of Ficat stage III and 8 cases of Ficat stage IV; 10 cases of steroid-induced ANFH, 7 cases of alcohol ic ANFH, and 2 cases of unexplained ANFH), and 18 cases of fresh femoral neck fracture (group C). There was no significant difference in the general data among 3 groups (P gt; 0.05). The bone mineral density (BMD) at weight-bearing area of the femoral head was measured with dual energy X-ray absorptiometry. The pathological changes were observed by using optical microscope and scanning electron microscope. The percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The expressions of VEGF, bFGF, and BMP-2 mRNA in femoral head were detected by use of in-situ hybridization technique. Results The BMD in groups A and B were significantly lower than that in group C (P lt; 0.05), and there was significant difference between group A and group B (P lt; 0.05). In the necrosis area of groups A and B, the bone trabecula was rarefactive and not of integrity, with a great number of empty bone lacuna. In healthy area, more fiber hyperplasia was observed in group A, the prol iferated and hypertrophic fat cells in the medullary cavity in group B. Scanning electron microscope showed that many osteocytes underwent fatty degeneration and necrosis, and that the prol iferation of fat cells in bone matrix was observed in groups A and B. While in group C, the femoral head had intact articular cartilage and intact bone trabeculae, and osteocytes were clearly seen. The percentage of empty bone lacuna was significantly higher (P lt; 0.05) and the percentage of trabecular bone area was significantly lower (P lt; 0.05) in groups A and B than group C; and there was significant difference in the percentage of empty bone lacuna between groups A and B (P lt; 0.05). The expressions of VEGF, bFGF, and BMP-2 mRNAwere significantly lower in groups A and B than group C (P lt; 0.05), and the expressions of BMP-2 and bFGF mRNA in group A were significantly higher than those in group B (P lt; 0.05). There were positive l inear correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and the BMD and percentage of trabecular bone area, respectively. While there were significantly negative correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and percentage of empty bone lacuna. Conclusion The repair capacity of local femoral head in traumatic ANFH is ber than that in non-traumatic ANFH. The expressions of VEGF mRNA, bFGF mRNA, and BMP-2 mRNA decl ine in traumatic and nontraumatic ANFH.