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 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 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 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 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 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.
Objective To study the biological activity of recombinant adeno-associated virus vector (rAAV) coexpressing human vascular endothel ial growth factor165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis. Methods The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7(experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions ofhVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbil ical vein endothel ial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineral ization of BMSCs detected by ALP staining and al izarin red staining. Results With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P lt; 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the exprimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, prol iferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P lt; 0.05). The ALP staining showed more bly stained granules in experimental group than in control group. There was significant difference in the number of the mineral ized nodules between two groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
摘要:目的: 探讨PCNA,VEGF在胃癌组织中的表达关系及其意义。 方法 :免疫组织化学法检测正常胃和胃癌组织中PCNA和VEGF的表达,并分析其与胃癌临床病理特征的相关性。 结果 :胃癌组织中PCNA、VEGF的阳性表达率分别为850%、683%,二者在正常胃组织中均为隐性表达。PCNA的表达与肿瘤分期、淋巴结转移有关(〖WTBX〗P lt;005),而与年龄、性别、肿瘤大小、组织分化程度无关(〖WTBX〗P gt;005);PCNA的表达与肿瘤分期、淋巴结转移有关(〖WTBX〗P lt;005),而与年龄、性别、肿瘤大小、组织分化程度无关(〖WTBX〗P gt;005)。PCNA和VEGF在胃癌中的表达呈正相关。 结论 :PCNA和VEGF在胃癌组织中的高表达促进了胃癌的发生发展。Abstract: Objective: To study the significance and relationship of expression of proliferating cell nuclear antigen(PCNA) and vascular endothelial growth factor (VEGF) in gastric cancer. Methods : The expression of VEGF and PCNA in normal gastric tissues and gastric cancer tissues were detected by immunohistochemistry (SP),and clinic pathological correlation with gastric cancer was analyzed. Results : The positive exression rates of PCNA and VEGF in gastric cancer were 850%、683%, respectively, while negative expression was shown in normal gastric tissues. It was found that the expressions of PCNA and VEGF were both in significant relation to TNM stages and lymph node metastasis (P lt;005), but in no relation to age, gender, diameter of tumor and tumor cell differentiation (P gt;005). The expressions of PCNA and VEGF were positively correlated with each other. Conclusion : It was indicated that the highlevel expressions of PCNA and VEGF might corporately accelerate the progression of gastric cancer.