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Objective:To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results:Compared with the control group, cell viability ( t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate ( t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant ( t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant ( t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion:BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
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Objective:To screening differentially expressed genes (DEGs) in proliferative diabetic retinopathy (DR) patients to provide new biological therapeutic targets for proliferative DR (PDR) therapy.Methods:A basic research. A total of 3 PDR patients (group PDR) and 3 non-diabetic patients (control group) were enrolled in the study in Tianjin Medical University Eye Hospital in October 2020. In addition, 40 cases of PDR and non-diabetic patients were selected and divided into PDR validation group and control validation group. Peripheral blood validation test was performed in PDR validation group and control validation group; RNA sequencing was performed in PDR group and control group. Transcriptomics (RNAseq) sequencing technology was used to screen DEG in PDR group and control group. The selected DEGs were analyzed by gene ontology (GO) function enrichment analysis, signal pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction network (PPI). The gene expression database was used to find the high-throughput data related to PDR, and multi queue comparison analysis was carried out. The target genes of differentially expressed miRNAs were predicted through targetscan platform, so as to clearly screen the correlation between DEG and PDR. Reverse transcription polymerase chain reaction and Western blot were used to verify the expression of DEG mRNA and protein related to PDR. The relative expression of PDR related DEG mRNA and protein between PDR validation group and control validation group were compared by paired t-test. Results:A total of 1 337 DEGs were screened by RNAseq sequencing in the peripheral blood of patients with PDR, of which 419 genes were up-regulated and 918 down-regulated. Among them, direct inhibitor of apoptosis protein-binding protein with low isoelectric point ( DIABLO), zinc finger and BTB domain containing 10 ( ZBTB10), polo-like kinases 3 ( PLK3), regulatory subunit 1 ( PIK3R1) and B cell translocation gene 3 (BTG3) were differentially expressed in PDR patients. The function of GO was enriched from the analysis of molecular function, biological process and cellular composition. The results showed that DIABLO, ZBTB10, PLK3, PIK3R1, BTG3 were involved in the pathological process related to PDR. KEGG enrichment analysis showed that glucose metabolic pathways such as extracellular matrix receptors, cytokine regulatory pathway, p53 signal pathway and galactose metabolism may be involved in the process of differential genes. The analysis of PPI protein interaction network showed that the larger the DEG-associated protein node, the greater the number of associated nodes. Among them, DIABLO, ZBTB10, PLK3, PIK3R1 and BTG3 played significant roles in the formation of the action network. By comparing and analyzing the existing high-throughput data related to diabetic retinopathy in Gene Expression Omnibus database and predicting by Targetscan platform, it was found that some significant differences in miRNA reported in aqueous humor, vitreous fluid and plasma of DR patients can be regulated by the differential genes found in this study. Compared with the control verification group, the relative expressions of DIABLO, ZBTB10, PLK3, PIK3R1 mRNA and protein in peripheral blood of the PDR verification group were up-regulated, and the relative expression of BTG3 mRNA and protein was down-regulated. Conclusion:DIABLO, ZBTB10, PLK3, PIK3R1 and BTG3 are DEGs in patients with PDR, and they can participate in the disease process by regulating the biological processes of cell proliferation, fibrosis and oxidative stress.
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ObjectiveTo investigate the clinical features, pathological features, differential diagnosis, and prognosis of solid pseudopapillary neoplasm (SPN) of the pancreas. MethodsA retrospective analysis was performed for the clinical data of 14 patients with SPN who were treated in our hospital from January 2014 to February 2018, and related articles were reviewed. ResultsThere were 11 female and 3 male patients with an age of onset of 13-77 years (mean 33.1 years). Most of them attended the hospital due to lesions found by physical examination or the presence of upper abdominal pain. Radiological examination revealed space-occupying lesion in the pancreas. The maximum diameter of the tumor ranged from 0.6 cm to 22 cm. Histological examination showed that most tumors were composed of solid areas and pseudopapillary areas, with a microcystic structure in local lesion. Immunohistochemistry showed positive Vimentin and negative CgA ,Glucagon, Gastrin, and Insulin in all patients. Some patients were positive for β-catenin (13/14), CD10 (10/14), CD56 (9/14), Syn (8/14), AA-T (11/14), PR (8/14), CyclinD1 (9/14), CA19-9 (3/14), CK-pan (9/14), CEA (1/14), and P53 (1/14). Ki-67 index was ≤10% in all 14 patients. ConclusionSPN of the pancreas should be diagnosed with reference to clinical data, imaging examination, histological features, and immunohistochemistry. The microcystic structure has a certain value in the diagnosis of SPN.
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Objective To study the effect and mechanism of Dl-3-n-butylphthalide(NBP) on mem-ory and learning ability in type 2 diabetes model db/db mice. Methods Sixteen male db/db mice were ran-domly divided into the NBP group and diabetes group,with 8 mice in each group. Eight male db/m mice of the same age were treated as the control group. NBP group was given NBP by gavage,while diabetes group and control group were given equal volume vegetable oil by gavage. After 6 weeks treatment,Morris water maze was used to examine the spatial memory and learning ability of the mice in each group. The changes of long-term potentiation ( LTP) in the hippocampus area of mice were detected by electrophysiological tech-niques. RT-PCR and Western blot were employed to measure expressions of VEGF,caspase-3,and Aβ1-42 in hippocampus of mice. Results Compared with the control group((21. 49±4. 41)s),average escape la-tency of day 5 in the diabetes group and NBP group were increased significantly ((51. 69±5. 45)s,(26. 92± 4. 76)s,t=9. 21,2. 35,both P<0. 05). Compared with the control group(7. 00±0. 60),the number of cross-ing the target platform in the diabetes group and NBP group were decreased significantly(2. 34± 0. 27), (4. 95±0. 54),t=-6. 56,-2. 49;both P<0. 05). Compared with the control group((255. 90±54. 24)%), LTP level in hippocampus of the diabetes group and NBP group were significantly decreased ( 130. 97 ± 14. 08)%,(176. 17 ± 18. 96)%,t=-4. 25,-2. 38; both P<0. 05). The relative expression of VEGF, caspase-3,and Aβ1-42 in the diabetes group and NBP group were significantly increased compared with those of the control group (t=4. 59,8. 42;7. 36,3. 85;3. 84,2. 11;all P<0. 05). Compared with the diabetes group,the escape latency of day 5,the number of crossing the target platform and LTP level were improved in the NBP group (t=-7. 25,4. 06,3. 25;all P<0. 05). The decreased expression of caspase-3,Aβ1-42 and increased expression of VEGF were reversed after NBP treatment in db/db mice (t=-4. 14,-2. 31,3. 42;all P<0. 05). Conclusion NBP might improve cognitive function and LTP by exerting anti-apoptosis effect through inhibiting the deposition of Aβ1-42 and upregulating VEGF expression in hippocampus of db/db mice.