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Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.
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Objective: The aim of this study was to identify the molecular subtypes of chronic obstructive pulmonary disease [COPD] and to prioritize COPD candidate genes using bioinformatics methods
Materials and Methods: In this bioinformatics study, the gene expression dataset GSE76705 [including 229 COPD samples] and known COPD-related genes [candidate genes] were downloaded from the Gene Expression Omnibus [GEO] and the Online Mendelian Inheritance in Man [OMIM] databases respectively. Based on the expression values of the candidate genes, COPD samples were divided into molecular subtypes through hierarchical clustering analysis. Candidate genes were accordingly allocated into the defined molecular subtypes and functional enrichment analysis was undertaken. Pathway deviation scores were then analyzed, followed by the analysis of clinical indicators [FEV1, FEV1/FVC, age and gender] of COPD patients in each subtype, and prediction models were constructed. Furthermore, the gene expression dataset GSE71220 was used to bioinformatically validate our results
Results: A total of 213 COPD-related genes were identified, which divided samples into three subtypes based on the gene expression values. After intersection analysis, 160 common genes including transforming growth factor beta1 [TGFB1], epidermal growth factor receptor [EGFR] and interleukin 13 [IL13] were obtained. Functional enrichment analysis identified 22 pathways such as 'hsa04060: cytokine-cytokine receptor interaction pathways, 'hsa04110: cell cycle' and 'hsa05222: small cell lung cancer'. Pathways in subtype 2 had higher deviation scores. Furthermore, three receiver operating characteristic [ROC] curves [accuracies >80%] were constructed. The three subtypes in COPD samples were also identified in the validation dataset GSE71220
Conclusion: COPD may be further subdivided into several molecular subtypes, which may be useful in improving COPD therapy based on the molecular subtype of a patient
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Objective To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV).Methods Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation,and flatmount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining,and the location of SNAI1,MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1,MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR).The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5％ CO2condition and mix condition of 94％ N2,5％ CO2 and 1％ O2,respectively.The expression of SNAI1,MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells,and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively,and the migrating number of the cells was assayed by Transwell chamber assay.Results CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA:1.291 ±0.060 vs.0.759±0.074,P =0.001;MMP2 mRNA:1.610±0.424 vs.0.772 ±0.080,P =0.044).The expression of MMP9 mRNA was not significantly elevated between model group and control group (P＞0.05).The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P＜0.01).The relative expressions of hypoxic induced factor 1α (HIF-1α),SNAI1 and MMP2 at mRNA level and protein level in RF/6A cells were significantly higher in the hypoxia group than those in the normoxia group (all at P＜0.05) and no considerable difference was seen in MMP9 mRNA expression between the two groups (P＞0.05).The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105,and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+siSNAI1 group and only hypoxia group,respectively,with significant differences between them (P =0.002,0.003).The migrating number of the cells was (254.60 ±71.31)/field in the hypoxia+siSNAI1 group,which was significantly less than (534.10±96.21) /field in the control group (P =0.029).Conclusions The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1,which promotes the migration of vascular endothelial cells and therefore participates in CNV formation,and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process.
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Objective To investigate the apoptosis and toxicity of oncolytic virus H101 combined with radiation on apoptosis of A549 lung adenocarcinoma cells. Methods A549 lung adenocarcinoma cells in exponential growth phase were divided into four groups: control ( PBS) group, radiation ( IR) group, oncolytic virus (H101) group and radiation combined with oncolytic virus (IR+H101) group. The cells were double dyed with Annexin fluorescein isothiocyanate ( V-FITC/PI ) and then the apoptosis ratio of cells in every group was detected by the flow cytometry. The cytotoxic effect of cells in every group was detected by lactate dehydrogenase ( LDH) release test. The mRNA expression of oncolytic viruses H101 capsid protein Hexon was detected by real-time fluorescence PCR ( RT-PCR) to compare the oncolytic virus replication in each group. Results Cell apoptosis rate in H101 group (55. 37%) was significantly higher than that in PBS group (1.03%) (t =36.51, P <0.05). Cell apoptosis rate in IR + H101 group (93. 06%) was significantly higher than that in H101 group (55. 37%), IR group (12. 67%) and PBS group (1. 03%) (t=13. 51, 24. 14, 38. 99, P<0. 05). LDH releasing percentage in IR group and H101 group at different time after virus transfection was significantly higher than that in PBS group ( t=25. 84,39. 38, 32. 51, 78. 18, P<0. 05;t=31. 40, 2. 68, 23. 43, 60. 98, P<0. 05). LDH releasing percentage in IR+H101 group was significantly higher than that in PBS group (t=80. 71, 119. 74, 109. 80, 123. 94, P<0. 05), IR group (t=28. 80, 54. 34, 72. 34, 61. 91, P<0. 05) and H101 group (t=42. 02, 57. 45, 57. 01, 58. 83, P<0. 05). Compared with H101 group at the same time point, the mRNA expression of Hexon in IR + H101 group at 24, 48 and 72 h was increased by 16. 26, 28. 37 and 39. 58 times, respectively (t=54. 50, 33. 73, 29. 28, P<0. 05). Conclusions The oncolytic virus H101 plays a role of radiosensitization in tumor cells. Radiation also increases the replication of the oncolytic virus H101 and thereby enhances the oncolytic effect of the oncolytic virus H101. Therefore, oncolytic virus H101 combined with radiotherapy has synergistic effect on killing tumor cells.
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Objective To investigate the apoptosis and toxicity of oncolytic virus H101 combined with radiation on apoptosis of A549 lung adenocarcinoma cells. Methods A549 lung adenocarcinoma cells in exponential growth phase were divided into four groups: control ( PBS) group, radiation ( IR) group, oncolytic virus (H101) group and radiation combined with oncolytic virus (IR+H101) group. The cells were double dyed with Annexin fluorescein isothiocyanate ( V-FITC/PI ) and then the apoptosis ratio of cells in every group was detected by the flow cytometry. The cytotoxic effect of cells in every group was detected by lactate dehydrogenase ( LDH) release test. The mRNA expression of oncolytic viruses H101 capsid protein Hexon was detected by real-time fluorescence PCR ( RT-PCR) to compare the oncolytic virus replication in each group. Results Cell apoptosis rate in H101 group (55. 37%) was significantly higher than that in PBS group (1.03%) (t =36.51, P <0.05). Cell apoptosis rate in IR + H101 group (93. 06%) was significantly higher than that in H101 group (55. 37%), IR group (12. 67%) and PBS group (1. 03%) (t=13. 51, 24. 14, 38. 99, P<0. 05). LDH releasing percentage in IR group and H101 group at different time after virus transfection was significantly higher than that in PBS group ( t=25. 84,39. 38, 32. 51, 78. 18, P<0. 05;t=31. 40, 2. 68, 23. 43, 60. 98, P<0. 05). LDH releasing percentage in IR+H101 group was significantly higher than that in PBS group (t=80. 71, 119. 74, 109. 80, 123. 94, P<0. 05), IR group (t=28. 80, 54. 34, 72. 34, 61. 91, P<0. 05) and H101 group (t=42. 02, 57. 45, 57. 01, 58. 83, P<0. 05). Compared with H101 group at the same time point, the mRNA expression of Hexon in IR + H101 group at 24, 48 and 72 h was increased by 16. 26, 28. 37 and 39. 58 times, respectively (t=54. 50, 33. 73, 29. 28, P<0. 05). Conclusions The oncolytic virus H101 plays a role of radiosensitization in tumor cells. Radiation also increases the replication of the oncolytic virus H101 and thereby enhances the oncolytic effect of the oncolytic virus H101. Therefore, oncolytic virus H101 combined with radiotherapy has synergistic effect on killing tumor cells.
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Objective To investigate the effect of Celecoxib on human brain microvascular endothelial cells release6-keto-PGF1α,TXB2 and apotosis after irradiation.Methods The logarithmic growth phase cells were divided into control groups (Con),simple irradiation (IR) groups and combination groups (IR+C).CCK-8 and clone formation experiment were used to evaluate the effects of radiosensitivity and toxicity of celecoxib.The results were observed atthe time point of 6 h,12 h,24 h,48 h after irradiation.ELISA was used to test the contents of 6-keto-PGF1α and TXB2,which metabolized by PGI2 and TXA2 from culture medium after irradiation at different time points in different groups.TXB2/6-keto-PGF1αratios were calculated.Annexin V-FITC/PI double staining method was used to measure the apoptosis rates at different time points in different groups.Western blot was used to measure the protein expression.Paired t test difference.Results Compared with simple irradiation group,there were no significant radiosensitivity (SER=0.96) in combination groups incubated with30 μmol/L of celecoxib.Compared with the control group,the ratio of TXB2/6-keto-PGF1αincreased at each time point in IR and IR+C (P<0.05),and the apoptosis rates increased (P<0.05).Cox-2,P-JNK and Cleaved caspase-3 increased.Compared with IR,the ratio of TXB2/6-keto-PGF1αdecreased at each time point in IR+C (P<0.05),and the apoptosis rates decreased (t=3.34~6.38,P< 0.05).The protein expression of Cox-2,P-JNK and Cleaved caspase-3 decreased.Conclusions Celecoxib may help to protect HBMECs from releasing TXA2 and decreasing the ratio of TXB2/6-keto-PGF1α,and inhibitting apoptosis after irradiation.The mechanisms of apoptosis inhibition may be related to the inhibition of Cox-2 and P-JNK,caspase-3 Cleaved proteinexpressions.
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Objective To study the ultrasonographic features and differential diagnosis of fetal penoscrotal transposition.Two dimensional and three dimensional ultrasound were applied in the diagnosis of fetal penoscrotal transpositionto improve the detection rate. Methods Twenty cases of suspected penile scrotal transposition of the fetus in Shengjing Hospital affiliated to China Medical University fromJanuary 2015 to February 2017were included in present study. The ultrasound findings, fetal chromosome examination and clinical follow-up outcome were retrospectively summarized. Results Among the 20 suspected cases of penile scrotal transposition, 17 cases were diagnosed correctly. All the 17 cases were partial type of penile scrotal transposition. In the remaining 3 cases, 2 caseswere hermaphroditism with the karyotype of 46-XX, and the other 1 case was confirmed as normal female fetusesby clinical follow-up after birth. The ″tulip″signwas the typical ultrasonographic features offetal penoscrotal transposition. Conclusion 2D combined with 3D ultraosound is useful in diagnosis and differential diagnosis of fetal penile scrotal transposition.
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Objective To establish a high performance liquid chromatography(HPLC)method for determining the content and dissolution of soluble guanylate cyclase(sGC)-003 tablets. Methods Content and dissolution of sGC-003 tablets were deter?mined by HPLC. Phenomenex Luna C18 column(250 mm×4.6 mm,5μm)was used. The mobile phase was acetonitrile-water(40∶60,V/V), with a flow rate of 1.0 ml/min,and the detection wavelength was set at 214 nm. The column temperature was 40℃and the injection volume was 20μl(injection volume of dissolution was 80μl). Dissolution of sGC-003 tablets was determined by the second method described in Chinese Pharmacopoeia(ChP)2015. 900 ml of pH 4.5 acetic acid buffer,pH 6.8 phosphoric acid buffer and water were used as dissolution media at the rotation speeds of 50 and 75 r/min to select the dissolution condition. Results This method had high specificity. The average recovery was about 99.58%(RSD=0.75%,n=9). And the working solution was stable within 12 h. The calibra?tion curves were had good linearity(R2=1)within the concentration range of 0.25-50μg/ml. The method of dissolution tests for sGC-003 tablets was established that 900 ml pH 6.8 phosphoric acid buffer was used as dissolution medium and paddle rotation speed was 50 r/min. The dissolution would be 80%at 30 min. Conclusion The dissolution condition can be used to determine the dissolution of sGC-003 tablets. The HPLC method is convenient,fast,sensitive and accurate in determining the content and dissolution of sGC-003 tablets.
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Objective To explore the correlation between the expressions of matrix metalloproteinase -9 (MMP-9), Toll-likeReceptor 4 ( TLR4) and lung revascularization in patients with chronic obstructive pulmonary disease .Methods Lung tissues frompatients with chronic obstructive pulmonary disease (COPD) (COPD group,n =25) and those without COPD (non-COPD group,n =25) were obtained from surgically resected specimens .The ratio of the area of the wall to that of the pulmonary arterioles (WA %) andthe ratio of the thickness of the wall to the external diameter of the pulmonary arterioles (WT %) were analyzed by computer-based imageanalysis system.Immunohistochemical technique was applied to investigate the expressions of TLR 4, proliferative cell nuclear antigen(PCNA) and MMP-9 in vascular smooth muscle cells.Results ⑴ The inflammatory infiltration degree, WA %, and WT %were significantly higher than that of non -COPD group ( P <0.01), respectively.⑵Compared with non-COPD group, the expressionsof PCNA, TLR4, and MMP-9 in vascular smooth muscle cells were increased significantly ( P <0.01).⑶The expressions of TLR4,MMP-9 had a positive correlation with WA%, WT%, degree of inflammatory infiltration, and the expression of PCNA ( r =0.67,0.74,0.47,0.44;0.59,0.71,0.61,0.33, P <0.01), up-regulated expression of TLR4 was closely related with the expression of MMP-9 ( r =0.55, P <0.01).Conclusions The pulmonary arterioles of COPD patients showed marked inflammatory and arteriolemuscularization, the TLR4 might aggravate inflammation,induced upregulation of MMP-9 expression, played an important role in the pulmonary vascular remodeling process.
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BACKGROUND: Acidic fibrobiast growth factor can regulate cell proliferation, migration, differentiation and survival, also can down-regulate the known inhibitor of axon regeneration, such as proteoglycan, help axons overcome these inhibitory factors, and have significant role on the regeneration of nerve fibers.OBJECTIVE: To study the feasibility and effect of the acidic fibroblast growth factor combined with peripheral nerve transplantation in the treatment of high-level spinal cord injury in rats.METHODS: A total of adult 108 female SD rats were randomly divided into autologous nerve group, autologous nerve combined .with acidic fibroblast growth factor group, and high-level spinal cord injury group. The rat T_(8-10) spinous process and lamina were bite, revealing dural sac, high-level spinal cord was resected at a horizon level, cutting 3 mm, no nerve fibers were confirmed to be attached under the microscope. In the autogenous nerve group and autologous nerve combined with acidic fibroblast growth factor group, bilateral the 8~(th) to 10~(th) pairs of intercostal nerves were harvested 2 cm, then cross-transplanted into high-level spinal cord defect (proximal white matter and distal gray matter, distal white matter and proximal gray matter), fibrin gel and fibrin gel containing acidic fibroblast growth factor were used respectively to fix the implanted intercostal nerve, followed by dural suture.High-level spinal cord transection group was subjected to exclusion between stumps. At 90 days postoperation, somatosensory evoked potential and motor evoked potential were used to test nerve electrophysiological recovery. At 76 days postoperation,biotinylated dextran amine anterograde tracing was applied to observe the motor conduction bundle recovery. At 60 days postoperation, hindlimb motor function recovery was assessed by BBB score.RESULTS AND CONCLUSION: The somatosensory and motor evoked potential waveforms were not elicited in rats of high-level spinal cord transaction group, but did elicit in autogenous nerve group and autologous nerve combined acidic fibroblast growth factor group. The average latency and amplitude of somatosensory and motor evoked potentials, as well as BBB scores in autologous nerve combined acidic fibrobiast growth factor group were significantly superior to autologous nerve group (P ＜ 0.01).In the autogenous nerve group and autologous nerve combined acidic fibroblast growth factor group, many more biotinylated daxtran amine-positive nerve fibers passed in the damage zone, compared with high-level spinal cord transection group (P ＜0,01), the autologous nerve combined acidic fibrobiast growth factor group was more than autogenous nerve group (P ＜ 0.01). It is indicated that autologous peripheral nerve graft acidic flbroblast growth factor can better restore the limb motor functions of rats after high-level spinal cord injury.