Investigation of endothelial cell pathological characteristics in murine choroidal neovascularization model based on single-cell RNA sequencing / 中华实验眼科杂志

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.

Association between Cardiovascular Burden and Requirement of Intensive Care among Patients with Mild COVID-19

BackgroundInformation regarding the impact of cardiovascular disease (CVD) on disease progression among patients with mild coronavirus disease 2019 (COVID-19) is limited. MethodsThis study evaluated the association of underlying CVD with disease progression in patients with mild COVID-19. The primary outcome was the need to be transferred to intensive care due to disease progression. The patients were divided with and without CVD as well as stable and intensive care groups. ResultsOf 332 patients with mild COVID-19, median age was 51 years (IQR, 40-59 years), and 200 (61.2%) were female. Of 48 (14.5%) patients with CVD, 23 (47.9%) progressed to severe disease status and required intensive care. Compared with patients without CVD, patients with CVD were older, and more likely to have fatigue, chest tightness, and myalgia. The rate of requiring intensive care was significantly higher among patients with CVD than in patients without CVD (47.92% vs. 12.4%; P<0.001). In subgroup analysis, rate of requiring intensive care was also higher among patients with either hypertension or coronary heart disease than in patients without hypertension or coronary heart disease. The multivariable regression model showed CVD served as an independent risk factor for intensive care (Odd ratio [OR], 2.652 [95% CI, 1.019-6.899]) after adjustment for various cofounders. ConclusionsPatients with mild COVID-19 complicating CVD in are susceptible to develop severe disease status and requirement for intensive care. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the impact of coexisting cardiovascular diseases (CVD) on disease progression in patients with mild COVID-19? FindingsAlthough most patients with mild COVID-19 were discharged alive from hospital, approximately 47.9% patients with coexisting CVD developed severe disease status and required intensive care. CVD is an independent risk factor of intensive care among patients with mild COVID-19. MeaningCoexisting CVD is associated with unfavorable outcomes among patients with mild COVID-19. Special monitoring is required for these patients to improve their outcome.

Risk prediction for poor outcome and death in hospital in-patients with COVID-19: derivation in Wuhan, China and external validation in London, UK

BackgroundAccurate risk prediction of clinical outcome would usefully inform clinical decisions and intervention targeting in COVID-19. The aim of this study was to derive and validate risk prediction models for poor outcome and death in adult inpatients with COVID-19. MethodsModel derivation using data from Wuhan, China used logistic regression with death and poor outcome (death or severe disease) as outcomes. Predictors were demographic, comorbidity, symptom and laboratory test variables. The best performing models were externally validated in data from London, UK. Findings4.3% of the derivation cohort (n=775) died and 9.7% had a poor outcome, compared to 34.1% and 42.9% of the validation cohort (n=226). In derivation, prediction models based on age, sex, neutrophil count, lymphocyte count, platelet count, C-reactive protein and creatinine had excellent discrimination (death c-index=0.91, poor outcome c-index=0.88), with good-to-excellent calibration. Using two cut-offs to define low, high and very-high risk groups, derivation patients were stratified in groups with observed death rates of 0.34%, 15.0% and 28.3% and poor outcome rates 0.63%, 8.9% and 58.5%. External validation discrimination was good (c-index death=0.74, poor outcome=0.72) as was calibration. However, observed rates of death were 16.5%, 42.9% and 58.4% and poor outcome 26.3%, 28.4% and 64.8% in predicted low, high and very-high risk groups. InterpretationOur prediction model using demography and routinely-available laboratory tests performed very well in internal validation in the lower-risk derivation population, but less well in the much higher-risk external validation population. Further external validation is needed. Collaboration to create larger derivation datasets, and to rapidly externally validate all proposed prediction models in a range of populations is needed, before routine implementation of any risk prediction tool in clinical care. FundingMRC, Wellcome Trust, HDR-UK, LifeArc, participating hospitals, NNSFC, National Key R&D Program, Pudong Health and Family Planning Commission Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSeveral prognostic models for predicting mortality risk, progression to severe disease, or length of hospital stay in COVID-19 have been published.1 Commonly reported predictors of severe prognosis in patients with COVID-19 include age, sex, computed tomography scan features, C-reactive protein (CRP), lactic dehydrogenase, and lymphocyte count. Symptoms (notably dyspnoea) and comorbidities (e.g. chronic lung disease, cardiovascular disease and hypertension) are also reported to have associations with poor prognosis.2 However, most studies have not described the study population or intended use of prediction models, and external validation is rare and to date done using datasets originating from different Wuhan hospitals.3 Given different patterns of testing and organisation of healthcare pathways, external validation in datasets from other countries is required. Added value of this studyThis study used data from Wuhan, China to derive and internally validate multivariable models to predict poor outcome and death in COVID-19 patients after hospital admission, with external validation using data from Kings College Hospital, London, UK. Mortality and poor outcome occurred in 4.3% and 9.7% of patients in Wuhan, compared to 34.1% and 42.9% of patients in London. Models based on age, sex and simple routinely available laboratory tests (lymphocyte count, neutrophil count, platelet count, CRP and creatinine) had good discrimination and calibration in internal validation, but performed only moderately well in external validation. Models based on age, sex, symptoms and comorbidity were adequate in internal validation for poor outcome (ICU admission or death) but had poor performance for death alone. Implications of all the available evidenceThis study and others find that relatively simple risk prediction models using demographic, clinical and laboratory data perform well in internal validation but at best moderately in external validation, either because derivation and external validation populations are small (Xie et al3) and/or because they vary greatly in casemix and severity (our study). There are three decision points where risk prediction may be most useful: (1) deciding who to test; (2) deciding which patients in the community are at high-risk of poor outcomes; and (3) identifying patients at high-risk at the point of hospital admission. Larger studies focusing on particular decision points, with rapid external validation in multiple datasets are needed. A key gap is risk prediction tools for use in community triage (decisions to admit, or to keep at home with varying intensities of follow-up including telemonitoring) or in low income settings where laboratory tests may not be routinely available at the point of decision-making. This requires systematic data collection in community and low-income settings to derive and evaluate appropriate models.

Supplementing the National Early Warning Score (NEWS2) for anticipating early deterioration among patients with COVID-19 infection

BackgroundThe National Early Warning Score (NEWS2) is currently recommended in the United Kingdom for risk stratification of COVID outcomes, but little is known about its ability to detect severe cases. We aimed to evaluate NEWS2 for severe COVID outcome and identify and validate a set of routinely-collected blood and physiological parameters taken at hospital admission to improve the score. MethodsTraining cohorts comprised 1276 patients admitted to Kings College Hospital NHS Foundation Trust with COVID-19 disease from 1st March to 30th April 2020. External validation cohorts included 5037 patients from four UK NHS Trusts (Guys and St Thomas Hospitals, University Hospitals Southampton, University Hospitals Bristol and Weston NHS Foundation Trust, University College London Hospitals), and two hospitals in Wuhan, China (Wuhan Sixth Hospital and Taikang Tongji Hospital). The outcome was severe COVID disease (transfer to intensive care unit or death) at 14 days after hospital admission. Age, physiological measures, blood biomarkers, sex, ethnicity and comorbidities (hypertension, diabetes, cardiovascular, respiratory and kidney diseases) measured at hospital admission were considered in the models. ResultsA baseline model of NEWS2 + age had poor-to-moderate discrimination for severe COVID infection at 14 days (AUC in training sample = 0.700; 95% CI: 0.680, 0.722; Brier score = 0.192; 95% CI: 0.186, 0.197). A supplemented model adding eight routinely-collected blood and physiological parameters (supplemental oxygen flow rate, urea, age, oxygen saturation, CRP, estimated GFR, neutrophil count, neutrophil/lymphocyte ratio) improved discrimination (AUC = 0.735; 95% CI: 0.715, 0.757) and these improvements were replicated across five UK and non-UK sites. However, there was evidence of miscalibration with the model tending to underestimate risks in most sites. ConclusionsNEWS2 score had poor-to-moderate discrimination for medium-term COVID outcome which raises questions about its use as a screening tool at hospital admission. Risk stratification was improved by including readily available blood and physiological parameters measured at hospital admission, but there was evidence of miscalibration in external sites. This highlights the need for a better understanding of the use of early warning scores for COVID. KO_SCPLOWEYC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWMESSAGESC_SCPLOWO_LIThe National Early Warning Score (NEWS2), currently recommended for stratification of severe COVID-19 disease in the UK, showed poor-to-moderate discrimination for medium-term outcomes (14-day transfer to ICU or death) among COVID-19 patients. C_LIO_LIRisk stratification was improved by the addition of routinely-measured blood and physiological parameters routinely at hospital admission (supplemental oxygen, urea, oxygen saturation, CRP, estimated GFR, neutrophil count, neutrophil/lymphocyte ratio) which provided moderate improvements in a risk stratification model for 14-day ICU/death. C_LIO_LIThis improvement over NEWS2 alone was maintained across multiple hospital trusts but the model tended to be miscalibrated with risks of severe outcomes underestimated in most sites. C_LIO_LIWe benefited from existing pipelines for informatics at KCH such as CogStack that allowed rapid extraction and processing of electronic health records. This methodological approach provided rapid insights and allowed us to overcome the complications associated with slow data centralisation approaches. C_LI

The promoting effects of SNAI1 activating matrix metalloproteinase on choroidal neovascularization under hypoxia / 中华实验眼科杂志

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.

The effects of Celecoxib on human brain microvascular endothelial cells release 6-keto-PGF1α and TXB2 and apoptosis after radiation / 中华放射肿瘤学杂志

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.

Combination effect of oncolytic virus H101 and radiation on apoptosis induction and toxicity in A549 lung adenocarcinoma cells / 中华放射医学与防护杂志

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.

Combination effect of oncolytic virus H101 and radiation on apoptosis induction and toxicity in A549 lung adenocarcinoma cells / 中华放射医学与防护杂志

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.

Prenatal ultrasonographic diagnosis and differential diagnosis of fetal penoscrotal transposition / 中华医学超声杂志（电子版）

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.

Content and dissolution determination of sGC-003 tablet by HPLC / 国际药学研究杂志

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.

Relationships between the expressions of matrix metalloproteinase-9,Toll-like Receptor 4 and lung revascularization in pa-tients with chronic obstructive pulmonary disease / 中国医师杂志

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.

Autologous intercostal nerve plus acidic fibroblast growth factor transplantation for the treatment of high-level spinal cord injury / 中国组织工程研究

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.