Background Histopathological examination is an important approach to the basic and clinical study in ophthalmology.Different fix methods of retinal samples produce a large impact.The first fixed way often is used at home,and the last fixed way is preferred abroad.The comparative study between the two ways is lack.Objective This study was to compare rat retinal frozen sections following 4 different fix methods and confirm a simpler and better way.Methods Forty eyeballs were extracted from 40 SD rats and were randomly divided into four groups using random number table,ten for each group.The eyeballs were firstly fixed in 4％ paraformaldehyde overnight,4％ paraformaldehyde for 2-3 hours,formalin-acetic acid-alcohol (FAA) mixture solution,and then OCT embedding was performed in 3 groups.However,the samples were fixed in cold acetone following first liquid nitrogen frozen,OCT embeded in the forth group.Retinal serial sections of 4 groups were examined and compared after hematoxylin and eosin staining under the optical microscope.Results Retinal tissue was loosened interlayerly,and clustering of cells in the inner nuclear layer (INL) and outer nuclear layer (ONL) were seen in the 4％ paraformaldehyde overnight fix group.The arrangement of interlayer and cells was improved in the 4％ paraformaldehyde for 2-3 hours group compared with 4％ paraformaldehyde overnight fix group.The retinal structure was closer among the layers in the FAA fixed group.In the later cold acetone fixed group,retinal morphology was more clear with the intact structure and regularly arranged cells in the ONL,INL and ganglion cell layer(GCL),as an living histology structure.Conclusions The first fix of retina produces a large impact on retinal morphology and structure.However,cold acetone fixation following first liquid nitrogen frozen is simpler,less time-consuming and more efficient way for the histopathological examination of retina.

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133~+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133~+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133~+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144~+ cells in CD133~+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133~+ group on 7 d (P<0.01). Compared to CD133~+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133~+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that it's not a preferential way to obtain EPCs for clinic therapy.

Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133(+) enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133(+) sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133(+) enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144(+) cells in CD133(+) group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133(+) group on the day 7 (P<0.01). As compared with CD133(+) group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133(+) sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.

Objective: To explore the clinical value of plasma copeptin level on major adverse cardiovascular event (MACE) occurrence in patients with acute ST-segment elevation myocardial infarction (STEMI) during hospitalization.
Methods: Our research included 2 groups:STEMI group, n=80 and Control group, n=80 patients with stable coronary artery disease (CAD). All patients were treated in our hospital from 2012-06 to 2014-06. Plasma level of copeptin was detected by ELISA, other relevant examinations were conducted to study the MACE occurrence in STEMI patients.
Results: Plasma copeptin level in STEMI group (523.26 ± 142.69) pg/ml was higher than that in Control group (345.25 ± 89.36) pg/ml, P<0.05. In STEMI group, there were 28/80 (35%) patients suffered from MACE, compared with non-MACE patients, they had increased plasma copeptin, cardiac muscle protein I (cTnI), kinase isoenzyme (CK-MB) and left ventricular ejection fraction (LVEF), P<0.05. Multivariate regression analysis indicated that plasma copeptin, cTnI and LVEF were the independent risk factors for MACE occurrence. According to occurred area under the curve, compared with cTnI and CK-MB, plasma copeptin level had the higher predictive value to judge the ROC, positive/negative possibility, sensitivity and speciifcity for MACE occurrence in STEAMI patients, P<0.05.
Conclusion: Plasma copeptin level could effectively predict MACE occurrence in patients with acute STEMI during
hospitalization, it may predict their prognosis at certain point.

Objective To study the impact of tumor necrosis factor-α (TNF-α) and its antagonist on the expressions of intestinal mucosa claudin-1,Zonula Occludens-1 (ZO-1) and myosin light chain kinase (MLCK) in rat models of acute liver failure.Methods Fifty four healthy male SpragueDawley (SD) rats were randomly divided into normal control group,model group and intervention group according to a random number table.Rats in normal control (n=6) group were intraperitoneally injected with 0.9％ saline (12 mL/kg).Rats in model group (n=24) and intervention group (n=24) were intraperitoneally injected with a full dose of D-galactosamine (D-GalN) at a dose of 1 200 mg/kg to establish model of acute liver failure,while rats in intervention group were intraperitoneally injected with TNF-α antagonists (rhTNFR∶Fc) at a dose of 12.5 mg/kg before 24 hours given D-GalN.At each time point of hour 8,24,48 and 72,six rats in both model group and the intervention group were sacrificed,respectively,while the normal control group were all anesthetized and sacrificed at 72 h.Models were repeated five times.Serum liver function was detected by biochemical method,and serum TNF-α level was detected by enzyme-linked immunosorbent assay (ELISA).Hematoxylin-eosin (HE) stained sections of liver and terminal ileum were examined under an optical microscope for pathological changes;and protein expression of the terminal ileum Claudin-1,ZO-1 protein and MLCK were determined by immunohistochemistry and Western blot.Means among groups were compared with t test.Results Acute liver failure was successfully induced in the D-GalN injected rats.In the model group,alanine aminotransferase (ALT) began to decline,total bilirubin continued to rise,and enzyme-jaundice separation developed at hour 72.But total bilirubin in intervention group at hour 72 was decreased.Light microscope showed that at hour 72,villus lodged at terminal ileum in the model group with part of villus tip failing off in the model group.Villus mucosa and submucosa interstitial were edema and infiltrated with numerous neutrophils.The terminal ileum kept integrate in the intervention group,and villus mucosa and submucosa were mild edema and only infiltrated with a small amount of neutrophil.Expressions of tumor necrosis necrosis factor (TNF)-α in rats of model group and intervention group were gradually increased and peaked at hour 24 ([239.83 ± 15.81] and [182.71± 17.08] ng/L,respectively),which were significantly higher than that of the control group ([24.19±3.57] ng/L,t=22.68and 15.73,respectively;both P＜0.01).Expression of serumTNF-α in the intervention group was significantly lower than that of model group (t=4.58,P＜0.01).Expressions of Claudin-1 and ZO-1 in model group decreased gradually at an early stage and reached the lowest level at hour 24 (0.355 ± 0.068 and 0.387 ± 0.091,respectively),which were both significantly lower than that of control group (1.640±0.188 and 1.015±0.150,respectively;t=12.87 and 7.14,respectively;both P＜0.01).In the intervention group,expressions of Claudin-1 and ZO-1 also decreased to the lowest level at hour 24 (1.051 ± 0.370 and 0.642 ± 0.082,respectivley),which were both significantly lower than that of control group (t =2.84 and 4.36,respectively;both P＜0.05),but significantly higher than model group with stastically difference (t =3.70 and 4.15,respectively;both P＜0.01).MLCK protein levels in the model and intervention group were gradually increased,which peaked at hour 24 (1.298±0.194 and 1.033 ± 0.073,respectively),significantly higher than the control group (0.460±0.069,t=8.16 and 11.44,both P＜0.01);and MLCK in the intervention group was lower than that in the model group with statistically difference (t=2.56,P＜0.05).Conclusions Expression of serum TNF-α in rat model of acute liver failure increases,which leads to decreased expression of Claudin-1 and ZO-1,and increased expression of MLCK,makes cell shrunk and cell gap increased.TNF-α antagonist could significantly reduce the inflammation and liver cell apoptosis,improve liver function by inhibiting MLCK expression and preventing decrease of Claudin-1 and ZO-1 proteins.

OBJECTIVETo study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation.

METHODSForty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance.

RESULTSALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining.

CONCLUSIONThe function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.

Animals ; Bacterial Translocation ; Defensins ; Disease Models, Animal ; Galactosamine ; Injections, Intraperitoneal ; Intestines ; Liver Failure, Acute ; Male ; Muramidase ; Phospholipases A2 ; Protein Precursors ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

Two isolation methods for sorting of endothelial progenitor cells(EPCs): from peripheral blood mononuclear cells(PBMCs)and CD133+ enriched cells were compared,by defining the cell morphology,phenotype,reproductive activities and function in vitro,to provide a reference for clinical application of EPCs.PBMCs from healthy subjects were used either directly for cell culture or for CD133+ sorting.The two groups of cells were cultured in complete medium 199(M199)for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS.The proliferation of differentiated EPCs was studied by MTT assay,and the VEGF concentration was measured using an ELISA kit.ECM gel experiment and migration assay were performed in vivo.The results showed that PBMCs produced more colony-forming units(CFU)than CD133+enriched cells from the same volume of blood(P<0.01).From day 7 to 14,the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers,but CD144+cells in CD133+ group were less than in PBMCs group(P<0.01).PBMCs group secreted more VEGF than CD133+group on the day 7(P<0.01).As compared with CD133+ group,PBMCs group had more potent potential of proliferation and vascularization in vitro.It was concluded that CD133+sorted cells showed a lower capacity of differentiation,secretion,proliferation and vascularization in vitro,suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.

Objective:Analysis of the influencing factors of maternal oral health care behavior based on the health belief model.Methods:From July to December 2023, a cross-sectional survey was conducted in Peking Union Medical College Hospital on 316 pregnant women who received the health belief questionnaire and self-efficacy scale。 t test and χ2 test were used to analyze the factors affecting the oral health care behavior of pregnant women from the perspective of social psychology. Results:Among the 316 pregnant women included, 110(34.8%) had poor daily oral health care behavior, 120 (38.1%)did not have oral examination before or during pregnancy. The health beliefs of pregnant women in overall oral care were not high, with a score of 6.63+3.23, Median score is 7 (5).Perceived susceptibility to oral diseases ( OR=1.51, 95% CI:1.026-2.213), self-efficacy of daily living ( OR=2.64, 95% CI: 1.384-5.040), self-efficacy of oral examination ( OR=1.74, 95% CI:1.184-2.570) were independent factors of daily oral health care behavior in pregnant women. Health motivation ( OR=2.47, 95% CI:1.474-4.126) and self-efficacy of oral examination ( OR=4.17, 95% CI:2.626-6.619) were independent factors of oral examination behavior before and during pregnancy. Conclusion:Health beliefs of maternal oral health, especially perceived susceptibility, health motivation and self-efficacy are closely related to maternal oral health care behaviors. To improve the health belief and self-efficacy of pregnant women′s oral health care and avoid potential obstacles, which could be conducive to the effective promotion of oral health care for pregnant women.

Objective:Analysis of the influencing factors of maternal oral health care behavior based on the health belief model.Methods:From July to December 2023, a cross-sectional survey was conducted in Peking Union Medical College Hospital on 316 pregnant women who received the health belief questionnaire and self-efficacy scale。 t test and χ2 test were used to analyze the factors affecting the oral health care behavior of pregnant women from the perspective of social psychology. Results:Among the 316 pregnant women included, 110(34.8%) had poor daily oral health care behavior, 120 (38.1%)did not have oral examination before or during pregnancy. The health beliefs of pregnant women in overall oral care were not high, with a score of 6.63+3.23, Median score is 7 (5).Perceived susceptibility to oral diseases ( OR=1.51, 95% CI:1.026-2.213), self-efficacy of daily living ( OR=2.64, 95% CI: 1.384-5.040), self-efficacy of oral examination ( OR=1.74, 95% CI:1.184-2.570) were independent factors of daily oral health care behavior in pregnant women. Health motivation ( OR=2.47, 95% CI:1.474-4.126) and self-efficacy of oral examination ( OR=4.17, 95% CI:2.626-6.619) were independent factors of oral examination behavior before and during pregnancy. Conclusion:Health beliefs of maternal oral health, especially perceived susceptibility, health motivation and self-efficacy are closely related to maternal oral health care behaviors. To improve the health belief and self-efficacy of pregnant women′s oral health care and avoid potential obstacles, which could be conducive to the effective promotion of oral health care for pregnant women.

Objective To develop an expert consensus on subcutaneous injection for allergen-specific immunotherapy.Methods Relevant domestic and intemational literature was reviewed,and nursing experts who had experiences in subcutaneous injection of allergen-specific immunotherapy were interviewed to form the initial draft of the consensus.A total of 85 experts from 42 hospitals nationwide were invited to participate in discussions.2 rounds of expert consultations,adjustments,revisions,and improvements were made to the initial draft,and an online meeting was held to form the final version of the consensus.The content approved by more than 75％of the expert group is adopted,or it will be discussed or deleted.Results The expert consensus includes operational standards for subcutaneous injection of allergen-specific immunotherapy,identification and management of adverse reactions,and health education.Conclusion The consensus demonstrates strong scientific rigor and practicality,providing guidance for nursing practices in the field of clinical allergology.