Objective:To compare the effect of fluorescence staining and periodic acid-Schiff staining in the diagnosis of fungal keratitis (FK).Methods:A total of 147 corneal specimens from 147 FK patients treated in Eye Hospital of Shandong First Medical University from January 2017 to May 2019 with positive corneal scraping or fungal culture were collected.Among them, there were 84 cases with penetrating keratoplasty (PKP), 42 cases with lamellar keratoplasty (LKP) and 21 cases with lesion resection.Another 11 cases with herpes simplex virus keratitis served as negative control.The corneal tissue specimens were performed with fungal fluorescence staining and periodic acid-Schiff staining, respectively.The stained sections were placed under fluorescence microscope and optical microscope to observe fungal hyphae or spores, respectively.The positive rates of the two staining methods were compared, and the positive cases of the diagnosis of FK in corneal tissue samples obtained by different surgical methods and corneal infection caused by different strains of the two staining methods were compared.Written informed consent was obtained from each patient.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY-2016012).Results:The positive rate of periodic acid-Schiff staining and fungal fluorescence staining was 60.5% (89/147) and 79.6% (117/147), respectively.The positive rate of fluorescence staining in the diagnosis of fungal keratitis was significantly higher than that of periodic acid-Schiff staining ( χ2=28.00, P<0.01), and both the specificity of the two staining methods was 100%.The positive rate of specimens obtained by PKP with fluorescent staining was 85.7% (72/84), and the positive rate with periodic acid-Schiff staining was 65.5% (55/84), and the difference was statistically significant ( χ2=17.00, P<0.01). The positive rate of specimens obtained by LKP with fluorescent staining was 71.4% (30/42), and the positive rate with periodic acid-Schiff staining was 52.4% (22/42), and the difference was statistically significant ( χ2=8.00, P<0.01). The positive rate of resected foci specimens with fluorescent staining was 71.4% (15/21), and the positive rate with periodic acid-Schiff staining was 57.1% (12/21), and the difference was not statistically significant ( χ2=1.30, P=0.25). The positive cases of two kinds of staining were different among different fungal strains.Among them, the positive cases of Fusarium solani complex, Pythium insidiosum, Aspergillus fumigatus complex, Candida guilliermondii, Trichoderma and Nigrograna mackinnonii with fluorescence staining were 19, 5, 5, 1, 1 and 1, and the positive cases of periodic acid-Schiff staining were 11, 0, 3, 0, 0 and 0, respectively.The staining results of the 11 negative controls were negative. Conclusions:Fluorescence staining is more sensitive than periodic acid-Schiff staining in the detection of fungal components in paraffin-embedded corneal tissues, and it can significantly improve the fungal detection rates.

Objective To explore the relationship of methylenetetrahydrofolate reductase (MTHFR)gene C677T,factor V(FV)gene G1691A and prothrombin(PT)gene G20210A polymorphisms to unexplained recurrent early spontaneous abortion(URESA).Methods One hundred and twelve patients with URESA and 100 women with at least 1 normal pregnancy and without any miscarriage were analyzed for MTHFR,FV and PT gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Results MTHFR gene T/T genotype and T allele frequencies were increased in URESA patients[38.4%(43/112)and 59.8%(134/224)]versus controls[18.0%(18/100)and 43%(43/100),P

Objective To establish a flow-cytometric method to detect microvesicles (MVs) in rat peripheral blood, and to detect platelets-derived MVs (PMVs) and endothelial cell-derived MVs (EMVs) in blood from ischemic precondition-ing (IPC) treated rats. Methods Blood was withdrawn from rat abdominal aorta and anticoagulated with sodium citrate. Platelets-free plasma (PFP) was isolated through two centrifugations at room temperature. PFP was incubated with FITC-conjugated mouse anti-rat CD61 or PE-conjugated mouse anti-rat CD144. Standard beads in diameter of 1 and 2μm were used for calibration and absolute counting, respectively. Analysis was performed on flow cytometer. Results When 3.5%so-dium citrate was mixed with blood at volume ratio of 1∶4, clear supernatant was collected after centrifugation. Signals of parti-cles smaller than 1μm accounted for more than 99%of overall signals. PMVs and EMVs were CD61 positive and CD144 positive, respectively. Their diameters were both smaller than 1 μm. The concentration of PMVs and EMVs in peripheral blood from IPC treated rats was (4 053±1 987)/μL and (4 870±825)/μL, respectively. Conclusion The method for MVs de-tection by flow cytometry was successfully established and optimized, and verified through detecting PMVs and EMVs in pe-ripheral blood from IPC treated rats.

Objective: To determine total phenylethanoid glycoside and acteoside in Plantago Herba to provide reference for evaluating the quality of medicinal materials.Methods: With acteoside as the control sample, a UV visible spectrophotometric method was used to determine total phenylethanoid glycosides in Plantago Herba.An HPLC method was applied to determine acteoside in Plantago Herba , and the conditions were as follows: an ODS2 C 18 (150 mm× 4.6 mm ,5 μm) chromatographic column was used with acetonitrile-0.1% formic acid (13∶87) as the mobile phase at a flow rate of 1.0 ml·min-1 , the detection wavelength was 332nm, the column temperature was 30℃, and the sample volume was 10 μl.Results: The reference solution and the sample solution had the maximum absorption at 332 nm, and the linear relationship was good within the range of 0.003 1-0.155 0 mg·ml-1 (r=0.999 5).The content of total benzene alcohol glycosides in 3 batches of samples was 2.73% , 2.61% and 2.84% , respectively;acteoside over the range of 0.000 6-0.155 0 mg·ml-1 (r=0.999 1) showed a good linear relationship with peak area,the sample recovery was 98.5% and the RSD was 1.6% (n =6), and the acteoside content in 3 batches of samples respectively was 0.54% , 0.51% and 0.56%.Conclusion: The method is simple, accurate and reproducible, and can be used for the determination of total phenylethanoid glycosides and acteoside in Plantago Herba.

Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; immunology ; Endoglin ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Microvessels ; immunology ; Middle Aged ; Nitric Oxide Synthase Type II ; metabolism ; Receptors, Cell Surface ; immunology ; Uterine Cervical Neoplasms ; genetics ; metabolism

AIM:To explore the inhibitive effects of cervical lymphadenectomy on kerstoplasy after alkaline burns.METHODS:The Wistar rats' corneas were transplanted into Sprague-Dawley(SD)rats' eyes which were randomly divided into 4 groups:group A(control group);group B,the cervicallymphadenectomy group;group C,corneal transplantation after the alkali burn injury;group D,cervical lymphadenectomy following group C.Out of 6 rats in each group,the cornea of one rat Was used for mawophage immunohistochemistry at day 14 after the transplantstion,and the remaining 5 rats were used for studying corneal immune rejection with a slit lamp.The time when allograft rejection occurred was recorded and mean survival times(MST)were compared among the groups.RESULTS:Compared with the MST of group A(10.40±1.14 days),the MST of group B(46.30±9.46 days)Was significantly longer(P＜0.05).MST of grafts between group C(7.00± 1.58 days)and group D(15.00±3.39 days)was also significant (P＜0.05).At 14th day after the transplantation,there was no CD68 immunoreactivity in the graft of group B,and CD68 proteins were expressed to some extent in the grafts of group A and D.However,in the graft of group C,the expression of proteins Was dramatically up-regulated.CONCLUSION:Cervical lymphadenectomy therapy has a significant effect in preventing corneal allograft rejection in normal and alkali burned oorneai beds.

Objective To investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.Methods Small interfering RNA targeting at miR-10a (miR-10a-siRNA) was constructed,then it was transfected into pancreatic cancer AsPC-1 cells,and nonsense siRNA (Nc-siRNA) group and blank control group was established.Real time PCR assay was used to detect the expression of miR-10a in the 3 groups,and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells.The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.Results The miR-10a levels in control group,NC-siRNA group and miR-10a-siRNA group were 1.05 ±0.08,1.03 ±0.06,0.02 ±0.01 ; and the number of transmembrane cell were (150 ± 2.6),(145 ± 2.2),(62 ± 1.8),the levels of MMP-13 in the supernatant were (108.5 ± 2.8),(107.8 ± 2.5),(35.8 ± 1.5) pg/ml.The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P ＜ 0.01).The distance of cultured clone in miR-10a treated cancer cells (736± 18 μm) was significantly longer than those in the controls (385 ±5 μm) and NC-siRNA group (395± 13 μm,P＜0.01).Conclusions Down-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells,and the downregulated expression of MMP-13 may be one of the important mechanisms.

Abstract: The demand for reliable toxicological data of chemicals runs through every link of occupational health work. The prevention of occupational diseases involves high requirements for the standardization of chemical toxicity assessment in occupational health institutions. Good laboratory practice (GLP) emphasizes the integrity of the test process to trace and supervise the whole process of the test, which is conducive to the standardization of chemical toxicity identification. Therefore, the standardized construction of GLP laboratories is an important starting point for occupational health institutions to carry out chemical toxicity identification. In the construction and management process of GLP laboratories for chemical toxicity identification, occupational health institutions need to build a sound organization and operation system, carry out systematic training and assessment of personnel, establish standard operating norms and emphasize their importance, strengthen the management of facility environment and laboratory, pay attention to quality control and process supervision, and constantly improve their own ability level. To actively adapt to social development and market demand, to provide strong support for occupational health work.

Objective　To study the relationship between atherosclerotic plaques in carotid artery and ischemic cerebrovascular diseases. Methods　The extracranial carotid arteries (ECA) and middle cerebral artery (MCA) of 54 patients with transient ischemic attack (TIA) or cerebral infarction (CI) were examined with doppler ultrasound. The distribution of atherosclerotic plaque, degree of stenosis and ultrasounic classification of ECA and the mean velocity of blood flow in MCA were examined. Results　①Stenosis over middle-grade on asymptomatic side in extracranial internal carotid artery (EICA) in group of patients with TIA was significantly higher than symptomatic side(P<0.01). Stenosis over high-grade on asymptomatic side in ELCA in group of patients with CI was significantly higher than symptomatic side (P<0.01). ②Flat and soft plaque are most common in group of patients with TIA or CI, then are hard and ulcerative plaques. Incidence of soft plaques on asymptomatic side in group of patients with TIA or CI are significantly higher than symptomatic side (P<0.01); ③Among the group of patients with CI, mean velocity of MCA decreased on asymptomatic side in 31 cases (68.9%), and significantly higher than symptomatic side (P<0.01). Conclusion　Atheroclerotic plaques in carotid artery and intracranial hemodynamic characteristics are the important risk factors for ischemic cerebrovascular diseases. These findings have important values in predicting subsequent TIA or CI in asymptomatic subjects.

OBJECTIVETo investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes.

METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with 10 micromol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 microm latex beads and anti-PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining.

RESULTSEMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles (< 1 microm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner (P < 0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry.

CONCLUSIONMicrovesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.

Annexin A5 ; Apoptosis ; Calcimycin ; pharmacology ; Calcium ; metabolism ; Cell Line ; Cell Membrane ; drug effects ; Coculture Techniques ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Human Umbilical Vein Endothelial Cells ; Humans ; Myocytes, Cardiac ; drug effects ; Staining and Labeling