Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on immune function of peritoneal macrophages in mice. Methods The peritoneal macrophages were isolated from female BALBc mice in vitro. The macrophages were stimulated with different doses of HMGB1 (1, 10, 100, 1 000ng/ml) for 6 hours, or with 100ng/ml HMGB1 for different duration (0, 6, 12, 24, 48, 72 hours). The phagocytosis ability of macrophages was determined by the neutral red dye uptake assay and optical densities of samples were read at 540nm. The cytotoxicity to L1210 cells was measured with MTT assay and was expressed by absorbance at 570nm. The chemotaxis was assayed by using the Costar Transwell cell culture chamber inserts and the cells on the lower surface of the transwell membrane were fixed and counted. The cells were harvested at the indicated time points, fixed, and stained for indirect immunofluorescence, then the expressions of I-Ak MHC class Ⅱ alloantigen on macrophages were determined with flow cytometry. Results The effects of HMGB1 on phagocytic activities, cytotoxicity, chemotaxis and I-Ak antigen expression of macrophages were enhanced in a dose-dependent manner, starting at doses as low as 1ng/ml and with a maximal response at 100ng/ml (P

Objective To investigate the relationship between C-reactive protein and clinical significance of Tokyo Guidelines for acute cholangitis.Methods Analyzed 739 cases with acute cholecystitis in Xianyang Central Hospital of Shaanxi province.Records of patients diagnosed with acute cholecystitis were screened between September 2013 and October 2015.The criteria of Tokyo guidelines were used in grading the severity of acute cholecystitis.Patients were divided into 3 groups,Group Ⅰ (n =450),Group Ⅱ (n =260) and Group Ⅲ (n =29).C-reactive protein values at the time of admission were analyzed and compared among the groups.Results Mean C-reactive protein levels of groups were found to be significantly different,18.74 mg/L in Group Ⅰ,133.67 mg/L in Group Ⅱ,and 257.43 mg/L in Group Ⅲ.C-reactive protein values among the groups were found to be highly and significantly correlated with the disease grade (P ＜ 0.01).After evaluating C-reactive protein levels according to the grade of the disease,Group Ⅱ was distinguished from Group Ⅰ with a cutoff C-reactive protein level of 72.54 mg/L,and from Group Ⅲ with a value of 195.85 mg/L.Those results were found to be statistically significant (P ＜ 0.01).Conclusion C-reactive protein can be accepted as a strong predictor in classifying different grades of the disease,and treatment can be reliably planned according to this classification.

AlM: To investigate the efficacy of photodynamic therapy ( PDT) and intravitreal injection with ranibizumab on macular choroidal neovascularization ( CNV ) of pathologic myopia ( PM) .
METHODS: There were patients ( 32 eyes ) who were diagnosed as PM with CNV. Randomly selected 16 cases ( 16 eyes ) which were given the PDT treatment ( PDT group ) . The remaining were given both PDT and intravitreal injection with ranibizumab ( combination group) . There is no significant difference on macular edema between two groups. We analyzed the changes in the best corrected visual activity ( BCVA) , optic coherence tomograph ( OCT ) and fundus fluorescein angiography (FFA) before and 1, 6mo after treatment.
RESULTS:One month after the treatment in PDT group:the BCVA increased while the CMT decreased compared with that of pretreatment (P<0. 05). One month after the treatment in combination group: the BCVA increased while the CMT decreased significantly compared with that of pretreatment (P<0. 01);the changes of BCVA and CMT showed statistically significant between the two groups ( P<0. 05). Six month after the treatment in PDT group: the BCVA increased while the CMT decreased compared with that of pretreatment ( P <0. 05 ). Six month after the treatment in combination group: the BCVA increased while the CMT decreased significantly compared with that of pretreatment ( P <0. 01 ); compared with changes of BCVA and CMT in two groups, the difference was significant after treatment (P<0. 05). Compared 1mo with 6mo after treatment:there was no significant difference in the BCVA and CMT changes (P>0. 05). One month after treatment: in PDT group, FFA showed no leakage or reduced leakage of CNV in 11 eyes (69%), and the fundus remained leaky in 5 eyes ( 31%); in combination group, FFA showed no leakage or reduced leakage of CNV in 13 eyes (81%);the fundus remained leaky in 3 eyes (19%). Six month after treatment:in PDT group, FFA showed no leakage or reduced leakage in 10 eyes ( 62. 5%); the fundus remained leaky in 4 eyes ( 25%); two eyes ( 12. 5%) relapsed leakage; in combination group, FFA showed no leakage or reduced leakage of CNV in 15 eyes (94%);the fundus remained leaky in 1 eye (6%).
CONCLUSlON: Not only PDT but also PDT and intravitreal injection with ranibizumab can block CNV of pathologic myopia completely or partly, and reduce the danger causing descent of vision. Effects and the stability of the combination therapy is superior to PDT treatment.

Objective To obtain the Hiwi gene encoding full-length and construct human Hiwi adenoviral vectors carrying green luorescence protein (GFP), and to establish foundation for a further study on Hiwi function and mechanism of inducing leukemia stem cell differentiation and apoptosis.Methods All coding areas of human Hiwi gene full length were amplified using method of overlapping extension PCR technology, and the full length coding aeras were inserted into the vector of Flag-IRES-hrGFP carrying GFP with Gateway technology to construct pDown-Hiwi-3 × flag-IRES-hrGFP. The cloning vector pDown-Hiwi-3 × flag-IRES-hrGFP and expression vector pAV. Des1d were used for homologous recombination reaction to obtain recombinant adenovirus vector pAV.Ex1d-Hiwi-3× flag-IRES-hrGFP.The positive clones were selected by PCR to extract the recombinant adenovirus plasmid and to pack into recombinant Ad-Hiwi-3 × flag-IRES-hrGFP adenovirus. Results The human recombinant Hiwi was successfully cloned and the recombinant adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was found to be successfully constructed via restriction enzyme digestion and sequencing methods. The adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was transfected into the HEK293A cells using lipofectamine mediated gene transfection method. Under fluorescence microscope, the transfected cells with green fluorescence could be observed.Conclusion The expression plasmid of adenovirus vector pAV.Ex1d-Hiwi-3 × flag-IRES-hrGFP is successfully constructed and it can express in HEK293A cells.

Objective To identify novel mutations in the GATA6 gene associated with congenital atrial septal defects (ASD).Methods This was a case-control study.A cohort of 220 unrelated Han-race patients with congenital ASD and 200 unrelated ethnically matched healthy individuals used as controls,who were admitted to Tongji University Affiliated Tongji Hospital from January,2007 to October,2011,were recruited.The peripheral venous blood samples from the participants were prepared.All the coding exons and their flanking sequences of the GATA6 gene were amplified by polymerase chain reaction and sequenced using the di-deoxynucleotide chain termination technique.The acquired sequences were aligned with the sequences derived from GenBank by BLAST to identify the sequence variations.The software ClustalW was used to analyze the conservation of the altered amino acids.Results Three novel heterozygous missense GATA6 mutations,c.250G ＞A (p.A84T),c.649G ＞C (p.G217R) and c.1270A ＞C (p.S424R),were identified in 3 of 220 ASD patients,respectively.None of the three mutations was detected in 200 healthy control individuals.A cross-species alignment of GATA6 encoded protein sequences showed that the mutated amino acids were relatively conserved evolutionarily.Conclusion The identification of novel GATA6 mutations associated with ASD contributes to the reveal of the mechanism involved in the pathogenesis of ASD.

Objective Intranuclear forkhead/winged helix transcription factor p3(Foxp3) plays a key role in T cell-mediated immunosuppression.The present study was performed to investigate the effects of high mobility group box-1 protein(HMGB1) on Foxp3 gene as well as protein expressions in splenic regulatory T cells(Tregs) and their potential regulating mechanisms in mice.Methods CD4+CD25+Tregs isolated from the spleens of male BABL/c mice by magnetic beads were seeded on 96-well(1?105 cells/well) cell culture plates coated with anti-CD3(1 ?g/ml) and soluble anti-CD28(1 ?g/ml),and the cells were stimulated with HMGB1 at various intervals or at different concentrations.After being stimulated,the Foxp3 mRNA/protein expressions in the Tregs were determined.The time-dependent and dose-dependent responses between HMGB1 and intranuclear Foxp3 expression were analyzed by flow cytometry,and the expressions of Foxp3 mRNA of Tregs were analyzed by quantitative PCR of SYBR GREEN.Results After stimulation with HMGB1,the intranuclear Foxp3 protein and mRNA expressions of splenic Tregs in mice were markedly down-regulated in 24 h to 72 h(P

Objective This study aims to understand the characteristics of the time sequence of ICR mouse first mandibular molar tooth germ development through dynamic observation.Methods Tooth germ of Embryos (E11.5,E12.5,E13.5,E14.5,E15.5,E16.5,E17.5 and E18.5) and postnatal (PN1,PN2) mice were obtained.The heads (E11.5-E15.5) and mandibles (E16.5-PN2) of mice were dissected,fixed and embedded for serial sections and HE staining.All the results were assessed under light microscopy.Results The tooth germ underwent various development stages including the bud,cap and bell stages.Mouse odontogenesis was initiated at E11.5.Proliferation of oral epithelium formed the bud stage at E13.5.Then the cap stage was observed at E14.5-E15.5 and the bell stage was appeared beginning from E16.5.The pre-dentin was observed at PN1,as well as the dentin at PN2.Conclusions Establishing the regular development pattern of the first mandibular molar of ICR mice will provide a reliable basis for the future use in the specific tooth germ developmental research.

Critical care medicine has the characteristics of complex,a wide range of knowledge,and interdisciplinary,so standardized training is difficult.Intensive Care Unit explored a standardized training mode and evaluation system suitable for standardized training residents in the Intensive Care Unit,including generalizing training target,developing step by step teaching plans,establishment of entrance education,basic theory,basic skills,scientific thinking and doctor-patient communication training with diversity of teaching methods,including lecture-based learning,problem-based and casebased learning,implement the flexible and strict evaluation system.

Objective:This study aims to ensure the private hospitals development strategy in the background of new healthcare reform and put forward some scientific suggestions to help promote their sustainable development. Methods:The SWOT analysis method was adopted to analyze the private hospitals’ internal strength, weakness, exter-nal opportunity and threats. Results: The results showed a clear property right, advanced management system and other outstanding advantages in the private hospitals. Opportunities like the improved policy environment and people’s medical demand expansion are also very prominent. Meanwhile, the private hospitals development also faces some disadvantages and challenges such as illegal operation, disorder in the market competition, vague orientation, etc. Conclusions:It suggests the strengthening of the personnel and hospital culture construction, adoption of the differen-tiation strategy, improvement of the diagnosis and treatment ability, etc.

OBJECTIVE:To study the effect of polysaccharides of Radix scrophulariae on immune function in mice under nor-mal physiological and hypoimmunical state. METHODS:80 mice were randomly divided into normal group,model group,treat-ment high-dose,medium-dose,low-dose groups and physiological high-dose,medium-dose,low-dose groups,10 in each group. Mice in normal group and model group were received distilled water (10 mL/kg) intragastrically,treatment high-dose,medi-um-dose,low-dose groups and physiological high-dose,medium-dose,low-dose groups were administrated 0.16,0.08,0.04 g/kg drug,ig,once a day,for 7 d. From the third day,mice in model group and treatment high-dose,medium-dose,low-dose groups were respectively received cyclophosphamide(100 mg/kg),ip,for 4 d to induce hypoimmunical model. Carbon clearance test was adopted to determine the carbon clearance indexes and organs(chest,spleen)indexes of mice. Another 80 mice were grouped with the same administration,serum half hemolytic value (HC50) was determined after compressed Mianyang red blood cell sensitiza-tion. Then another 80 mice were grouped with the same administration,1% dinitrofluorobenzene method was used to induce de-layed hypersensitivity in mice;its ear swelling was determined,as well as IL-2,IL-4,immunoglobulin G(IgG),immunoglobulin M(IgM),γ-interferon(IFN-γ)contents in serum,and the proliferation in vitro of splenic lymphocytes were detected. RESULTS:High-dose polysaccharides can promote the increasing of immune organ and carbon clearance indexes(P<0.05);enhance the inten-sity of delayed type hypersensitivity (HC50 increasing)(P<0.01) and improve the decreasing of serum hemolysin in model mice (P<0.01);promote the splenic lymphocytes proliferation and increase IL-2,IL-4,IgG,IgM,IFN-γ contents in serum(P<0.05 or P<0.01). CONCLUSIONS:Polysaccharides can enhance the immune function in mice under normal physiological and hypoim-munical state induced by cyclophosphamide.