Objective To study the prokaryotic expression, puriifcation and properties of recombinant human Chymotrypsin. Methods The protein was highly expressed in E.coliBL 21 (DE 3) as inclusion body. After refolding and activated with trypsin, the activated protein was obtained and purified with ion-exchange chromatography(CM-FF), some properties of the recombinant human chymotrypsin was investigated. Results The molecular weight of chymotrypsinogen was about 30 KD in SDS-PAGE, total activity recovery rate of CM-FF puriifcation was 93.7%. The recombinant chymotrypsin kept stable from pH 3 to pH 5, and owned good temperature stability. Km was 0.067 mmol/L with BTEE as a substrate. The UV maximum absorption wavelength was 281 nm.Conculsion The recombinant human enzyme was expressed successfully, and a feasible production method to get a high activity of the recombinant human chymotrypsin was established.

Background In recent years,incidence of drug-induced keratopathy is increasing highly.Druginduced keratopathy is lack of typical clinical features and offen confused with the primary disease.Therefore,summarizing and concluding the clinicals feature and standard treatments of drug-induced keratopathy are key problem need to be solved urgently for us.Objective This study was to retrospectively analyze the clinical features and therapeutic procedure of drug-induced keratopathy.Methods A retrospective case series analysis method was adopted.The clinical data of 36 eyes (31 patients) with drug-induced keratopathy were collected by Shandong Eye Hospital from 2008 to 2012,including eye disease history,medication history,medication dosage and duration.A series of relevant examinations were performed,including best corrected visual acuity (BCVA) before and 1 month after treatment,Schirmer test Ⅰ (S Ⅰ t),tear film break-up time (BUT),meibomian gland findings,the location of the keratopathy,the characteristics of keratophthy before and after fluorescein staining.The treating were given,including cessating of the original drugs,applying corneal repair promotion and anti-inflammatory drugs as well as the comprehensive treatment for meibomian gland embolization and dry eye,such as the hot packs and massage in the eyes with meibomian gland dysfunction and a tear dot embolization therapy in the eyes with S Ⅰ t ＜ 5 mm and BUT＜5 s.Paired t test and repeated measured one-way analysis of variance in SPSS 17.0 software were used to compare the BCVA,BUT and S Ⅰ t outcomes.The correlation between corneal repair duration and S Ⅰ t results was analyzed by Pearson linear correlation analysis.Results The primary cause of drug-induced keratopathy was irrational use of drugs,including antiviral drugs,antibiotics,hormone,antiallergic,lowering-intraocular pressure drugs,turn for 23 eyes,12 eyes,10 eyes,1 eye and 1 eye,respectively.Improper route of administration included 25 cases of overuse of eye drops and 6 cases of subconjunctival injection.BCVA was 0.69 ± 0.28 1 month after treatment,which was significantly improved in comparison with before treatment (0.32 ± 0.26) (t =11.02,P ＜ 0.01).Clinical manifestations included corneal epithelial diffusive and point-like roughness,corneal epithelial defect and even corneal ulcer for severe cases,corneal edema,Descemet membrane folds and partially visible corneal filiform.Drug-induced keratophthy was mainly located in the central and lower cornea.Comprehensive therapy was effective with the treating duration about 1 week to 8 weeks.A negative correlation was found between the corneal restore duration and S Ⅰ t results (r =-0.835,P＜0.01).Conclusions Corneal changes secondary to topical medications may affect all layers of the cornea.Clinicians should be mindful of drug-induced ocular disorders.The early diagnosis of druginduced keratopahthy depends on the medical history and clinical features.A comprehensive treating based on ocular surface conditions is available.

ObjectiveTo evaluate the daily vitamin intakes in diets of diabetic patients.MethodsUsing a food questionnaire,the contents of 63 type Ⅱ diabetics(group A) and 64 non diabetics(group B) were recorded and analysised by computer.The daily vitamin intakes were assessed and compared with the Recommend Dietary Allowance(RDA).ResultsThe daily intakes of vitamin B1 and vitamin B2 were both obviously deficient,less than 50% of the RDA.The daily intakes of vitamin E and A were significantly different between group A and group B.ConclusionsThe vitamin intakes of the senile diabetics in diets were inadequate.

ObjectiveTo investigate the clinical significance and histological origin of glomerular epithelial proliferative lesion in patients with focal segmental glomerulosclerosis (FSGS). MethodsSeventy-four patients with idiopathic FSGS hospitalized in Peking University First Hospital from Jan. 2000 to Dec.2005 were enrolled in this study. Patients were classified into two groups according to with or without glomerular epithelial proliferative lesion. Estimation of active and chronic pathological scores was carried out using a semi-quantitative grade system by two pathologists. Clinical and pathological characteristics were compared between two groups. Immunohistochemical studies were performed to analyze the histological origin of glomerular epithelial proliferative lesion. ResultsThirty-one patients with glomerular epithelial proliferative lesion showed shorter interval from presentation to biopsy (P<0.05), higher percentage of nephrotic syndrome (NS) (P<0.05), higher frequency of segmental glomerulosclerosis(P<0.05), higher pathological active scores (P<0.05) and lower pathological chronic scores (P<0.05)as compared to 43 patients without glomerular epithelial proliferative lesion. Twenty-nine patients were followed up and renal survival rate in patients with glomerular epithelial proliferative lesion (39.7%) was significantly lower than that in patients without glomerular epithelial proliferative lesion (83.3%) (P=0.049). The frequency of glomerular epithelial proliferative lesion and the serum creatinine (Scr) level at biopsy were independent predictors of ESRD (OR value was 1.204, 1.008 respectively ). Glomerular epithelial proliferative lesion did not express mature podocyte markers including WT-1 and pedocalyxin, but stained positive for PCNA, PAX-2 and CK-8. ConclusionsGlomerular epithelial proliferative lesion represents the pathological change of acute stage and active lesion of FSGS, and also may be the pathological marker of severe clinical presentation and worse renal survival. Glomerular epithelial proliferative lesion may be derived from proliferation of parietal epithelial proliferation or de-differentiated podocytes.

Objective To investigate the association between podocytes and proteinuria in children with Alport syndrome. Methods Twenty-one children including 13 boys and 8 girls with Alport syndrome were divided into 3 groups according to 24-hour urinary protein, ＜30 mg/kgas the mild proteinuria group (10 patients), 30 to 50 mg/kg as the moderate proteinuria group (4 patients) and ＞50 wg/kg as the heavy proteinuria group (7 patients). The correlation between foot process width and the degree of proteinuria was analyzed. By immunoperoxidase staining on renal tissue, the key slit diaphragm molecules nephrin, podocin and podocyte cytoskeleton-associated molecule synaptopodin were studied. Results The foot process width (420-2270 nm) in patients with Alport syndrome was positively correlated with proteinuria significantly (r=0.765, P＜0.01).Foot process width was significantly lower in patients with mild proteinuria [475 (420-900) nm)compared with that in patients with heavy proteinuria [1520 (480-2270) nm] (P＜0.05). In Alport syndrome children with heavy proteinuria, the distribution of nephrin and podocin changed dramatically. In two children with shorter proteinuria period (1 year), dramatic distribution change of nephrin and podocin occurred, however, the foot process along with synaptopodin preserved.Conclusions Podocyte foot process effacement and injury of slit diaphragm participate in the mechanism of proteinuria in Alport syndrome. The injury of slit diaphragm seems to be an early event in the development of foot process effacement in Alport syndrome, which may guarantee early treatment.

Objective To construct a shRNA lentiviral vector targeting the gene encoding tumor necrosis factor alpha-induced protein 8 (TNFAIP8) in RAW264. 7 cells, a mouse macrophage cell line, and to investigate the effects of TNFAIP8 gene silencing on the functions of mouse macrophages. Methods The shRNA sequence targeting TNFAIP8 gene was designed and DNA oligos containing small hairpin frame was synthesized. The double-stranded DNA was cloned into pLKO. 1-TRC vector after annealing. The recombi-nant vector was verified by using double enzyme digestion and gene sequencing. Lentiviruses were prepared by transfecting the constructed vector into 293T cells. Fluorescent quantitative RT-PCR and Western blot as-say were performed to detect the expression of TNFAIP8 at mRNA and protein levels after infecting the RAW264. 7 cells with lentiviruses. Flat dish adhesion experiment and wound-healing assay were used to evaluate the effects of TNFAIP8 gene silencing on the adhesion and migration of RAW264. 7 cells. Results The recombinant lentiviral vector was successfully constructed as indicated by double enzyme di-gestion and gene sequencing analysis. The expression of TNFAIP8 in RAW264. 7 cells at both mRNA and protein levels were significantly down-regulated after lentivirus infection (P<0. 05). Moreover, TNFAIP8 gene silencing significantly impaired the cell adhesion ability of RAW264. 7 cells after 15 min, 30 min or 2 hours of culture. Compared with the cells in control group, the RAW264. 7 cells harboring silenced TN-FAIP8 gene looked round with a smaller number of cellular extensions. The wound-healing assay showed that less TNFAIP8 gene-silenced RAW264. 7 cells migrated into the wounded area as compared with the cells in control group after 24 hours of culture (P<0. 05). The wound-healing rates of the experimental and control groups were 25% and 50%, respectively. Conclusion The recombinant lentiviral vector containing shRNA targeting the TNFAIP8 gene was successfully constructed. Transfecting the RAW264. 7 cells with the con-structed vector significantly silenced the expression of TNFAIP8 gene and inhibited the adhesion and migra-tion of these cells.

OBJECTIVE:To observe clinical efficacy and safety of rivaroxaban in the treatment of pulmonary embolism. METHODS:100 patients with pulmonary embolism were selected and randomly divided into observation group and control group, with 50 cases in each group. Both groups were given nursing intervention for rehabilitation. Observation group was treated with riva-roxaban 15 mg,bid,for consecutive 3 weeks,3 weeks later adjusting to 20 mg,qd;control group was given low molecular weight heparin 1 mg/kg,bid,combined with warfarin sodium 5 mg,qd,for more than 5 days,stopped taking low molecular weight heparin until the INR reached the target 2-3 and given warfarin alone orally. Treatment course of 2 groups lasted for 3 months. Clinical efficacy of 2 groups were observed,and PaO2,PaCO2 and D-dimer levels were observed in 2 groups before and af-ter treatment;the improvement of clinical symptoms as cough,pectoralgia,dyspnea,and the incidence of ADR were recorded in 2 groups. RESULTS:The total effective rate of observation group (90.0%) was significantly higher than that of control group (74.0%),with statistical significance(P<0.05). After treatment,PaO2,PaCO2 and D-dimer levels of 2 groups were increased signif-icantly,compared with before treatment;the observation group was higher than the control group,with statistical significance(P<0.05). The remission rate of clinical symptoms as cough,pectoralgia,dyspnea in observation group was significantly higher than in control group,with statistical significance (P<0.05). The incidence of ADR in observation group (12.0%) was significantly lower than in control group (30.0%),with statistical significance (P<0.05). CONCLUSIONS:Rivaroxaban supplemented by reasonable nursing intervention can effectively improve clinical efficacy of patients with pulmonary embolism with low incidence of ADR.

Objective To verify the validity and feasibility of national early warning score (NEWS) in evaluation of death risk in elderly patients with critical illness,in order to find out which scoring method is more suitable for elderly critical illness patients.Methods A prospective case-control study was conducted.The critical illness patients aged over 60 years old with the length of hospital stay over 24 hours,and admitted to Department of Emergency of Qingdao Municipal Hospital from January to December 2015 were enrolled.The clinical data including in emergency and the actual outcome of patients were collected,and the patients were divided into death group and survival group according to 30-day outcome.Patients in the two groups were assessed by using NEWS and risk classification according to the first results of vital signs monitoring.Multivariate logistic regression model was used to analyze the relationship between the NEWS classification and the risk of death in elderly critical ill patients.Results 1 950 emergency elderly patients with critical illness were enrolled,with 78 cases (4.0％) dead within 30 days and 1 872 survived (96.0％).Compared with the survival group,patients in death group were older (years:79.8 ± 10.8 vs.75.3 ± 8.9,t =4.335,P ＜0.001),and had higher acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score (22.9± 4.6 vs.18.2 ± 4.8,t =8.487,P ＜ 0.001),lower Glasgow coma scale (GCS) score (12.2 ± 4.5 vs.13.4 ± 5.2,t =-2.007,P =0.045),higher incidence of respiratory system diseases (29.5％ vs.17.9％,x 2 =12.742,P =0.013),higher NEWS score (11.2 ± 5.5 vs.3.9 ± 2.7,t =22.063,P ＜ 0.001),as well as higher proportion of patients with NEWS classification of high risk and very high risk (65.4％ vs.15.8％,x 2 =263.125,P ＜ 0.001).With the increase of NEWS risk classification,mortality rate was also increased,and the mortality rate in the patients with low,medium,high and very high risk were 0.81％ (9/1 108),3.63％ (18/496),5.83％ (13/223),30.89％ (38/123),respectively,with statistically significant difference (x 2 =179.741,P ＜ 0.001).It was showed by logistic regression analysis that the NEWS score of elderly patients with critical illness were positively correlated with 30-day death.The 30-day death risk of patients with middle risk,high risk and very high risk was 4.600,9.052 and 54.598 folds of the patients with low risk respectively.Conclusion NEWS score can be used to assess the risk of death in emergency elderly patients with critical illness.NEWS risk classification can quantify and classify the risk of death in the elderly patients with critical illness.

Objective:To explore the relationship among blood glucose,blood lipid levels,peripheral blood white cells and slow coronary flow (SCF).Methods:Clinical data of 183 patients receiving angiography in our hospital from Apr 2010 to Apr 2013 were retrospectively analyzed.Patients with TIMI grade 2 or lower coronary blood flow were defined as SCF;patients were divided into SCF group (n=93)and normal control group (n=90).Levels of blood lipid,glycosylated haemoglobin (HbA1c),hematocrit,and peripheral blood white cell levels were measured and compared between two groups.Results:Compared with normal control group,there was significant reduction in high density lipoprotein cholesterol (HDL-C) level [(1.21 ± 0.26)mmol/L vs.(1.12 ± 0.28)mmol/L,P =0.043],and significant rise in HbA1c level [(5.41±0.50)% vs.(5.83±0.45)%,P =0.01],hematocrit [(0.41 ±0.04)vs.(0.43±0.07),P =0.01]and white blood cell count [(6.1±1.6)109/L vs.(6.7±1.7)109/L,P <0.05]in SCF group.Logistic analysis indicated that white cell count was the risk factor of SCF (OR 1.920,95% CI 1.234~2.987,P =0.004).Conclusion:Levels of high density lipoprotein cholesterol,glycosylated haemoglobin, hematocrit and white blood cell count are related to slow coronary flow,and elevated white blood cell count may be a risk factor aggravating slow coronary slow.

Objective To integrate the self-developed Web-based early warning management system into EMR system to decrease average medical fee as well as the occurrences of cost overrun,arrearage for self-paid fee and etc.Methods The method mapped into image for users' browse based on using the user-defined URL extension characteristic of EMR system,combining the advantages of Web platform in handling Http request,applying Java as well as SQL technology to obtain source data,integrating statistical analysis for multi-dimensional comparative analysis as well as visualization technology.Results This system changed the behavior pattern of early warning and supervision in course,relieved work burden of medical staffs,and helped hospital achieve high economic management benefit.Conclusion The application of crossplatform system realizes high effect and provides approaches for expanding other functions.