With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Objective To explore the effects of beraprost sodium (BPS) on angiotensin Ⅱ(AngⅡ)-induced podocyte apoptosis and the relationship between BPS and peroxisome proliferator activated receptor δ(PPARδ). Methods Differentiated mouse podocytes were exposed to AngⅡ, further treated with BPS (or GSK0660) for 24 h. Podocyte apoptosis was detected by flow cytometry and AnnexinV-FITC staining. Bax , Bcl-2 and PPARδ mRNA were assessed by RT-PCR. Results AngⅡ promoted podocyte apoptosis and Bax mRNA expression significantly, down-regulated Bcl-2 and inhibited PPARδ mRNA (P < 0.05). Conversely, treatment with BPS reduced AngⅡ-induced podocyte apoptosis and Bax mRNA expression and promoted Bcl-s expression significantly resulting in an increase of PPARδ mRNA expression (P < 0.05). With the inhibition of PPARδ, podocyte apoptosis and Bax mRNA expression increased while Bcl-2 mRNA expression reduced. Conclusion BPS can effectively reduce AngⅡ-induced podocyte apoptosis , which is associated with the activation of PPARδand regulation of Bcl-2/Bax mRNA expression.

Objective To examine the effects of nitroglycerin tolerance on the following myocardial ischemia reperfusion injury (MI/R). Methods Male Sprague-Dawley rats were rardomly subjected to either one of the following treatments: GTN 600 ?g/(kg/h) or saline infusion for 12 h. GTN tolerance was determined with the measurement of mean arterial blood pressure (MABP) and isolated arterial tension. All the GTN-tolerated and the control rats were subjected to 40 min of ischemia followed by 4 h of reperfusion. Myocardial apoptosis (TUNEL analysis), myocardial infarction size (Evans blue-TTC stain), plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activity were determined. Results Nitrate tolerance aggravated MI/R injury as evidenced by increased myocyte apoptosis and infarction size and plasma CK, LDH activity. However, sole GTN tolerance without MI/R injury failed to show any deleterious effect on rat heart. Conclusion GTN tolerance may exert a “potential" deleterious effect on the ischemic hearts.

OBJECTIVE:To prepare weishu emulsion and to establish its quality control method.METHODS:Weishu emulsion was prepared with sodium carboxymethylcellulose,etc.as the adjuvants and the content of dyclonine hydrochoride was determined by UV-spectrophotometry.RESULTS:The linear range of dyclonine hydrochloride was2.0～12.0?g/ml(r=0.9999)and the average recovery rate was99.38%,RSD=0.47%,(n=6).CONCLUSION:The emulsion is feasible in preparation method and controllable in quality.

Objective To evaluate the effect of community systemic management on quality of life in patients with hypertension.Methods A cross-sectional study was used to survey 224 patients with essential hypertension systemically managed in community and 205 non-management patients using the questionnaire including demographic information,behaviour,years of hypertension,treatment,Chinese version SF-36 short form of quality of life and the symptom distress checklist.Student-t test,?2-test,and covariance analysis was used in data analysis.Result The managed patients' mean score of knowledge(6.40?1.31)was significantly higher than that in non-managed patients'(5.02?1.82,t=8.82,P

Objective The non intrusive ultrasound combined with western medicine in the treatment of chronic prostatitis prostate/research and analysis of the effect of chronic pelvic pain syndrome.Methods96 cases of chronic prostatitis patients who were treated in our hospital from September 2016 to were randomly divided into the observation group, the control group 1 and the control group2.The observation group was treated with western medicine in the treatment of non intrusive prostate ultrasound;1 patients in the control group were given a single non intrusive prostate ultrasound treatment;control group 2 given a single treatment with Chinese and Western medicine.The clinical symptoms and NIH-CPSI scores were evaluated before and after treatment in each group.ResultsComparison between groups of patients before treatment NIH-CPSI score, no significant difference;after treatment, the NIH-CPSI score has decreased, there are obvious differences (P<0.05);the patients in the observation group were decreased significantly, the total score before treatment (26.21±2.20), the total score after treatment (11.77±1.90), there are obvious the difference (P<0.05).The treatment groups were compared with the efficiency, the total efficiency of observation group was 93.75%, the control group of 1 patients with the total effective rate was 75%, the control group of 2 patients with the total effective rate was 68.75%, there was significant difference in the treatment effect, with statistical significance (P<0.05).ConclusionNon intrusive ultrasound combined with western medicine in the treatment of prostate in compared with non intrusive ultrasound therapy and Western medicine alone in the treatment of prostate, has more significant treatment effect, should strengthen the clinical application and promotion.

Objective: The primary aim of this research is to systematically sort out and analyze available documents for yang deficiency syndrome in traditional Chinese medicine, providing innovative ideas and methods for statistical study of yang deficiency syndrome. Methods: Based on the China National Knowledge Internet (CNKI) and Chongqing VIP databases, using "yang deficiency", "deficiency cold", and "patient" as keywords, we searched literature of clinical researches and used "ratio-comparison" and "integration-comparison" to analyze objective data after data preprocessing. Relative importance value of physiological and biochemical parameters were reflected by P value calculated by ratio-comparison or Q value by integration-comparison. Results: With calculation of relative importance values of clinical indicators among various systems, we got the ranking of relative importance of different indexes. Triiodothyronine and urine 17-hydorxycorticosteroid of endocrine system, immunoglobulin A of immune system, and the ratio of cyclic adenosine monophosphate to cyclic guanosine monophosphate, which had great |P| (or |Q|) values and negative P (or Q) values, had a clearly lower level in yang deficiency patients than in the control; hemorheological indicators such as plasma viscosity, whole blood reduced viscosity and erythrocyte electrophoresis time had small |P| (or |Q|) values. These indicators, however, showed less importance for reflecting yang deficiency. Conclusion: Ratio-comparison method and integration-comparison method employed in this study have merits of efficiency and robustness. After detailed compiling and mining of all available data, we have made reasonable advice on the most relevant index of yang deficiency for further study of the essence of this syndrome.

Cornea is the major refractive components of the eye. As a viscoelastic tissue, cornea exhibits complicated biomechanical properties: non - linear elasticity, anisotropy and viscoelasticity. The biomechanical properties play an important role in keeping the normal structureand function. Changes in biomechanical properties are always earlier than the clinical symptoms. So quantitative measurement of the biomechanical properties benefits the early diagnosis and treatment of diseases. Different methods to measure the biomechanical properties of cornea were reviewed in detail, including classic ex vivo destructive tests, commercially available in vivo measuring methods and other emerging methods with the potential for clinical application but not validated for in vivo measurement. The operating principles, advantages as well as limitations of these methods were also described.

Partial deletion of the long arm of chromosome 7 is a rare disease and is prone to missing the diagnosis or being misdiagnosed.Here we present a case of a 13-year-old boy that showed symptoms such as growth-retardation,moderate intellectual disability,hypotelorism,microcephaly,epicanthal folds,genu varum and lumbar vertebral cleft,but it did not show serious symptoms like cleft lip,urogenital malformation and hypotonia.He was eventually diagnosed as partial deletion of the long arm of chromosome 7 syndrome through gene analysis.Considering the rare incidence of this disease and more rarely for being hospitalized to endocrine ward due to growth retardation,this case report can provide more information for clinic diagnosis and differential diagnosis for growth retardation.

Objective To explore the methylation status of hMLH1 and hMSH2 promoter region in the epithelial ovarian cancer and its role in oncogenesis.Methods Methylation status of hMLH1 and hMSH2 promoter region was assayed in 20 normal ovarian tissues,25 benign epithelial tumor,56 malignant epithelial tumor and cell lines SKOV3,3AO by methylation-specific PCR (MSP).SKOV3 and 3AO were analyzed before and after 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment.In addition,an alterations of mRNA expression of hMLH1 and hMSH2 was observed by reverse transcription polymerase chain reaction (PT-PCR).Results No methylation of hMLH1 and hMSH2 promoter was found in normal ovarian tissues. CPG islands methylation of hMLH1 and hMSH2 was observed in 4% (1/25),8% (2/25) respectively in benign epithelial tumor,30.4% (17/56),51.8% (29/56) respectively in malignant epithelial tumor. Methylation status in promoter showed obvious correlation with pathological grade and lymph node metastasis (P