A protease from the culture supernatant of marine bacteria Pseudomonas sp. 7~11 was discovered. One component of the protease was purified to homogeneity by ammonium sulfate precipitation, dialysis, ion exchange chromatography. The specific activity of the enzyme was raised from 19.46U?mg -1 to 13953115U?mg -1 ,which was 717.02 times that of the culture supernatant with a yield of 1.24%.The molecular mass of purified enzyme was estimated to be 3000Da about by SDS PAGE. The optimum temperature for the activity was observed to be 45℃ using casein as substrate.The optimum pH for activity the enzyme was 8.0 and it was stable between pH6～7 and below 50℃.The activity of the enzyme was inhibited by EDTA and IAA strongly. But was not inhibited by PMSF. the enzyme was inhibited Mn 2+ , Hg 2+ , Fe 2+ , Zn 2+ , Cu 2+ ,Pb 2+ and SDS. The inactivity of the enzyme with EDTA could be recovered partially by Mg 2+ .while Ca 2+ , Mg 2+ and (NH 4) 2SO 4 was activator for the activity of the enzyme. The activity of the enzyme was fairly stable in the presence of ethanol and urea, and was resistant to Tween 20.

OBJECTIVEThis study aimed to investigate the effects of in vitro continuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix (ECM).

METHODSEnzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passages in vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the mRNA changes in the marker genes, routine genes, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities.

RESULTSAfter continuous expansion in vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased (P < 0.05), and it was almost negatively expressed by P5 chondrocytes. By contrast, the down regulation of routine genes was insignificant. The gene expression levels of MMPs and TIMPs both decreased (P < 0.05), but the change in MMP-1 and TIMP-1 was not statistically significant (P > 0.05). Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes (P < 0.05).

CONCLUSIONSerially passaged chondrocytes dedifferentiated and lost specific phenotypic characteristics during in vitro expansion culture. Simultaneously, the anabolism and catabolism of the cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0-P2 chondrocytes should be used.

Animals ; Cartilage ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; physiology ; Cytoskeleton ; Extracellular Matrix ; Gelatinases ; Gene Expression ; Hyalin ; physiology ; Matrix Metalloproteinase 1 ; Matrix Metalloproteinases ; Mice ; RNA, Messenger ; Tissue Engineering ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinases

Objective To analyze the Ag NORs in T cells of different patients to find clues for the diagnosis and monitoring of cancer.Methods A KL 2 imaging system was used to analyze the areas of Ag NORs in T cells. T cells from peripheral blood were cultivated and stimulated, and silver staining was performed. The areas of Ag NORs were analyzed.Results A total of 1 046 nomal adults, 393 patients with non malignant diseases and 706 patients with malignant diseases were analyzed. Their average IS% values were (7.81?0.69)%, (7.85?0 72)% and 5.17?0.87 respectively. The IS% values above 6% were 99.8%, 94.7% and 10% to 35% respectively. No differences were observed in different kinds of cancers except breast cancer and rectum cancer whose IS% were (5.61?0.86)% and (6.10?0.92)% respectively, 75% and 26% of their IS% were less than 6% respectively. 83% to 90% of the IS% in the patients with other cancers were below 6%. In comparison with serum CEA, AFP and the like, Ag NORs in T cells were more powerful in diagnosis and monitoring of cancers.Conclusion The value of Ag NORs of T cells was significantly lower in patients with cancers and was statistically different from that in nomal adults and patients without malignant diseases. The results demonstrated that the analysis of Ag NORs might play an important role in the diagnosis, differenciation and monitoring of cancer.

Objective To investigate the interference effects of orexin A on cell proliferation of the insulin?secreting beta?cell line(INS?1 cells) through the orexin receptor 1(OX1R)and the AKT/PKB signaling pathway. Methods INS?1 cells were exposed to different concentrations of orexin A in vitro,and treated with OX1R antagonist(SB334867),PI3K antagonist(wortmannin),or AKT antagonist(PF?04691502). The INS?1 cell proliferation and apoptosis,insulin secretion,OX1R protein activity and AKT phosphorylation level were determined. Results Orexin A(10-10 to 10-6 mol/L)stimulated the proliferation and activation of INS?1 cells,prevented apoptpsis,and increased insulin secretion. Additionally,AKT phosphorylation was stimulated by orexin A(10-10 to 10-6 mol/L). The OX1R antagonist SB334867(10-6 mol/L),the PI3K antagonist wortmannin (10-8 mol/L)and the AKT antagonist PF?04691502(10-6 mol/L)weakened the effects of orexin A. Conclusion Orexin A activated the AKT sig?naling pathway through the mediation of orexin A?OX1R,and promoted cell proliferation in INS?1 cells.

The P2Y12 receptor antagonist is used widely in prevention and treatment of cardiovascular and cerebrovascular disease. Monitoring changes of platelet function after treatment can improve the prognosis of patients. The platelet function test is the important way to evaluate high residual platelet reactivity after antiplatelet treatment, including light transmission aggregometry (LTA), whole blood impedance aggregometry assay (WBIA), vasodilator- stimulated phosphoprotein (VASP), thrombelastogram (TEG), platelet function analyzer- 100 (PFA-100) and VerifyNow system (VerifyNow). It is very different for the reflecting ability with residual reactivity of platelets among these tests after anti-platelet therapy, and also significant difference for assessment effect. Among them, LTA is a classic method for the curative effect evaluation of anti-platelet agents, which is convenient and cheap, but it is susceptible to the operating and environment interference. The clinical application of WBIA is less, and which lacks threshold value for assessment. VASP is sensitive for the changes of platelet function, but the test is complex and expensive. TEG can monitor the inhibition ratio of drugs on anti-platelets, but it needs to verify the safety of treatment. It is not clear for sensitivity and specificity with monitoring anti-platelet agent by PFA-100. VerifyNow is effective and reliable, but the cost is high. The evidence of clinical study shows that LTA, VASP and VerifyNow can reflect the effect of platelet inhibition of P2Y12 receptor antagonists sensitively, and is associated with the risk of major adverse cardiac events (MACE) in patients with cadiovascular diseases.

Objective To explore the clinical efficacy of anti-helicobacter pylori (HP) treatment on patients with type 2 diabetes mellitus and HP infection.Methods A total of 112 type 2 Diabetes patients were diagnosed with HP infection in Combine Traditional Chinese and Western Medicine Hospital of Taizhou from April 2012 to June 2016.These patients were divided into control and treatment group based on the preprandial blood glucose averages from lower to higher hierarchy.Thus,there were 56 patients in each of the two groups.The control group was given Motilium and Talcid for gastric symptoms in addition to anti-diabetes treatment,while the treatment was administered the same treatments as well as the quadruple anti-HP therapy (omeprazole,amoxicillin,clarithromycin and colloidal-bismuth-subcitrate,with Talcid stopped while colloidal-bismuth-subcitrate was administered).The anti-HP lasted for 14 days.The two groups were compared the gastric symptoms,the blood glucose levels,and the HbA1c one month after treatment.Half a year and one year post treatment,the two groups were compared the gastric mucus signs under gastroscopy.Results The improvement rates after treatment with the treatment group in abdominal pain,bloating,regurgitation,belching and diarrhea/constipation were 88.5％ (23/26),83.3％ (25/30),74.1％ (20/27),83.9％ (26/31),82.6％ (19/23),respectively,and with the control group being 29.2％(7/24),32.1％(9/28),28％(7/25),30.3％(10/33),18.2％ (4/22),respectively.The differences between the two groups were significant (x2=8.06,6.62,3.92,7.65,6.66,P＜0.05 or P＜0.01).The control group did not show significant changes in preprandial glucose levels,the glucose levels two hours post meals and the HbA1c(P＞0.05) while the treatment group showed statistically significant changes(P＜0.05 or P＜0.01).The differences in the three indicators after treatment between the two groups were significant (t =4.07,7.85,4.16,P＜ 0.05).The Gastric mucus signs under gastroscopy showed improvements in both groups after treatment.The improvement rates with the treatment group were 86.2％(25/29),86.7％ (13/15),77.8％ (14/18),72.7％ (8/11) respectively,with the control group being 36％ (9/16),27.3％ (3/11),13.3％ (2/15),14.3％ (1/7),respectively.The differences between the two groups were significant (x2 =6.71,4.12,4.38,3.85,P ＜ 0.05 or P＜ 0.01).The effectiveness rate,which was based on combined improvements in gastric symptoms,glucose levels and gastric mucus signs,was 76.8％ with the treatment group and 32.1％ with the control group.The difference was statistically significant (x2 =6.78,P＜0.01).Conclusion Anti-HP treatment can relieve the gastric symptoms,stabilize the glucose levels,and help to reverse the changed gastric mucus.All these can reduce the complications of the diabetes and improve the prognosis of the patients.

Objective:To introduce a modified method for umbilical reconstruction after single-incision laparoscopic surgery.Methods:Between June 2012 and June 2015,173 patients underwent single-incision laparoscopic cholecystectomy at our institution.All the patients adopted our modified method for umbilical reconstruction.Results:All the surgeries were successful.The umbilical incision healed well with good cosmetic result.Conclusion:The modified method for umbilical reconstruction can improve cosmesis and decrease the possibility of incision hernia after single-incision laparoscopic surgery.More studies are needed to evaluate definitive clinical advantages of this technique.

Objective To investigate the effects of salvianolic Acid A and B(Sal A,Sal B)on acute myocardial ischemia of rats.Method The rat model with acute myocardial ischemia was established by coronary ligation and intravenous injection of posterior pituitary.The effect of Sal A 10,5,2.5 mg/kg and Sal B 10 mg/kg on the electrocardiogram of different time points,serum creatine kinase(CK),lactate dehydrogenase(LDH)and myocardial infarction area were observed.Results To the rat model was established by coronary ligation,Sal A 10,5,2.5 mg/kg and Sal B 10 mg/kg could lower the ST-segment elevation,reduce infarct size and serum CPK,LDH.The effect of Sal A 10,5 mg/kg was significantly higher than that of Sal B.The role of Sal A 2.5 mg/kg was equivalent to Sal B 10 mg/kg.To the rat model was established by intravenous injection of posterior pituitary,Sal A 10,5,2.5 mg/kg and Sal B 10 mg/kg could lower the ST-segment elevation.The effect of Sal A 10,5 mg/kg was significantly higher than that of Sal B.The role of Sal A 2.5 mg/kg and sal B 10 mg/kg had no significant difference.Conclusion Treatment with Sal A 10,5 mg/kg was more effective in the rat models with acute myocardial ischemia than Sal B 10 mg/kg.Sal A 2.5 mg/kg improved acute myocardial ischemia in rats was equivalent to Sal B 10 mg/kg.

Objective To analyze the clinical features and treatment and prognosis of Paget's diseases.Method 7 cases of Paget's disease of the extramammary have been encountered. Results 3 patients with groin matastases were died within 5 years, the patients without groin matastases were all surrived.Conclusions Surgery is the treatment of first choice for Paget's disease. Prognosis of patient with groin matastases is very poor.

OBJECTIVE:To explore quality control of plasma concentration monitoring for sodium valproate and to improve the quality of pharmaceutical monitoring.METHODS:The statistical and continuous analysis of sodium valproate control data from our hospital of 2008 were conducted using fluorescence polarization immunoassay(FPIA).RESULTS:Mean recovery and RSD were 98.37% and 1.94% for low control samples,99.33% and 2.88% for medium control samples,98.17% and 4.10% for high control samples.The RSD of sample was lower than 5% and in line with the requirement for biological sample determination in Chinese Pharmacopoeia.CONCLUSION:FPIA is a comparatively accurate and stable method for plasma concentration monitoring of sodium valproate.