Objective: To highly express rh-Endostatin from Pichia pastoris and purify it to homogeneity. Methods: Constructed Pichia pastoris X33/pLW202 was amplified and inoculated to ferment media. The supernatant of the strain was collected after induction. Through ultrafiltration, affinity and gel chromatography, rh-Endostatin with high purication was obtained. Results: 60mg/L rh-Endostatin was obstained from supernatant. HPLC showed its purity was above 98%. Conclusion: High level expression of secreted rh-Endostatin has been successfully achieved in Pichia pastoris expression system.

[ ABSTRACT] AIM:To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS:Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice.Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model.The levels of interleukin-1β( IL-1β) , interleukin-10 ( IL-10) and tumor necrosis factor-α(TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).Real-time PCR was used to evaluate the mRNA expression of the cytokines.RESULTS:Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1βand TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group.The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40μmol/L) inhibited the expression of IL-1βand TNF-αat both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels.CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1βand TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.

Objective To study the imaging appearances of congenital splenorenal venous shunt,and to evaluate their clinical significances.Methods Of 127 283 patients who underwent upper abdominal CT scanning,6 patients were diagnosed to have congenital splenorenal venous shunt.The imagings were studied retrospectively.Plain scanning,enhanced CT and Doppler ultrasonography were performed on all these patients.Results The incidence was about 47/million.The six patients were all females,the age was between 32 to 67 years.The mean age was 48.8 years.Enhanced CT demonstrated that there were twisted and dilated shunting blood vessels between the splenic and renal veins.The diameters in four patients were larger than the splenic and portal veins.In the remaining two patients,they were smaller shunting blood vessels.One patient had an associated absence of right portal vein.Two patients had associated dysplasia of portal veins and splenic veins.MPR,CPR,MIP and VR could three-dimensionally depict the courses,the beginnings and the ends of the splenorenal shunts.Doppler ultrasonography showed counterflow between the portal and splenic veins,and showed the blood in the splenic vein to flow into the left renal vein.Conclusions A congenital splenorenal venous shunt is one of the rare form of congenital extrahepatic portosystemic venous shunts.Enhanced MSCT scan combining with its post-processing techniques could clearly demonstrate the shunt vessels and the associated lesions.Doppler ultrasonography could further demonstrate the shunt direction.

Aim To explore inhibitory effects and mechanism of engineering endostatin on growth and metastasis of melanoma cells in mice. Methods Melanoma cells(2× 106/mouse)were inoculated sabcutaneously to C57BL/6 mice. After tumorigenesis,endostatin(8mg/kg.d)was administrated to tumor-bearing mice,once a day ,twenty-one in all.Dietetic state and weight change of the tumor-bearing mice were observed and tumorous sige was measured during administration of endostatin. On 26thday,the tumor-bearing mice were sacrificed,subcutaneously tumorous weight was weighed and brain,lung ,liver,spleen and kidney were excised and sections were made to supply the pathological examination. Results Area under curve in the endostatin-treated group was obviously less than that in tamor control group(P∨ 0.01). Pathological study revealed that lavge areal necrosis arose in tumor and newborn cappillaries around the tumor disapeared. Conclusion Endostatin possosses strongly inhibitory effects on growth and metastasis of mouse melanoma and formation of newborn capillaries around tumor.

Objective To characterize the expressions of androgen receptor (AR) and estrogen receptor (ER) in penis skin of patients with congenital hypospadias. Methods Dorsal prepuce, ventral prepuce, and urethral plate were harvested from 30 patients with congenital hypospadias. The expressions of AR and ER in epidermal cells and dermal fibroblasts were assessed respectively by automated immunohistochemistry. Image Pro plus 6.0 was used to analyze the optical density (OD) value of AR and ER in different parts of epidermal cells and dermal fibroblasts. Results AR and ER were located mainly in nucleis of the squamous basal cells and prickle cells, and were also found in nucleis of subcutaneous fibroblast cells. The expression of AR was lower in epidermis of urethral plate than that of dorsal prepuce and ventral prepuce (P<0.05), but no significant difference was detected in dermal fibroblasts. The expression of ER was higher in epidermis of dorsal prepuce than that of urethral plate and ventral prepuce (P<0.05). The dermal expression of ER in fibroblast cells was increased successively in dorsal prepuce, ventral prepuce and urethral plate (P<0.05). Conclusion Lower expression of AR in urethral plate may contribute the development of hypospadias. Disorder of ER in dermal fibroblast cells of prepuce may play an important role in hypospadias.

AIM: To investigate the effect of diltiazem on mean pulmonary arterial pressure(mPAP) and nitric oxide synthase(NOS) in arterioles in chronic hypoxic hypercapnic rats. METHODS: Twenty-four rats were randomly divided into three groups: control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+ diltiazem group (C), constitutive endothelial NOS(ceNOS) were observed in arterioles of rats using the technique of immunohistochemistry,ceNOS mRNA were observed by the technique of in situ hybridization . RESULTS: (1)mPAP was significantly higher in rats of B group than that of A and C group( P 0 05),but mCAP was lower in rats of C group than that in B group.(2)Light microscopy showed WA/TA (vessel wall area/total area) was significantly lower in rats of C group than that of B group ( P

Objective Detrusor overactivity (DO) is one known cause of overactive bladder (OAB) symptoms in benign prostatic hyperplasia (BPH).In this study,OAB symptoms suggestive of DO in BPH were treated with α1-blocker monotherapy or α1-blocker and antimuscarinics add-on therapy,and the efficacy and safety were assessed.Methods BPH patients who suffered from OAB symptoms for at least 3 month were enrolled in a prospective self-control study from August 2010 to April 2012.The inclusion criteria are total international prostate symptom score (IPSS) ≥8,OAB Symptom Score (OABSS) ≥3,OABSS urgency score ≥2,Postvoid residual volume (PVR) ＜ 100 ml,and maximum urinary flow rate (Qmax) ≥ 5 ml/s.All the patients who met the inclusion criteria were treated with α1-blocker ( tamsulosin 0.2 mg/day or doxazosin 4 mg/day) for 2 weeks.After 2 weeks,patients with no symptomatic improvement ( OABSS≥3) underwent pressure-flow test,and those whose Pdet≥ 40 cm H2O and DO presented more than one time were added antimuscarinics (solifenacin 5 mg/day or tolterodine 4 mg/day) for an additional 2 weeks.OABSS,IPSS,QOL,Qmax and PVR were re-evaluated every 2 weeks.Results Ninety-four cases of BPH/OAB patients met the inclusion criteria and completed 4 weeks treatment.The baseline of total OABSS was 7.0 ± 1.3,IPSS was 17.0 ± 1.7,QOL was 5.0 ±0.7,Qmax was (8.8 ±2.5) ml/s and PVR was (86.0 ± 16.5) ml.After 2 weeks treatment with α1-blocker alone,OABSS was 5.2 ± 0.8,IPSS was 14.2 ± 1.9,QOLwas4.O±0.8,Qmaxwas (11.4±2.4) ml/s and PVR was (67.9±12.9) ml.After another2 weeks treatment with α1-blocker plus antimuscarinics,OABSS was 3.1 ± 0.8,IPSS was 11.1 ± 1.9,QOL was 3.1 ± 0.7,Qmax was ( 10.8 ± 2.4) ml/s and PVR was (71.8 ± 11.9 ) ml.Compared with baseline values,OABSS,IPSS,QOL,Qmax and PVR significantly improved (P ＜ 0.01 ) in α1-blocker monotherapy group and α1-blocker plus antimuscarinic group.The improvement of OABSS,IPSS,QOL scores of α1-blocker plus antimuscarinic group were greater than α1-blocker monotherapy group (p ＜ 0.05 ),while Qmax and PVR showed no differences between the two groups.No acute urinary retention (AUR) was deteted.Conclusion Both of α1-blocker monotherapy and α1-blocker with antimuscarinics add-on therapy can improve OAB symptoms.

Objective To evaluate the clinical value of multi-slice spiral CT (MSCT)in the screening of coronary artery lesions in elderly patients with diabetes mellitus.Methods MSCT and coronary angiography(CAG)were performed in 136 elderly patients(68 patients with diabetes and 68 patients without diabetes).The number of diseased coronary segments and the plaque type (noncalcified,mixed and calcified)for each patient were determined.The characteristics of coronary lesions were compared between patients with and without diabetes.The sensitivity and specificity of MSCT were evaluated.Results More noncalcified and calcified plaques and few ermixed plaques were observed in patients with diabetes,compared with patients without diabetes(noncalcified plagues:10 cases or 14.7％ vs.4 cases or 5.9％;calcified plagues:47 cases or 69.1％ vs.39 cases or 57.4％)(P＜ 0.05).In addition,diabetic patients showed a significantly higher frequency of multivessel disease(P＜0.05).Furthermore,MSCT was able to clearly show stenosis and plaque type in the trunk and branches of the coronary artery,with a sensitivity of 91.0％,a specificity of 95.0％,a positive predictive value of 93％ and a negative predictive value of 96.0％.Conclusions Diabetes is associated with a higher risk of coronary artery disease.MSCT can effectively detect the characteristics of coronary artery disease in diabetic patients and is an effective,noninvasive,and safe screening method.

Objective To summerize computed tomography (CT) and magnetic resonance imaging (MRI) appearances of the adrenal oncoctyoma(AOC),and to improve the diagnostic accuracy.Methods The 11 cases imaging materials of AOC confirmed by pathology from March 2006 to August 2017 were analyzed retrospectively.There were 3 males and 8 females,aged from 24 to 65 years old(mean 46.8 years).There were 11 cases with CT unenhanced scan in which 9 cases performed enhancing scan,8 cases underwent unenhanced and enhanced MRI scan.Results Of 11 cases (13 lesions),10 cases were singular,1 was unilaterally multiple(3 lesions).5 cases were located in the right side,6 in the left side.10 lesions presented oval,3 were round.The diameters of all lesions ranged from 1.4 to 9.9 cm,with a mean of 3.9 cm.All lesions were shown well-defined soft-tissue density on plain CT scan,69.2％ (9/13) of whom were homogeneous,30.8％ were heterogenous.The CT value ranged from 32.6 to 48.6 HU,with a mean of 37.9 HU.In 9 cases,5 masses were markedly enhanced,2 were slightly enhanced and 2 were moderately enhanced,and all of them depicted prolongedly enhanced on triple-phase scan.The masses of 8 cases appeared isointensity or slightly hypointensity on T1WI,hyperintesity on fat-suppressed T2WI.The signal did not change on opposed-phase imaging.The lesions demonstrated heterogeneously prolonged enhancement to some degree on enhanced TI WI images.There was no peritumoral adjacent invasion,enlarged lymph nodes and metastasis.Conclusions AOC usually presents soft-tissue density on unenhanced CT,no changing on MRI opposed-phase images.The tumors are characterized by markedly heterogeneously prolonged enhanced on enhanced CT and MRI.

OBJECTIVETo optimize the experimental model of nitric oxide (NO) production in mouse peritoneal macrophages in response to lipopolysaccharides (LPS) stimulation.

METHODSMouse resident peritoneal macrophages were collected by lavaging the peritoneal cavity of mice with Hank's solution and stimulated with Pseudomonas aeruginosa LPS for NO production. NO concentration in the culture supernatants was measured with Griess Reagent. The influences of cell density, LPS concentration, LPS stimulation duration and culture medium volume on NO production were investigated. Finally, the feasibility of the model was confirmed with specific anti-inflammatory drugs.

RESULTSThe density of macrophages produced the most significant effect on NO production (P<0.001), and optimal results were obtained at the macrophage density of 6×10(6) cells/ml with a volume of 100 µl in each well in 96-well plate. At a LPS concentration below 1 µg/ml, NO production increased proportionally with the increment of LPS concentration (P<0.001), but the increment of NO production declined obviously at LPS concentrations beyond 1 µg/ml, and the peak NO production occurred at a LPS concentration of 10 µg/ml. NO production also increased significantly with the prolongation of LPS stimulation (P<0.05), and the increments were greater within 24-48 h than those in 48-72 h. NO content in the culture supernatant was associated with the medium volume, and the highest level occurred in a system volume of 100 µl. Aspirin (1 mmol/L), dexamethasone (10 µmol/L), and cyclosporin A (10 µmol/L) all significantly inhibited LPS-stimulated production of NO in mouse resident peritoneal macrophages (P<0.001).

CONCLUSIONSMacrophage density, LPS concentration, and the duration of LPS stimulation are the main factors affecting LPS-stimulated NO production in mouse resident peritoneal macrophages. The optimal results can be obtained with a macrophage density of 5×10(6) cells/ml (100 µl per well), LPS concentration of 10 µg/ml, LPS stimulation duration of 24 h or 48 h, and a culture medium volume of 100 to 200 µl.

Animals ; Cells, Cultured ; Female ; Lipopolysaccharides ; pharmacology ; Macrophages, Peritoneal ; drug effects ; secretion ; Male ; Mice ; Mice, Inbred Strains ; Nitric Oxide ; biosynthesis