Objective To establish a method of quantiying trace α-galactosidase from Bacteroides fragilis in enzymatic conversion of blood group B to O red blood cells ( B-ECO RBCs) .Methods BALB/c mice were immunized with purified recombinant B.fragilisα-galactosidase ( the purity>90%) to prepare monoclonal antibodies.The ascites were prepared using hybridoma cell lines stably secreting antibody and purified by HiTrap rProtein A column.The antibody titer and spe-cificity were detected by ELISA and Western blotting, respectively.Purified monoclonal antibody and rabbit polyclonal an-tibody were applied to detect residual enzyme in B-ECO RBCs and the washing solution was analyzed by indirect ELISA. Results A high titer and purity antibody was obtained.Western blotting showed that the antibody specifically reacted with B.fragilisα-galactosidase.Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residualα-gal-actosidase at the concentration of 1 ng/ml.After four repeat washing cycles with 1∶4 ( v/v) phosphate-buffered saline, the amount of residual enzyme in B-ECO RBCs was less than 10 ng/ml.Conclusion An effective method of detecting the min-imal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs prepara-tion by enzymatic treatment.

Objective To reduce immunogenicity of porcine skin by removingα-Gal epitopes expressed in cell surface and extracellular matrix using recombinant α-galactosidase produced by Bacteroides fragilis.Methods The porcine skin was harvested from healthy 2-month-old pigs without any skin disorders before being sterilized by iodine and 75%alcohol, respectively.Enzymatic removal of α-Gal antigen was followed by washing with PBS.The α-Gal antigen in the prepared porcine skin was measured with immunofluorostaining of cryosections and the residual enzyme was measured with a double-antibody sandwich ELISA method.Enzymatic removal procedures were optimized by detecting residual enzyme and the effi-cacy ofα-Gal removal under different enzymatic and washing conditions.Results Efficient enzymatic and washing methods were established to removeα-Gal antigen.Theα-Gal removal efficacy was above 90% and residual enzyme was undetect-able (αprescribed minimum ofα-galactosidase detection with indirect ELISA was 1 ng/ml) .Conclusion It is feasible to efficiently removeα-Gal antigen under these enzymatic and washing conditions, and a method of producing low-immunoge-nicity pig skin dressing for burn is established.

Objective To explore the clinical efficacy of laparoscopic inguinal hernia repair (LIHR) in elderly patients.Methods The retrospective cohort study was adopted.The clinical data of 3 203 patients with inguinal hernias (3 847 sides) who were adnitted to the Ruijin Hospital of Shanghai Jiaotong University School of Medicine between January 2001 and December 2013 were collected.Of 3 203 patients,979 (1 107 sides) with age ＜ 60 years and 2 224 (2 740 sides) with age ≥ 60 years were respectively allocated into the under 60 years group and 60 years or older group.The surgical procedures including transabdominal preperitoneal (TAPP) approach,total extraperitoneal (TEP) approach and intraperitoneal onlay mesh (IPOM) approach were selected and performed by doctors in the same team.There were light-weight and heavy-weight patches.Observation indicators included (1) overall operation situations,(2) surgical comparison between the 2 groups,(3)comparison of postoperative indicators between the 2 groups,(4) follow-up.Follow-up using telephone interview and outpatient examination was performed to detect the recovery time of non-restricted activity,recurrence of hernia and complications.Measurement data with normal distribution were represented as ~ ± s and comparison between groups was done by the t test.Comparisons of count data were analyzed using the chi-square test or Fisher exact probability.Ranked data were compared by the nonparametric rank sum test.Results (1) Overall operation situations:3 203 patients with inguinal hernias (3 847 sides) underwent LIHR,including 1 475 (1 677 sides) using TAPP approach,1 718 (2 154 sides) using TEP approach and 10 (16 sides) using IPOM approach (6 using TAPP and IOPM approaches in each side).The light-weight patch was used in 2 206 sides and heavy-weight patch was used in 1 641 sides.Operation time was (31 ± 12) minutes in all 3 203 patients,(27 ±9)minutes in 2 559 patients with unilateral hernia and (44 ± 12)minutes in 644 patients with bilateral hernia,respectively.Duration of postoperative hospital stay was (1.5 ± 1.2) days.(2) Surgical comparison between the 2 groups:TAPP approach,TEP approach,IPOM approach,light-weight patch and heavy-weight patch were performed to 567,538,2,751,356 sides in the under 60 years group and 1 110,1 616,14,1 455,1 285 sides in the 60 years or older group,respectively,with statistically significant differences in above indicators between the 2 groups (X2 =37.976,70.022,P ＜ 0.05).Operation time in unilateral hernia and bilateral hernia and total operation time were (27 ± 9)minutes,(42 ± 10)minutes,(29 ± 10)minutes in the under 60 years group and (27 ± 10)minutes,(44 ± 12)minutes,(3 1 ± 13)minutes in the 60 years or older group,respectively,with no statistically significant difference between the 2 groups (t =-0.106,-1.768,-4.445,P ＞ 0.05).(3) Comparison of postoperative indicators between the 2 groups:the pain score at postoperative day 1 and duration of postoperative hospital stay were 2.4 ± 1.1,(1.5 ± 1.1) days in the under 60 years group and 2.3 ± 1.0,(1.5 ± 1.3) days in the 60 years or older group,respectively,with no statistically significant difference between the 2 groups (t =1.419,-0.126,P ＞0.05).(4) Follow-up:all the patients were followed up for 23-60 months,with a median time of 43 months.Cases with non-restricted activity recovery at postoperative week 2 and 4 were 973,978 in the under 60 years group and 2 208,2 222 in the 60 years or older group,respectively,showing no statistically significant difference between the 2 groups (X2=0.113,P ＞0.05).The recurrence of hernia,severe complications,serum tumescence,paresthesia and enteroparalysis were detected in 1,0,49,5,1 sides in the under 60 years group and 11,3,132,16,2 sides in the 60 years or older group,respectively,with no statistically significant difference between the 2 groups (x2=1.556,0.269,0.254,P ＞ 0.05).The urinary retention in the under 60 years group and 60 years or older group was respectively detected in 6 and 44 sides,showing a statistically significant difference between 2 groups (x2=6.956,P ＜ 0.05).Conclusion LIHR is safe and effective in elderly patients,and it can achieve good clinical efficacy under selecting reasonable operation procedures and patches.

In the basis of a large amount of literatures, this article sumed up the characteristics and application of eggshell membrane.

Objective To investigate the clinical efficacy of hemocoagulase for severe hemorrhagic cystitis (HC) following allogeneic hemotopoietic stem cell transplantation (HSCT). Methods Twenty patients undergoing allogeneic HSCT developed severe HC with an onset time of 14 to 70 days, all patients received the treatment of hemocoagulase (1 U ivgtt q12 h × 5 d). The urine speciments reserved before and after hemocoagulase were examined by naked eye and microscope to evaluate the efficacy. Results Twenty patients received the treatment of hemocoagulase. The HC was cured in 18 patients, improved in 1 patient and uncontrolled in 1 patient. For the patients with response, macroscopic hematuria disappeared at a median of 28 days (4-127 days) after the treatment. All procedures were tolerated well and no severe adverse effect was observed. Conclusion Hemocoagulase seems to be a safe and effective drug for severe HC following HSCT.

To investigate the delivery mechanism of micro-porous osmotic pump tablets ( MPOP), taking tramadol hydrochloride ( TR) as the model drug, tramadol hydrochloride micro-porous osmotic pump tablets (TR MPOP) were prepared with compressible starch as diluent, cellulose acetate as coating material, polyethylene glycol 400 as pore-forming agents. The equilibrium solubility and osmolality of TR were determined. The effects of fillers in tablet cores, coating levels, and osmotic pressures of release media on expansion behavior of preparations were described. The influences of the category, osmolality, and pH value of release media, release methods, and release conditions on release curves of tablets were evaluated. Based on several models, the delivery pattern of TR MPOP was fitted. The equilibrium solubility in water and osmolality of TR were (775.8 +/- 17.7) g x L(-1) and 4.036 Osmol x kg(-1), respectively. During the drug-release period, it was observed that the tablets expanded markedly in response to the expansion characteristics of compressible starch and the osmotic pressure difference across the membrane. When osmotic pressure of release media increased, the significant change of the equilibrium solubility of TR was not found, but the release rates of TR MPOP decreased significantly. The delivery rate was not influenced by the pH of release mediums, dissolution methods and paddle stirring rates. The drug release profile conformed to the model of zero order in 8 h. The pore-forming agents were dissolved in release medium, which caused micro-pores. The expansion of tablets made the size of micropores bigger, and then the drug-releasing pores were obtained. It was proved that the drivers of drug delivering from TR MPOP were mainly the difference of osmotic pressure, and secondly the difference of solubility. TR MPOP were the controlled-release preparation.
Analgesics, Opioid ; administration & dosage ; chemistry ; Cellulose ; analogs & derivatives ; chemistry ; Delayed-Action Preparations ; Drug Carriers ; Drug Delivery Systems ; methods ; Drug Stability ; Osmosis ; Osmotic Pressure ; Polyethylene Glycols ; chemistry ; Porosity ; Solubility ; Starch ; chemistry ; Tablets, Enteric-Coated ; Technology, Pharmaceutical ; methods ; Tramadol ; administration & dosage ; chemistry ; pharmacokinetics

OBJECTIVETo observe the effect of Yirong Oral Liquid (YROL) on reperfusion injury in rats with cerebral infarction undergoing thrombolysis.

METHODSClinical reperfusion under thrombolysis was simulated by applying thrombolysis on reversible local cerebral ischemic rat model. In the rat model, effect of YROL on parameters concerning anti-oxidation capability, cerebral edema and ultrastructure of brain were observed.

RESULTSYROL could alleviate the cerebral edema after reperfusion, markedly increase the activity of superoxide dismutase in blood plasma, decrease the content of malonyldialdehyde, inhibit the post-reperfusion lipid peroxidation, and significantly reduce the ischemia/reperfusion injury of nerve cells in brain of rat.

CONCLUSIONYROL has definite protecting effect on brain.

Animals ; Cerebral Infarction ; drug therapy ; pathology ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Male ; Malondialdehyde ; blood ; Neuroprotective Agents ; therapeutic use ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Superoxide Dismutase ; blood ; Thrombolytic Therapy ; Urokinase-Type Plasminogen Activator ; therapeutic use

Objective:To investigate the effects and potential mechanisms of resveratrol on obesity in elderly mice.Methods:In this study, 3 groups were randomly formed for 32-week-old mice and for 48-week-old mice.The normal diet group received regular chow and 0.3 ml saline by gavage once a day, the high-fat diet group received a high-fat diet(containing 21% fat and 1.25% cholesterol)and 0.3 ml saline once a day, and the high-fat diet plus resveratrol group received a high-fat diet and resveratrol(22.4 mg/kg, dispersed in 0.3 ml saline)by gavage once a day.After 12 weeks, body weight and adipose tissues were measured.Plasma leptin concentrations were determined by an enzyme-linked immunosorbent assay(ELISA), and values for hypertrophic obesity-related indexes of mice were obtained by quantitative real-time PCR.Results:The body weight and the proportion of subcutaneous fat tissues were lower in the high-fat diet plus resveratrol group than in the high-fat diet group[(34.43±3.23)g vs.(53.16±2.16)g, (3.21±1.58)% vs.(4.86±0.64)%, P<0.01], and were similar to those in the normal diet group.Resveratrol had a more obvious inhibitory effect on leptin in elderly mice than in middle-aged mice.In elderly mice, the plasma leptin concentration was lower in the high-fat diet plus resveratrol group than in the high-fat diet group[(0.015±0.009)g/L vs.(0.100±0.027)g/L]and the normal diet group( F=19.85, P=0.001), and it was similar to that in the middle-aged mice on a normal diet.Resveratrol significantly increased the expression of peroxisome proliferator-activated receptor gamma(PPARγ)and glucose transporter 4(GLUT4)and reduced the expression of tumor necrosis factor-α(TNF-α)( F=10.79, 9.31 and 7.02, P=0.003, 0.006 and 0.010). Conclusions:Resveratrol can significantly improve hypertrophic obesity in elderly mice, and the inhibition of leptin secretion and up-regulation of PPARγ may be the key mechanisms.

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.
Actins ; chemistry ; Animals ; Cell Adhesion ; Cell Movement ; Cells, Cultured ; Collagen ; chemistry ; Drug Combinations ; Extracellular Matrix ; chemistry ; Laminin ; chemistry ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Proteoglycans ; chemistry ; Pulmonary Artery ; cytology ; Rats

OBJECTIVETo explore the effects of safflower injection on prevention and treatment of hypoxic pulmonary hypertension and clarify the function of the endoplasmic reticulum stress apoptosis pathway during the process.

METHODSThirty male SD rats were randomly grouped as normal control group, hypoxia-hypercapnia group and hypoxia+safflower group. The latter two groups were put in the cabin with oxygen concentration ranged from 9% to 11% and carbon dioxide concentration from 5% to 6%. The pulmonary artery pressure and the index of right ventricular hypertrophy were determined after hypoxia exposure (8 h/dx28 d). Changes in morphology of lung tissue were observed by electron microscopy. To explore the possible mechanisms, we also detected apoptosis and apoptosis-related genes/proteins in lung tissue by TUNEL reactivity and PCR and Western blot.

RESULTSCompared with the normal control group, pulmonary artery pressure and the index of right ventricular hypertrophy in hypoxia group were 45% and 33.4% higher, respectively. Tiny blood vessel wall of lungs was thickened and edema, and proliferation of collagen fibers was obvious under the electron microscope. TUNEL staining of apoptotic cells in lung tissues showed more high brightness green fluorescence (+-++), but less green fluorescence showed in the pulmonary vascular smooth muscle cell layer, and apoptosis index (AI) value was 150% higher; gene and protein expression levels of endoplasmic reticulum stress pathway were increased. Compared with hypoxia-hypercapnia group, pulmonary artery pressure and the index of right ventricular hypertrophy in the hypoxia+safflower group were 18% and 15.6% lower, respectively; collagen fibers were decreased, and smooth muscle cells and epithelial cells were got apoptotic-like changes under the electron microscope. TUNEL staining of apoptotic cells in lung tissues showed brighter green fluorescence (++-+++); the high brightness green fluorescence showed in pulmonary vascular smooth muscle cell layer, and apoptotic index (Al) value was 40% higher; gene and protein expressions of endoplasmic reticulum stress pathway were significantly upregulated.

CONCLUSIONOur findings demonstrate that safflower injection could activate endoplasmic reticulum stress-induced apoptosis and especially promote apoptosis in pulmonary vascular smooth muscle cells.

Animals ; Apoptosis ; drug effects ; Carthamus tinctorius ; chemistry ; Endoplasmic Reticulum Stress ; drug effects ; Hypercapnia ; physiopathology ; Hypertension, Pulmonary ; drug therapy ; physiopathology ; Hypoxia ; physiopathology ; Lung ; cytology ; physiopathology ; Male ; Myocytes, Smooth Muscle ; drug effects ; Rats ; Rats, Sprague-Dawley