2.Soluble Expression and Purification of Snake Venoms Fihrino(geno)lytic Emzyme Alfimeprase in E.coli
Shou-Tao ZHANG ; Yan-Sheng ZHOU ; Xue-Hua LAI ; Xing-Feng BAO ; Ai-Guang GUO ;
China Biotechnology 2006;0(03):-
Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.
3.Studies on Difference between Sporoderm-broken and Nonbroken Spores of Ganoderma lucidum (Leyss. ex Fr.) Karst.by Polysaccharide Analysis
China Journal of Chinese Materia Medica 2001;26(5):326-328
Objective:To compare the release ability of water-soluble polysaccharides in sporoderm-broken and nonbroken spores of Ganoderma lucidum, and establish a comparatively correct method for the determination and analysis of polysaccharide contents in Chinese herbs.Method:The release ability of water-soluble polysaccharides was determined on the basis of phenol-sulfuric acid modification in different conditions.Result:The release ability of polysaccharides of sporoderm-broken spores was much grerter than that of nonbroken spores; and the phenol-sulfuric acid modified cation method proved excellent in accuracy and reproducibility, with a relative error less than 1.5%.Conclusion:The spores should be wall-wracked if used as a nutriment, or for extraction and analysis of their effective components. The method can be successfully used for the determination of polysaccharide contents in Chinese herbs or nutriments.
4.The level of serum lipids,body-fat content of obesity rats,and the relationship between intracellular calciumand ventricular arrhythmia
Lin-chuan, LANG ; Xing, JIANG ; Yan-kun, HAO ; Yun-Long, BAI ; Yan-jie, LV ; Bao-feng, YANG
Chinese Journal of Endemiology 2008;27(6):632-635
Objective To study the level of serum lipids and body-fat content of high-fat diet induced pbesity rats(DIO).explore the relationship between intracellular caleium and ventrieufar arrhythmia.Methods Sixty male Sprague-Dawley(SD)rats were divided into control group(15)and experiment group(45),high-fat diet was administrated for 12 weeks to established obesity model,15 rats were selected into obesity group according to their body weight gain.The standard 2-lead electrocardiograph was used to detect the incidence and scores of arrhythmia induced by barium chloride(BaCl2,0.1 mg/kg)for 1 hour on every 8 rats from different groups respectively.Body-fat content.the level of serum total cholesterol(TC),triglyceride(TG),low density lipopmtein cholesterol (LDL-C) and high density lipopmtein cholesterol (HDL-C)were measured.The epididymal(EP),retroperitoneal (RE) and mesenteric(ME)white adipose pads was measured to obtain the body fat content.Single ventricular myrocytes of rats were isolated by enzymatic dissociation.The confocal laser scanning microscope was used to record basic intraceUular calcium level([Ca2+]i).Results The body-fat content in obesity group[(7.71±0.74)%] was significantly higher than control group[(4.69±0.37)%](t=3.650,P<0.05).The level of serum TC,TGand LDL-C were significantly higher(t=3.801,2.778,3.536.P<0.05) in obesity group[(1.26±0.04),(0.58±0.10),(0.51±0.04)mmol/L]than those in control group[(0.92±0.08),(0.29+0.03),(0.31±0.04) mmol/L].The level of serum HDL-C wa8 decreased gradually from control group[(0.53±0.05)mmol/L] toobesity group[(0.52±0.02)mmol/L],but there waft no significant difference between them(t=0.186,P>0.05).The incidence of arrhythmia induced by BaCl2(0.1 mg/kg)in obesity group was significantly higher than controlgroup(X2=5.333,P<0.05),and the scores of arrhythmla was increased in obesity group(2.5±0.6)too.The fluorescence intensity standing for[Ca2+]i was increased significantly(t=2.409,P<0.05)from obesity group(247.96±20.03)to control group (174.25±23.13).Conclusion As the free cytosolic calcium begin to accumulate,the arrhythmia morbidity is increased in obesity rats.
5.Preliminary study of an intracavitary convex array probe for detecting distal extracranial internal carotid artery by transoral carotid ultrasonography.
Xing-xing ZHOU ; Zi-hai DING ; Bao-ping WANG ; Li-hong LUO ; Feng-lin WU
Journal of Southern Medical University 2010;30(4):895-897
OBJECTIVETo explore the feasibility of using an intracavitary convex array probe for detecting the distal extracranial internal carotid artery (ICA) by transoral carotid ultrasonography (TOCU).
METHODSForty patients underwent examinations with bilateral ICA inspected with an intracavitary convex array probe by TOCU to observe the internal diameter, visible length, peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistance index (RI).
RESULTSEight of the 40 patients were excluded from the observation for the presence of carotid plaques. The examination was terminated in two patients due to sensitive throat and severe pharyngeal reflex. The rest of the patients completed the examination of the internal diameter, visible length, PSV, EDV and RI, which showed no statistically significant differences among them (P>0.05).
CONCLUSIONUsing intracavitary convex array probe, the distal extracranial ICA disease can be diagnosed with higher accuracy.
Adult ; Aged ; Carotid Artery, Internal ; diagnostic imaging ; Female ; Humans ; Image Processing, Computer-Assisted ; methods ; Male ; Middle Aged ; Mouth ; diagnostic imaging ; Ultrasonography, Doppler, Duplex ; methods
6.Partial laryngectomy for ninety-eight patients with glottic carcinoma of the larynx.
Zhi-chun HUANG ; Feng-bing ZHANG ; Jian-xing GU ; Xu FENG ; Bao-bin SUN
Chinese Journal of Oncology 2005;27(11):685-687
OBJECTIVETo investigate the therapeutic results after various kinds of partial laryngectomy for patients with glottic carcinoma of the larynx.
METHODSFrom January 1980 to Mar 2004, 98 patients with glottic carcinoma of the larynx were treated by various kinds of partial laryngectomy. The types of operation varied according to stage of the lesion: vertical or frontovertical partial laryngectomy for T1b, T2 and T1a with invasion of anterior commissura, Tuker's operation or supracricoid partial laryngectomy for T1b, T2 or those with invasion of over half of contralateral cord, and extended vertical partial laryngectomy with resection of arytenoids for T3, or with invasion of ipsilateral arytenoids cartilage without involving postcricoid mucosa or posterior commissura. Totally, vertical partial laryngectomy was carried out for 76 patients, extended vertical partial laryngectomy for 7, supracricoid partial laryngectomy for 3 and Tucker's operation for 12. One stage unilateral neck dissection was performed in 5 patients and postoperative radio-therapy was supplemented to 5 (dose 60 Gy). For laryngeal function restoration, pectoro-hyomyo-flap and cricohyoidoepi glotopexy procedures were also performed for some patients. The clinical data of these patients were retrospectively analyzed.
RESULTSThe 3- and 5-year survival rate was 86.5% (64/74) and 81.7% (49/60), respectively. The total decannulation rate was 94.9% (93/98), though fifteen patients had had symptoms of aspiration. Swallowing function of all patients recovered to the normal level. The phonation of all patients was restored to various degrees and they were able to communicate socially.
CONCLUSIONThe 3- and 5-year survival rates of partial laryngectomy for patients with glottic carcinoma of the larynx are comparable to the results achieved by total laryngectomy, but the quality of life of the patents much better. Complete resection of the tumor and simultaneous preservation of respiratory and vocal function whenever possible by partial laryngectomy is very important for improving patients' life quality. With a suitable size and good blood supply, the sternohyoid muscle flap of unipedical and half-thickness instead of whole thickness is ideal in the reconstruction of laryngeal defect after vertical partial laryngectomy.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; surgery ; Female ; Glottis ; surgery ; Humans ; Laryngeal Neoplasms ; surgery ; Laryngectomy ; methods ; Male ; Middle Aged ; Quality of Life ; Retrospective Studies ; Treatment Outcome
7.Preparation and ectopic osteoinduction study of macroporous bone substitute with calcium phosphate cements and rhBMP-2 loaded gelatin microspheres.
Meng LI ; Xu-dong LIU ; Xing-yan LIU ; Bao-feng GE
China Journal of Orthopaedics and Traumatology 2011;24(5):411-415
OBJECTIVETo prepare macroporous bone substitute composed of calcium phosphate cements and rhBMP-2 loaded gelatin microspheres, and to investigate ectopic osteoinduction of the composite.
METHODSAfter being prepared by improved emulsified cold-condensation method and crosslinked by 5% genipin solution,gelatin microspheres (GMs) were observed by scanning electron microscope (SEM) and loaded with rhBMP-2 by adsorption. Macroporous bone substitute was developed by mixing calcium phosphate cement (CPC) with 2.5% GMs, being as the experimental group,and CPC with rhBMP-2 was the control group. After the both composites had been soaked in the sodium chloride for 1 week or 3 weeks, compressive strength of the composites were tested, and the cross-sections were observed by SEM. Concentrations of rhBMP-2 in the solutions at different time by ELISA method and the cumulative drug release amount was calculated. The composites had been implanted in the muscle bags of the mouses for 3 weeks. Then the tissues around the materials were collected, stained by hematoxylin and eosin, and Ca and ALP in the tissues were also measured.
RESULTSGelatin microspheres were spherical with diameters of (62 +/- 18) microm. Macropores appeared in the experimental materials 1 week and 3 weeks after being soaked,and total porosity, macroporosity, cumulative release amount of rhBMP-2 in the experimental group were higher than that in the control. But compressive strength of the experimental group was lower than that of the control group 3 weeks after being soaked. Results of HE stain showed chondral formation in both groups, but there were more chondral tissues in the experiment group, and so were the concentrations of Ca and ALP.
CONCLUSIONMacroporous calcium phosphate cement can be prepared by using rhBMP-2 loaded gelatin microspheres, and it is an excellent bone substitute due to it's proterty of promoting rhBMP release and powerful ectopic osteoinduction.
Animals ; Bone Cements ; chemistry ; Bone Morphogenetic Protein 2 ; chemistry ; Bone Substitutes ; chemistry ; pharmacology ; Calcium Phosphates ; chemistry ; Gelatin ; chemistry ; Humans ; Male ; Mice ; Microspheres ; Osteogenesis ; drug effects ; Porosity ; Recombinant Proteins ; chemistry
8.Case-control study on bone setting manipulation for the treatment of over degree II supination-eversion fractures of ankle joint.
Yue-Feng QI ; Fa-Lin CHEN ; Shu-Ren BAO ; Cheng-Huan LI ; Xing-Wei ZHAO ; Shi-Ming LIU ; Wen-Xue CHEN ; Ye LI ; Peng WANG
China Journal of Orthopaedics and Traumatology 2012;25(8):634-638
OBJECTIVETo explore therapeutic effects of bone setting manipulation for the treatment of over degree II supination-eversion fractures of ankle,and analyze manipulative reduction mechanism.
METHODSFrom 2005 to 2008, 95 patients with over degree II supination-eversion fractures of ankle were treated respectively by manipulation and operation. There were 43 cases [11 males and 32 females with an average age of (44.95 +/- 12.65) years] in manipulation group, and 2 cases were degree II, 11 cases were degree III, and 30 cases were degree IV. There were 52 cases [21 males and 31 females with an average age of (39.96 +/- 13.28) years] in operative group,and 6 cases were degree II, 18 cases were degree III, and 28 cases were degree IV. Bone setting manipulation and hard splint external fixation were applied to manipulative group. Operative reduction internal fixation was performed in operative group. X-ray was used to evaluate reduction of fracture before and after treatment, 2 months after treatment. Ankle joint function was evaluated according to Olerud-Molander scoring system after 6 months treatment.
RESULTSAll patients were followed up with good reduction. Three cases occurred wound complication in operative group, but not in manipulative group. In manipulation group, 19 cases got excellent results, 20 cases good and 4 cases fair; while in operative group, 30 cases got excellent results, 20 cases good and 2 cases poor. There were no significant differences in fracture reduction and ankle joint function recovery between two groups (P > 0.05). Efficacy of operative treatment was better than that of manipulative treatment at degree IV fracture (P < 0.05).
CONCLUSIONBone setting manipulation is a good method for treating supination-eversion ankle joint fractures, which has advantages of simple and safe operation, reliable efficacy. For ankle join fracture at degree IV, manipulative reduction should be adopted earlier, and operative treatment also necessary
Adult ; Ankle Injuries ; physiopathology ; therapy ; Ankle Joint ; physiology ; Case-Control Studies ; Female ; Fractures, Bone ; physiopathology ; therapy ; Humans ; Male ; Manipulation, Orthopedic ; methods ; Supination
9.Detection and clinical significance of phospholipase A2 in semen of male infertile patients.
Shu-Kui WANG ; Yu-Feng HUANG ; Bao-Tong LI ; Xing-Yi XIA ; Zi-Zheng WANG
National Journal of Andrology 2003;9(2):90-93
OBJECTIVESTo explore the clinical application of anti-human seminal plasma phospholipase A2 (PLA2) monoclonal antibody (McAb) for male infertility.
METHODSEnzyme-linked immunoabsorbent assay (ELISA), immunocytochemistry(ICC), as well as flow cytometry (FCM) analysis were established using two strains anti-human seminal plasma PLA2 McAb prepared by our laboratory to detect the PLA2 content in human seminal plasma and the anterior head region of spermatozoa, respectively. Then the PLA2 content in male infertile patients were compared with that in normal control with fertility. The seminal routine analysis was performed by computer-assisted semen analysis (CASA).
RESULTSThe PLA2 content of infertile groups were (31.13 +/- 14.49) ng/ml in azoospermic patients, (17.71 +/- 12.45) ng/ml in oligospermic patients and (16.46 +/- 11.31) ng/ml in patients with normal sperm density, which were all higher than that of normal controls [(8.09 +/- 3.15) ng/ml, P < 0.01]; There was significantly negative correlation between PLA2 content in seminal plasma and sperm density(r = -0.602, P < 0.05), while there was insignificant correlation between PLA2 and sperm motility or percentage of motility. The PLA2 content in the anterior head region of spermatozoon of male infertile groups was significantly lower than that of normal controls by ICC and FCM(P < 0.01).
CONCLUSIONSPLA2 in human seminal plasma is closely related to male fertility, and the PLA2 deficiency in the head of spermatozoa may be one of the reasons causing male infertility. The methods detecting PLA2 content in seminal plasma and the head of spermatozoa can provide powerful evidences for exploring the mechanism of male infertility.
Adult ; Humans ; Infertility, Male ; enzymology ; Male ; Phospholipases A ; analysis ; Phospholipases A2 ; Semen ; enzymology
10.An analysis of surgical outcome and influencing factors in patients of congenital basilar invagination with atlantal-axial dislocation: report of 120 cases.
Xing-wen WANG ; Feng-zeng JIAN ; Zan CHEN ; Hao WU ; Yu-hai BAO
Chinese Journal of Surgery 2013;51(3):207-210
OBJECTIVETo investigate the surgical outcome and its influencing factors in patients of congenital basilar invagination (BI) with atlanto-axial dislocation (AAD).
METHODSFrom May 2004 to August 2010, 120 patients who had BI with AAD were surgically treated with direct posterior intraoperative distraction-reduction and fixation technique, 93 patients were successfully followed up by means of questionnaire survey, telephone and clinical evaluation. Pre- and postoperative dynamic cervical X-rays, computed tomographic scans, 3-dimentional reconstruction views and magnetic resonance imaging were performed. Pre- and postoperative Japanese Orthopaedic Association (JOA) score, distance between odontoid tip and Chamberlain's line and atlantodental interval were measured to evaluate the surgical result. Statistical analysis was performed by means of paired t test and Pearson Correlation analysis.
RESULTSThere were 93 cases were followed up for 24-99 months with an average of 46.5 months. Until the final follow-up, clinical symptoms were improved in 79 patients (84.9%), and were stable in 7 patients (7.5%) and deteriorated in 4 patients (4.3%). Three patients died postoperatively (3.2%). Patients without intramedullary signal intensity change (ISIC) had better surgical outcome. Patients with compression from anterior odontoid tip and posterior bone margin of occipital foramen had the worst surgical outcome (F = 3.987, P < 0.01). Overall, good decompression and bone fusion were shown on postoperative image in 87 patients (93.5%). There were 3 deaths in this series because of basilar artery thrombosis, posterior fossa hematoma and unknown reasons each.
CONCLUSIONSThe direct posterior intraoperative distraction-reduction and fixation technique is an effective simple and safe method for the treatment of BI with AAD. Anterior compression from odontoid tip and posterior compression from bone margin of occipital foramen-atlantal posterior arch play important roles in its developing mechanism. ISIC on MRI is a predictive factor for the worse surgical outcome.
Adolescent ; Adult ; Atlanto-Axial Joint ; surgery ; Bone Screws ; Child ; Decompression, Surgical ; Female ; Follow-Up Studies ; Humans ; Joint Dislocations ; complications ; surgery ; Male ; Middle Aged ; Platybasia ; complications ; surgery ; Root Cause Analysis ; Spinal Fusion ; methods ; Young Adult