ObjectiveTo investigate the effect of immunoglobulin G (IgG) extravasated from blood circulation on the expression of toll-like receptor 4 (TLR4) induced by peripheral lipopolysaccharide (LPS) in rat brain. Methods The rats were divided into four groups in random, 5 rats in each. Group one received LPS 100μg/kg by intraperitoneal administration, normal saline was given by intravenous injection 6 hours later; group two was injected with adrenalin (AD) 15μg/kg intravenously; group three was treated with LPS intraperitoneally, AD was injected 6 hours later; group four was injected normal saline intravenously as control. For all groups, the animals were sacrificed 30 min after the last injection, and the brains were taken for investigation of the TLR4 expressions by immunofluorescence staining and RT-PCR. Result Immunofluorescence staining showed that IgG immunoreactive product was patch-like, distributed in the brain parenchyma in all the animals that received AD. In the LPS+normal saline group, IgG was found merely around the blood vessels. Meanwhile, in LPS+AD animals, TLR4 immunoreactive product coexisted with microglia marker Iba-1 within the IgG extravasated area. The double-labeled cells dispersed in the brain parenchyma and near to the cerebral vessels. In the LPS+saline group, TLR4 positive cells were endothelial-like. RT-PCR results indicated that the expression level of TLR4 in the LPS+AD group were significantly higher than that in the LPS+saline group or AD group or the saline control (P<0.01). Conclusion Extravasated circulating IgG may enhance the TLR4 expression in the rat brain induced by peripheral LPS.

Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.

BACKGROUND: To avoid acute rejection,it is necessary to use imunosuppressive drug regimen for long term to control immune state.However,imunosuppressive drug regimen of allogenic organ transplantation increases infection incidence of recipients,and induction of allograft immunological tolerance might be an ideal method for solving these problems.The long-term immunologic tolerance has been able to be induced in the experimental rodent models.Among these protocols,donor bone marrow cell (DBMC) infusion exerts an important role in the induction of allograft immunological tolerance.OBJECTIVE: To investigate effects of combination of cyclosporine A (CsA) with DBMC infusion on heterotopic rat cardiac allograft survival time.DESIDN: A randomized controlled animal experiment.SETTING: Renal Disease Center,First Affiliated Hospital of Zhejing University School of Medicine.MATERIALS: This study was performed at the Laboratory Animal Center,Zhejiang University School of Medicine between March 2002 and December 2005.Inbred male Lewis rats (n=40,serving as donors) and male BN rats (n=60,serving as recipients) of SPF grade were used in this study.The protocol was approved by the Hospital's Ethic's Committee.METHODS: Forty rats prepared for heterotopic rat cardiac allograft were randomly divided into 4 groups,with 10 rats in each: control group,in which,rats received no treatment,CsA group,in which,rats received CsA infusion for 7 days successively; CsA +DBMC group,in which,rats received DBMCs during and 6 days after the surgery and additional 7 successive days of CsA infusion,and a DBMC group,in which,rats received DBMCs infusion during and 6 days after the surgery.In addition,BN rats that received beterotopic rat cardiac allograft served BN controls.The survival time of heteroropic rat cardiac allograft was investigated.Serum interleukin-2 level and tumor necrosis factor-α mRNA expression level in the transplanted cardiac allograft were measured. The percentage of antigen presenting cells (APC) from donor,CD3+CD25+ cells,CD4+CD25+ cells,CD86+ cells,and the ratio for CD4+CD45RC+ and CD4+CD45RC- in the recipient peripheral blood karyocytes were measured by flow cytometry 6,12 and 18 days after surgery.MAIN OUTCOME MEASURES: The survival time of beteruropic rat cardiac allograft,serum interleukin-2 (IL-2)level,tumor necrosis factor- α (TNF- α ) rnRNA expression level, rejection grading,the percentage of DBMCs in the recipient peripheral blood karyocytes,CD3+CD25+ cells,and CD4+CD25+ cells,as well as CD86 expression,and the ratio for CD4+CD45RC+ and CD4+CD45RC.RESULTS: Forty Lewis male rats and sixty male BN rats were all included in the final analysis. The heterotopic rat cardiac allograft survival time was longer in the CsA +DBMC group than in the control group and DBMC group (P ＜ 0.05). Serum IL-2 level and TNF- α mRNA expression were respectively lower in the CsA +DBMC group than in the control group and DBMC group ( P ＜ 0.05).The rejection was milder in the CsA +DBMC group than in the remaining 3 transplantation groups.In the CsA +DBMC group,CD 86 expression in the recipient peripheral blood karyocytes was markedly inhibited,and 6 and 12 days after surgery,the ratio for CD4+CD45RC+ and CD4+CD45RC- and the percentage of CD3+CD25+ were respectively lower compared to control group and DBMC group.DBMCs in the recipient peripheral blood karyocytes were more in rats that received DBMC infusion compared to rats that received no BDMC infusion.CONCLUSION: Short-term CsA treatment combined with DBMC infusion can lower acute rejection of heterotopic rat cardiac allograft and prolongssurvival time of cardiac allograft.

Objective To evaluate two methods of selecting region of interest (ROI)to measure the apparent diffusion coefficient (ADC)of breast lesions on diffusion-weighted imaging (DWI).Methods MRI findings (including plain MRI,dynamic contrast-en-hanced MRI and DWI)of 5 3 cases with breast mass confirmed by pathology were analyzed retrospectively.Two observers independ-ently measured the ADC of breast lesions by the method of fixed ROI with the size of 10 mm2 ±2 mm2 and the routine method.The mean ADCs of benign and malignant breast lesions were compared between two methods and two observers by using paired samples T-test.The values of ADC diagnosing breast cancer were compared between the two methods by using the receiver operating charac-teristic (ROC)curve.Results Among 62 lesions of 53 cases,33 lesions were malignant,and 29 lesions were benign.For the first observer,the mean ADCs of benign and malignant lesions were (1.647±0.359)×10-3 mm2/s and (1.136±0.227)×10-3 mm2/s measured by the fixed ROI method,respectively.The area under the ROC was 0.897.The mean ADCs of benign and malignant le-sions were (1.603±0.415)×10-3mm2/s and (1.251±0.237)×10-3 mm2/s measured by the routine method,respectively.The area under the ROC was 0.768.For another observer,the mean ADCs of benign and malignant lesions were (1.647±0.357)× 10-3 mm2/s and (1.130±0.250)×10-3 mm2/s measured by the fixed ROI method,respectively.The mean ADCs of benign and malignant lesions were (1.590± 0.390)×10-3 mm2/s and (1.250±0.237)×10-3 mm2/s measured by the routine method,respec-tively.There was no significant difference for the mean ADC of benign lesions between the two methods.However,there was sig-nificant difference for the mean ADC of malignant lesions between the two methods.The diagnostic value of ADC measured by the fixing method was better than that of the routine method (P=0.008).There were no significant differences for ADCs of benign and malignant lesions between the two observers.Conclusion DWI has an important value for differential diagnosis of benign and malignant breast lesions.The measurement of ADC by the fixed and smaller ROI is better than that by the routine ROI for dis-tinguishing malignant from benign breast lesions.

Objective To describe a novel surgical technique for male long-segment urethral stric- ture after pelvic trauma using the intact and pedieled pendulous urethra to replace the bulbar and membra- nous urethra,and then reconstructing anterior urethra.Methods Three patients with long-segment post- traumatic bulbar and membranous urethral strictures with short left pendulous urethras who had undergone several failed previous surgeries were treated with staged pendulous-prostatic anastomotic urethroplasty fol- lowed by reconstruction of the anterior urethra.This procedure was divided into 3 stages.The first-stage sur- gery was mobilization of anterior urethra down to the coronary sulcus and then re-routing the prostatic urethra followed by pendulous-prostatic anastomotic urethroplasty with transposition of penis to perineum.The sec- ond-stage surgery was transecting the anterior urethra at the site of coronary sulcus 6 months later when it was re-vaseularized,then straightening the penis and performing urethroperineostomy.The third-stage surgery was reconstruction of anterior urethra 6 months later.Results Case 1 reported satisfactory voiding postopera- tively.Retrograde urethrography showed that the urethra was patent with no post-voiding residual urine (PVR),and bilateral vesicoureteral reflux almost disappeared.The Qmax was 18.8ml/s,and 18ml/s after the third stage surgery and at 2-year follow-up.Case 2 also had satisfactory voiding.A 22F urethral catheter could smoothly pass through the urethra,and Qmax was 19.5 ml/s with no PVR at 2-year follow-up.Case 3 underwent the first stage surgery through perineal and pubic routes.The urethrorectal and urethroperineal fis- tulas were excised and repaired simultaneously.After operation the fistulas healed,but the stenostomia resul- ting from wound infection needed further treatment.Conclusions This procedure is effective for men with complex long-segment post-traumatic bulbar and membranous urethral strictures,especially for those undergo- ing failed previous surgical treatment.

Sanjie Zhentong capsules were scanned by using a near infrared spectra probe with different drug mass fraction and the spectral information of capsule shells and contents in it were obtained. Then partial least squares (PLS) models were developed for the prediction of mass fraction of Fritillariae Thunbergii Bulbus and Resine draconis in Sanjie Zhentong capsules. The correlation coefficient (r9c)) and root mean standard error( RMSEC) of 0.949 5, 0.958 2 and 4.742 4, 4.135 7. The models obtained correlation coefficient (r(v)) of 0.919 2, 0.936 7 and root mean square error (RMSECV) of 6.158 9, 5.037 3 respectively in the training set. The paired T test analysis of statistics showed that there were no significant difference between predictive values and measure values. The established models reflected a strong prediction performance and can meet the needs of the production.
Capsules ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Least-Squares Analysis ; Spectroscopy, Near-Infrared ; methods

This study is to analyze and identify the water soluble components of water buffalo horn (Bubali Cornu, WBH), and also establish a method for investigating these components. Shotgun proteomic analysis identified proteins in WBH aqueous extraction: keratin, collagen, desmoglein, etc. Ultrafiltration and LC-MS/MS were used to separate and identify the peptides in WBH aqueous extract, as a result, identified peptides were mainly derived from nonspecific degradation products of keratin and collagen, which including C-terminal peptides and non C-terminal peptides. Hypoxanthine, uridine, guanosine, and adenosine were identified by comparing with the standards. The strategy in present study could be used in analyzing water soluble components of animal horn derived TCM. It provides a reference for investigation of the material basis of animal horn derived TCM.
Animals ; Buffaloes ; Chromatography, Liquid ; Guanosine ; Horns ; chemistry ; Hypoxanthine ; Mass Spectrometry ; Medicine, Chinese Traditional ; Peptides ; Proteomics ; Tandem Mass Spectrometry ; Uridine

Objective To investigate the ability of single photon emission computed tomography (SPECT) and MRI in detecting skull-base invasion in nasopharyngeal carcinoma. Methods Sixty-one patients with nasopharyngeal carcinoma received whole body and skull-base tomography SPECT, and nasopharynx and skull-base MRI before radiotherapy. The results were double-blind compared and evaluated. Results The overall positive rates of skull-base invasion detected by SPECT and MRI were 51% and 46% (P=0.508). In paitents with headache, cranial nerve palsy or both, the rates were 83% and 86% (P=1.000) ,80% and 80% (P=1.000), 88% and 94% (P=1.000), respectively. In patients with T1+T2 and T3+T4lesions,the rates were 22% and 0(P=0.031) ,74% and 82% (P=0.250) ,repectively. In patients with N0+N1and N2+N3lesions,they were 50% and 48% (P=1.000) ,53% and 40% (P=0.500) ,respectively. The conformation rate between SPECT and MRI was 85%. Binary Logistic regression analysis showed that T stage was a risk factor for positive SPECT(χ2=4.23,P=0.040, OR=3.04). Headache tended to be a risk factor for both positive SPECT and positive MRI (χ2=3.13, P=0.077, OR=4.54;χ2=3.64,P=0.056,OR=12.00). Conclusions The detection sensitivity of SPECT in skull-base invasion in nasopharyngeal carcinoma is equivalent to that of MRI. The consistency between SPECT and MRI is good. Moreover, there is a good correlation between SPECT and symptoms, signs and stage. SPECT of skullbase tomography is necessary for patients with severe headache, negative CT and those who can not receive MRI. When SPECT result is positive,skull-base should be considered to be invaded and should be defined as gross tumor volume in radiotherapy planning.

BackgroundGlaucoma associates with apoptosis of retinal ganglion cells ( RGCs).Research showed that ciliary neurotrophic factor (CNTF) can repair optic nerve trauma,but it has not reported whether CNTF has a protective effect on glaucomatous optic neuropathy.ObjectiveThis experimental study was to explore the protective effect of CNTF on RGCs in rat eyes with acute ocular hypertension.MethodsOcular acute hypertension models were induced in bilateral eyes of 24 clean Wistar rats by forced perfusion of a balanced salt solution into the anterior chamber.Two days before molding,0.5 μg recombinant human CNTF (5 μl) was injected intravitreously in the left eyes,and 5 mmol/L sodium phosphate solution (5 μl) was injected in the same way as the control group.Three other Wistar rats were used as the normal control group.The animals were sacrificed by excessive anesthesia and the retinal sections were prepared 1,3,7,14 days after molding.The morphology of the retina was examined and RGCs counting was performed by hematoxylin & eosin staining and observed under a light microscope.The expression of glutamic acid in RGCs was assessed by immunochemistry.ResultsRegular retinal structure with clearly defined cell layers were observed in normal control rats.Changes in vacuoles in RGCs were seen in the model control group.The number of degenerative RGCs decreased in the CNTF group.Compared with the model control group,number of normal RGCs increased from 1 day to 14 days after molding with a significant difference between the two groups (all P=0.000).Immunochemistry assay showed that the numbers of positive cells for glutamic acid were 5.50±1.04 and 6.00±1.41 for the average of 3 fields at 3 days or 7 days in the CNTF group,and those in the model control group were 9.00±2.91 and 10.83 ± 1.94,respectively,showing significant differences between them ( all P =0.000 ).However,no comparable differences were found in the numbers of positive cells for glutamic acid at 1 day and 14 days between the two groups( P=0.578,0.180).ConclusionsCNTF down-regulates the expression of glutamic acid in RGCs and offers neuroprotection for RGCs in an acute glaucoma rat model.

Objective To evaluate the efficacy of lateral femoral approach to continuous sciatic nerve block for patient-controlled analgesia after foot and ankle surgery.Methods One hundred American Society of Anesthesiologists physical status Ⅰ-Ⅲ patients of both sexes,aged 18-60 yr,weighing 50-100 kg,with body height of 145-190 cm,scheduled for elective foot and ankle surgery,were divided into 2 groups (u =50 each) using a random number table:patient-controlled nerve block analgesia (PCNA) group and patient-controlled intravenous analgesia (PCIA) group.In group PCNA,the lateral femoral approach to sciatic nerve block was performed under the guidance of ultrasound and a neurostimulator,0.2％ ropivacaine 20 ml was injected after successful location,the catheter was inserted,and 0.2％ ropivacaine 10 ml was injected again.In group PCIA,0.2％ ropivacaine 30 ml was injected after successful location of the sciatic nerve.General anesthesia was performed using laryngeal mask airway in both groups.In group PCNA,PCNA was performed with 0.2％ ropivacaine (diluted to 200 ml in normal saline) at the end of surgery,and the PCNA pump was set up with a 0.5 ml bolus dose,a 15-min lockout interval and background infusion at a rate of 5 ml/h.In group PCIA,PCIA was performed with sufentanil 100 μg,tramadol 500 mg and tropisetron 10 mg (diluted to 200 ml in normal saline) at the end of surgery,and the PCIA pump was set up with a 0.5 ml bolus dose,a 15-min lockout interval and background infusion at a rate of 2 ml/h.The visual analog scale score was maintained≤ 3,and postoperative analgesia lasted until postopera-tive 72 h.When visual analog scale scores ≥ 4,tramadol 100 mg was intramuscularly injected as rescue analgesic.The requirement for rescue analgesia and development of adverse effects such as nausea and vomiting,insomnia,puncture site infection and bleeding were recorded within 72 h after surgery.Results The requirement for rescue analgesia and incidence of nausea and vomiting were significantly lower in group PCNA than in group PCIA (P＜ 0.05).Conclusion The lateral femoral approach to continuous sciatic nerve block can be safely and effectively used for patient-controlled analgesia after foot and ankle surgery.