Objective:To evaluate the effect of iliac bone graft and radial forearm flap in the reconstrucion of maxilla.Methods:Maxilla defects were reconstructed using iliac bone graft and radial forearm flap in 4 patients.The effects were evaluated clinicaly.Results:In all the 4 cases,palatal defects resulted from maxillectomy were optimally reconstructed with non-vascularized iliac graft and radial forearm flap.The masticatory function of the upper jaw,intelligible speech,swallow and natural facial appearance were recovered.As a result,quality of life of the patients was improved.Conclusion:Iliac bone graft and radial forearm are feasible in the reconstruction of maxilla defects.

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective:To explore the effects of cysteine-rich 6 1 (Cyr6 1 )on biological behavior of human adenoid cystic carcinoma ACC-LM and ACC2 cells.Methods:The chemically synthesized Cyr6 1-siRNA was transfected into ACC-LM and ACC2 cells.Cell proliferation was measured by the MTT method,the invasive ability was evaluated by Transwell chamber assay,and cell apoptosis was analyzed using flow cytometry by double staining with Annexin V and propidium iodide.Results:Cyr61-siRNA significantly down-regu-lated Cyr61 protein expression in ACC-LMand ACC2 cells.Cyr61-siRNA markedly inhibited the proliferation and invasion of the cells, however,there was no significant difference in cell apoptosis between Cyr6 1-siRNA and control groups.Conclusion:Cyr6 1 promote the proliferation and invasion of adenoid cystic cancer cells.

Objective:To explore the effect of siRNA targeting myeloid cell leukemia-1(Mcl-1)on the biological behavior of salivary adenoid cystic carcinoma cells.Methods:The chemically synthesized Mcl-1-siRNA was transfected into salivary adenoid cystic carci-noma SACC-2 cells.The expression levels of Mcl-1-mRNA and Mcl-1protein were examined by Real-time PCR and western blotting respectively.MTT assay,transwell chamber and flow cytometry were used to determine the effect of Mcl-1-siRNA on SACC-2 cell pro-liferation,migration and apoptosis.Results:Compared with the control group,liposome group and NC-siRNA group,SACC-2 cell proliferation rate of Mcl-1-siRNA group was obviously slowed down.48 h after transfection,the migration of SACC-2 cells in Mcl-1-siRNA group(39 ±9.0)were lower than that in control group(69 ±6.0).The apoptosis rate of Mcl-1-siRNA group(8.6%)was sig-nificantly higher than that in control group(1.9%).Conclusion:Silence Mcl-1 can inhibit cell proliferation and migration and pro-mote apoptosis of salivary adenoid cystic carcinoma cells.

Objective To establish a brand new model of bladder outflow obstruction (BOO) at single cell level in vitro to offer a more stable and scientific experimental base. Methods Recurrent mechanical stretch generated by vacuum facility was applied to cells attached to flexible membrane of special culture plate, which led to 10%, 20% and 30% elongation of them. Immunocytochemistry was used to analyze the expression of ?-actin (sign of contractile phenotype) and RT-PCR was performed to detect the dynamic changes of mRNA expression of PCNA and Western blotting to protein expression of PCNA and cdk2 kinase. MTT assay was used to observe the changes of proliferation of cells. Results In the case of gradual elongation, the expression of ?-actin rose at first and fell afterwards; the mRNA expression of PCNA and protein expression of PCNA and cdk2 kinase rose gradually. Changes in 30% elongation group were greater than the control (P

Objective To establish a stable animal model of bladder outlet obstruction(BOO) and to clarify the effects of BOO on the biomechanical properties of detrusor and the mechanisms. Methods BOO animal model was established by partial ligation of the proximal urethra of male Wistar rats forming, a urethra stricture with internal diameter of 1 mm. The active contraction of detrusor muscle stripes to carbachol was recorded with transfusor and organ bath tube. The compliance and maximum volume of bladder were examined by filling cystometry. Results The bladders demonstrated tropical post obstruction alterations at week 2 after partial ligation of the proximal urethra. The maximum volume (3 3?1 9) ml and bladder wet weight (10 4?2 8) g increased significantly in BOO group as compared with those in sham operation group [(1 6?0 8)ml and (7 2?3 5)g]. The detrusor contractile force of detrusor instability(DI) group was significantly lower than that in the control group. However, the contractile force in detrusor stability (DS) group was higher at week 2 but lower at week 4 after BOO than that in the sham operation group. Conclusion BOO animal model established by partial ligation of the proximal urethra of adult rats is simple and can give high survival rate and good stability, suitable for the studies of bladder outlet obstruction. There are two types of the detrusor contraction after BOO: DI group with impaired contraction and DS group with bidirectional change which was higher at early stage(week 2) but lower at later stage(week 4). The changes of bladder compliance are related to the stability of bladder, decrease in DI but increase in DS.

Objective To provide experimental basis for further study of the mechanism of urinary storage dysfunction of diabetic cystopathy (DCP). Methods Steptozotocin(STZ) induced and sucrose induced diabetic rats were employed as DCP and diuresis models. Normal rats served as the control. The changes of calcitonin gene related peptide(CGRP) were analyzed by immunocytochemical method and computer assisted image analysis software. Compliance of the whole bladder prepared in vitro was evaluated by bladder irrigation. The messenger molecule cAMP of detrusor relaxation was determined by radioimmunological assay. Results Compared with those in other groups, the CGRP immunoreactive positive nerve fibers and content of this neurotransmitter significantly decreased in the bladder wall, especially in the submucosa. Significantly higher bladder compliance was observed( P

Four salts of ticagrelor, ticagrelor-3,5-dinitrobenzoic acid, ticagrelor-pyrazinamide, ticagrelor-D-proline and ticagrelor-L-proline were prepared by solvent suspension and liquid-assisted grinding to improve the solubility of ticagrelor. The compounds were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, nuclear magnetic resonance spectroscopy, elemental analysis, and the intermolecular salt-bonding forces were analyzed. The equilibrium solubility of salts and pure drug in hydrochloride buffer pH 1.2 and phosphate buffer pH 6.8 were measured by high-performance liquid chromatography. Ticagrelor was salted with 3,5-dinitrobenzoic acid, pyrazinamide, D-proline, L-proline all in a stoichiometric ratio of 1∶1; with the exception of ticagrelor-D-proline, the solubility of the other three salts provided significantly improved solubility in hydrochloride buffer pH 1.2, and the equilibrium solubility of ticagrelor-3,5-dinitrobenzoic acid was increased by approximately 1.7 folds as compared to pure drug. Salt-forming technology is convenient and can improve the solubility of ticagrelor.

Objectives To investigate the prenatal diagnosis method of spinal muscular atrophy with amniotic fluid sample.Methods Totally 1 064 amniotic fluid samples from mid-trimester pregnant women were enrolled during January 2015 and January 2016 in 4 hospitals.Genetic analysis was performed for detecting potential contamination of maternal tissue by a genetic technique based on short tandem repeat ( STR) markers.Deletion of SMN1 gene was detected in 1 062 uncontaminated amniotic fluid samples by real-time PCR and multiplex ligation-dependent probe amplification ( MLPA) respectively.Results Two contaminated amniotic fluid samples were detected within 1 064 mid-trimester pregnant women by STR genotyping.The other 1 062 uncontaminated amniotic fluid samples were tested by real-time PCR.There were 37 samples with heterozygous deletion of Exon 7 of SMN1 gene ( 3.67%) , 34 samples with heterozygous deletion of Exon 8 of SMN1 gene (3.2%) and two samples with homozygous deletion of Exon 7 and Exon8 of SMN1 gene ( 0.19%) respectively , while other samples observed with no deletion of Exon 7 and Exon8 in SMN1 gene.Totally 41 samples with heterozygous or homozygous deletion of SMN 1 gene and 55 samples with undetected deletion of SMN 1 gene were confirmed by MLPA and the results showed 100%consistence with that of real-time PCR.Conclusions Both real-time PCR and MLPA are suitable for detecting the deletion of SMN 1 gene with amniotic fluid sample . Real-time PCR exhibits less sample requirement and time compared with MLPA .

Objective To establish a system using CK activity assessing with follow-up Duchenne muscular dystrophy (DMD) gene testing to newborn screening for DMD.This study provided a pathway to improve the health outcome for individuals with DMD.Methods Tests for CK were performed with Beckman original reagent on a Beckman Coulter AU 5800.Preliminary studies established a population-based range of CK in newborns using 5 892 deidentified anonymous blood samples,which were collected from Shanghai Changning Maternity and Infant Health Hospital between November 2013 and July 2014.Mutation analysis used multiplex PCR-denature high-performance liquid chromatography (PCR-DHPLC) method for screening large duplications and deletions and Sanger DNA sequencing for screening point DMD gene mutation.Results DMD gene mutations (point mutation,exon60,c.9072G ＞ A) were found in 1 of 5 892 newborn subjects,which had CK level ＞ 2 000 U/L large duplications and deletions in DMD gene were not found.Conclusions A system of analysis for newborn screening for DMD has been established.This path for newborn screening fits our health care system and minimizes the false-positive results for predicting DMD gene mutations by use of CK levels in blood