To prepare rivastigmine liposome, rivastigmine was loaded into liposome via ammonium sulfate gradient method. Its pharmacokinetic profile in rats was evaluated after intranasal administration. The size, zeta potential, entrapped efficiency and release of rivastigmine from the liposome in vitro were determined. Plasma concentration of rivastigmine was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS) using antipyrine as internal standard. The pharmacokinetic parameters were calculated by DAS 2.0. The entrapped efficiency of rivastigmine liposome was (33.41 +/- 6.58) %, with the mean diameter of 154-236 nm and zeta potential of (-10.47 +/- 2.41) mV. The release behavior of rivastigmine was fitting the first order equation in vitro. The pharmacokinetic studies indicated that the C(max), T(max) and AUC(0-infinity), of rivastigmine liposome were (1.50 +/- 0.15) mg x L(-1), 15 min and (89.06 +/- 8.30) mg x L(-') x min, respectively. Rivastimine liposome was absorbed rapidly, and could reach a certain concentration in rat plasma after intranasal delivery.
Administration, Intranasal ; Animals ; Area Under Curve ; Chromatography, Liquid ; Drug Carriers ; Drug Compounding ; Liposomes ; Male ; Neuroprotective Agents ; administration & dosage ; blood ; chemistry ; pharmacokinetics ; Particle Size ; Phenylcarbamates ; administration & dosage ; blood ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Rivastigmine ; Tandem Mass Spectrometry

Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes.Methods The resistance fragment, including upstream and downstream homologous arms of targeted regions, was reamplified by asymmetric PCR.The amplicons were introduced into Y.pestis harboring plasmid pKD46.With the induction of L-arabinose,the recombinant related enzymes: Exo, Beta and Gam, were expressed to guide the homologous recombination.A donor plasmid, pKSI-1, which carried the desired modification fragment flanking by I-SceⅠ recognition sites, was introduced into Y.pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains:△waaA and waaA(△9nt), were successfully constructed for Y.pestis strain 201.Scarless modification introduces no extra modification to the genome, and it is ideal for comprehensive functional genomic studies.

The clinical application of orbital magnetic resonance (MR) T2-mapping imaging in detecting the disease activity of Graves' ophthalmopathy (GO),and the predictive values of therapy response to intravenous glucocorticoid (ivGC) were investigated.Approved by the local institutional review board (IRB),106 consecutive patients with GO were included in this prospective study.All subjects were divided into two groups according to the patients' clinical activity score (CAS):the CAS positive group (CAS ≥3) or the CAS negative group (CAS ＜3).T2 relaxation time of extraocular muscles (T2RT;ms) and the areas of four extra-ocular muscles (AEOMs;mm2) were measured by 3D T2-mapping MR sequence before and after methylprednisolone treatment,so as the CAS and some ophthalmic examinations including visual acuity,intra-ocular pressure,eyeball movement,diplopia and proptosis.In addition,24 healthy volunteers were recruited as the control group.The mean T2RT and AEOMs in CAS positive group were higher than those in CAS negative group.Both CAS positive and negative groups had significantly higher mean T2RT and AEOMs than the control group (P＜0.01).There was a positive correlation between T2RT and AEOMs values in GO patients,both of them had a positive correlation with CAS and the ophthalmic examinations.It was concluded that to evaluate the activity of GO,CAS was mostly related to inflammation symptoms of ocular surface,more than that,T2RT and AEOMs were also related to abnormal findings of the ophthalmic examinations including high ocular pressure,impaired eyeball movement,diplopia and proptosis.T2RT and AEOMs can reflex the inflammation state of ocular muscles better.CAS combined with 3D T2-mapping MR imaging could improve the sensitivity of detection of active GO so as the prediction and evaluation of the response to methylprednisolone treatment.

The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467C > T and 539G > C) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.
ABO Blood-Group System ; genetics ; Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; ethnology ; Female ; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase ; genetics ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Point Mutation

OBJECTIVETo investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population.

METHODSThe polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying C35T variant was established by using PCR to amplify a 125 bp FUT1 gene fragment, including C35T variant sequence, and the PCR product was digested by Hae III restriction enzyme. One hundred and fifty-eight random samples from Chinese blood donor were screened by PCR-RFLP.

RESULTSAmong 158 Chinese individuals with normal ABO and H blood group phenotypes, 8 and 83 were homozygous with 35T/T and 35C/C, respectively, while 67 were heterozygous with 35C/T. The allele frequencies were compatible with Hardy-Weinberg equilibrium.

CONCLUSIONThe C35T substitution of FUT1 gene is not a mutation which gives rise to a non-functional h allele responsible for para-Bombay phenotype but a single nucleotide polymorphism in Chinese population.

ABO Blood-Group System ; genetics ; Alleles ; Female ; Fucosyltransferases ; genetics ; Gene Frequency ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVETo investigate the incidence of lymph node metastasis (LNM) in the right para-tracheal triangle (RPT) of esophageal carcinoma patients and the technique of dissection.

METHODSOn the top of double mediastinal and abdominal lymphadenectomy, 333 esophageal carcinoma patients received RPT lymphadenectomy through the right pleural apical approach from 1990 to 2001.

RESULTSIn these 333 patients, the lymph node metastasis (LNM) rate in the RPT was 36.40%. A total of 457 nodes among 2 159 nodes removed gave a metastasis degree of 24.96%. The LNM rates in RPT for cervical, upper third, middle third, and lower third segments of esophagus were 66.67%, 45.45%, 34.19% and 15.79% (P < 0.05), while their respective metastasis degrees were 44.44%, 27.04%, 24.32% and 18.92% (P > 0.05). The frequency of positive nodes in the RPT for PTI, PT1, PT2, PT3 and PT4 was 0, 17.24%, 28.7%, 45.16% and 53.57%, while those of metastasis degree were 0, 8.77%, 17.62%, 33% and 41.17% (P < 0.01). The frequency of LNM in the RPT in papillary, erosive, patch-like and covert type of early tumor was 40%, 3.85%, 0 and 0 (P < 0.05), while those of the metastasis degree were 29.41%, 1.82%, 0 and 0 (P < 0.01). Higher rate of LNM in progressive stenotic esophageal carcinoma was observed compared with those of the other gross types (56.52%, P < 0.05), so was the degree (P < 0.01). The frequency of LNM in the RPT for mono-focal and multi-focal tumor was 34.98% and 70% without significant difference (P > 0.05), while the degree was 24.29% and 53.33% (P < 0.05). Postoperative complications were: leak (0.6%), and recurrent laryngeal nerve injury (1.2%). No injury of vein or infra-clavicular artery, tracheal damage or mortality occurred.

CONCLUSION1. The lymph node metastasis from esophageal carcinoma has a tendency of wide spread and right para-tracheal triangle is an important region to be doomed. 2. With location, depth of tumor invasion and differentiation of tumor as major factors affecting LNM of esophageal carcinoma, dissection of this region should be paid more emphasis. 3. In early lesions, higher frequency of LNM in the RPT is found in papillary and erosive lesions than in the other macroscopic types. 4. Exposing the RPT, lymph node by dissection through a right pleural apical approach is very important and significant.

Adult ; Aged ; Cardia ; Esophageal Neoplasms ; pathology ; surgery ; Esophagectomy ; methods ; Esophagus ; pathology ; Female ; Humans ; Lymph Node Excision ; methods ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Mediastinum ; Middle Aged ; Neck ; Neoplasm Invasiveness

OBJECTIVETo explore the roles of intracellular cholesterol metabolism in neuroendocrine (NE) differentiation of prostate cancer based on an androgen-independent prostate cancer NE cell model induced by androgen deprivation.

METHODSLNCaP cells were cultured in androgen-depleted medium, and NE phenotypes were identified by observing the changes in cell morphology, molecular markers (SgIII, NSE and CgA) and cell proliferation. The expression and distribution of cholesterol and Sg III were determined by immunofluorescence staining. The expressions of the key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake were detected by semi-quantitative RT-PCR.

RESULTSThe LNCaP cells showed shrinking bodies and extending axons after androgen deprivation, and all the molecular markers, such as Sg III, NSE and CgA, significantly increased in a time-dependent manner, while the cell proliferation was obviously inhibited (P < 0.05). The cholesterol distribution in the LNCaP cells after NE differentiation presented remarkable aggregation at the axon terminals. However, there were no significant differences in the expression of cholesterol between the two types of cells, nor in the changes of the expressions of key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake (P > 0.05).

CONCLUSIONTransient androgen depletion could successfully induce NE differentiation of LNCaP cells, and the intracellular cholesterol could re-distribute into axon terminals to enhance the formation of neurosecretory granules.

Androgens ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Cholesterol ; metabolism ; Humans ; Male ; Neurosecretory Systems ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, LDL ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

OBJECTIVETo observe the effects of lithium chloride combined with human umbilical cord blood mesenchymal stem cell (hUCB-SCs) transplantation in the treatment of spinal cord injury in rats.

METHODSEighty female SD rats with complete T9 spinal cord transaction were randomized into 4 groups (n=20), namely the control group (group A), lithium chloride group (group B), hUCB-SCs group (group C) and hUCB-SCs(+) lithium chloride group (group D). On days 1 and 3 and the last days of the following weeks postoperatively, the motor function of the hindlimb of the rats were evaluated according to the BBB scores. At 8 weeks, all the rats were sacrificed and the spinal cords were taken for morphological observation. The spinal cord tissues at the injury site were observed with Brdu nuclear labeling to identify the survival and migration of the transplanted SCs. The regeneration and distribution of the spinal nerve fibers were observed with fluorescent-gold (FG) spinal cord retrograde tracing.

RESULTSBrdu labeling showed that the transplanted hUCB-SCs survived and migrated in the spinal cord 8 weeks postoperatively in groups C and D. FG retrograde tracing identified a small amount of pyramidal cells that migrated across the injury site in groups C and D. The BBB scores of the hindlimb motor function 8 weeks postoperatively were 4.11∓0.14, 4.50∓0.15, 8.31∓0.11 and 11.15∓0.18 in groups A, B, C and D, respectively.

CONCLUSIONLithium chloride can promote the survival and differentiation of hUCB-SCs into neural cells at the injury site. Lithium chloride combined with hUCB-SCs transplantation may accelerate functional recovery of the hindlimbs in rats with complete transection of the spinal cord.

Animals ; Cord Blood Stem Cell Transplantation ; Female ; Humans ; Lithium Chloride ; therapeutic use ; Rats ; Spinal Cord Injuries ; therapy

OBJECTIVE: To explore the feasibility and to sum up the experience of breast intraductal neoplasm resection under breast fiberoptic ductoscopy (FDS). METHODS: FDS was performed on 548 patients with nipple discharge from Sep.2004 to Nov.2006. The clinical data of breast intraductal neoplasm found by FDS in patients who underwent tumor resection were analyzed, and the breast intraductal neoplasm image characteristics, diagnosis, operative type and postoperative pathological results were analyzed. RESULTS: Of the 548 patients with nipple discharge, intraductal neoplasm was found in 187 cases (34.1%), intraductal papilloma in 159 cases (29.0%), intraductal papillomatosis in 12 cases (2.2%), and breast carcinoma in 16 cases (2.9%). One hundred thirty-five patients were operated on in our hospital, of whom 91 were performed tumor resection or segmentectomy under the localization by FDS, and the other 44 were performed segmentectomy after breast duct infusion of methylene blue. The diagnostic rate under FDS in the FDS group (97.8%) was higher than that in the breast duct infusion methylene group (86.4%) (chi2=6.96, P=0.008). CONCLUSION FDS is not only an accurate diagnosis for breast intraductal lesion, but also an assistance to localize the breast intraductal neoplasm and to remove them in the operation.
Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; diagnosis ; surgery ; Carcinoma, Ductal, Breast ; diagnosis ; surgery ; Endoscopy ; methods ; Female ; Fiber Optic Technology ; methods ; Humans ; Middle Aged ; Papilloma, Intraductal ; diagnosis ; surgery

Objective To investigate the activation of signal transducer and activator of transcription-3 (STAT3) in rats following focal cerebral ischemia/repeffusion (IR) and explore the correlation between STAT3 activation and the cerebral infract volume. Methods Ninety-nine SD rats were randomized into sham-operated group (n=9) and two IR groups with right middle cerebral artery occlusion with thread for 2 h (n=45) and 6 h (n=45) followed by reperfusion. At different time points after the end of the ischemia, the rats were sacrificed to obtain the brain tissue for triphenyltetrazolium chloride (TTC) staining to determine the infract volume. Immunohistochemistry and Western blot were used to detect the expressions of STAT3 and phosphorylated STAT3 (P-STAT3) in the brain tissue, and their correlations to the infarct volume were analyzed. Results Cerebral ischemia induced obvious infraction in the right hemisphere of the rats, where TTC staining was absent. Ischemia for 6 h resulted in more extensive areas without TTC staining than ischemia for 2 h (P<0.05). Reperfusion for 24 h after a 2-hour ischemia was associated with obviously reduced area free of TTC staining (P<0.05), whereas reperfusion for 24 h following a 6-hour ischemia only caused mild reduction of the TTC staining-free area. Immunohistochemistry of the brain tissue demonstrated the presence of STAT3 protein expression in the cytoplasm and P-STAT3 in the cell nuclei. IR was found to cause changes in the expression of P-STAT3 but not STAT3 protein, and the expression of P-STAT3 increased with the reperfusion time, reaching the peak level at 24 h of reperfusion. The activation level of STAT3 protein was inversely correlated to the dimension of the TTC staining-free area in the brain. Conclusion STAT3 is expressed in the cytoplasm and P-STAT3 in the cell nucleus of the brain tissue. Without causing obvious changes in STAT3 expression level, cerebral ischemia and reperfusion increases the phosphorylation level of STAT3 in inverse correlation to the size of the infarct area.