Objective To observe the expressions of inducible nitric oxide synthase(iNOS) in the progress of rat subchronic fluorosis,and analyse the mechanism of nitric oxide(NO) free radical injnry in bone.Methods Male wistar rats were divided randomly by body weight into two groups.i.e.sodium fluoride group and control group.Sodium fluoride group was given drinking water with 150 mg/L sodium fluoride,and control group was given tap water only.The animals were bred for 24 weeks.Every four weeks some rats were killed.The contents of serum and bone fluoride were examined and analyzed.The levels of serum NO were determined by Griess Reagent.The expressions of iNOS mRNA and protein were analyzed by RT-PCR and immunohistochemistry.Results The serum NO contents significantly increased(t=9.36,P＜0.01) in NaF-treated rats after 8 weeks[(19.94±3.04)nmol/L],but significantly decreased(t=10.47,4.46,P＜0.01) after 20 weeks[(11.55±3.54)nmol/L]and 24 weeks[(20.83±2.49)nmol/L],compared with control group[(9.11±1.21,31.13±3.93,33.10±7.37)nmol/L].The expression of iNOS mRNA significantly increased(t=13.09,4.82,14.23,4.64,7.82,5.29,P＜0.01)in rats treated with sodium fluoride[(1.87±0.11),(1.87±0.78),(1.90±0.29),(1.93±0.67),(1.88±0.38),(1.84±0.03)],compared with control group[(0.41±0.25),(0.30±0.17),(0.18±0.06),(0.63±0.15),(0.66±0.04),(0.65±0.55)],and these proteins mainly appeared in hyperplasie zone and hypertrophic zone cells of epiphyseal plate,cartilages,articular cartilage cells,osteoblasts and ligament cells.Conclusions High dose fluoride might persistentlv induce the expressions of iNOS and catalyze synthesis of NO,then regulates osteoblast and osteoclast activitv and finally influences bone turnover.

Objective To observe the expressions of matrix metal proteinases-13(MMP-13) mRNA and tissue inhibitor of metal protease- 1 (TIMP- 1) mRNA and analyse the molecular mechanism of bone matrix degradation in the progress of rat subchronic fluorosis. Methods Male Wistar rats were randomly divided into two groups according to body weight, i.e. sodium floride group and control group. Rats in the sodium fluoride group were given drinking water containing 150 mg/L F-, and the animals in the control group were given tap water. The animals were bred for 24 weeks. Every 4 weeks some rats were killed. The change of obsteoclst was observed by transmission electron microscope. The expression levels of MMP-13 mRNA and TIMP-I mRNA were analyzed by RT-PCR. Results The number of lysesome and the synthesis of lysosoma enzyme in osteeclast were decreased. The expression of MMP-13 mRNA was significantly increased(t=2.29,2.41,3.07,2.52, 3.15,2.22, P<0.05) in rats treated with sodium fluoride (1.87±0.67,1.87±0.75,1.90±0.73,1.93±0.86,1.88±0.61,1.84±0.53), compared with control group(1.24±0.39, 1.19±0.27,1.07±0.22, I. 15 ~ 0.17, 1.17±0.18, 1.20±0.62). The expression of TIMP-1 mRNA was significantly increased (t=2.69,2.19,2.68,2.46,2.43,2.96, P<0.05) in rats treated with sodium fluoride(1.89±0.77,1.70±0.85,1.61±0.82,1.81±0.84,1.70±0.74, 2.06±0.96), compared with control group (1.07±0.39,0.87±0.49,0.71±0.48,0.99±0.43,0.95±0.46,0.89±0.57). Conclusion High dose fluoride might persistently induce the expressions of MMP-13 mRNA and TIMP-1 mRNA and may be involved in bone turnover.

AIMTo prepare solid lipid nanoparticles by microemulsion technique.

METHODSStearic acid was used as the oil phase, lecithin as surfactant, alcohol as cosurfactant and distilled water as the aqueous phase. Microemulsion was prepared by mixing the above component in proper ratio. The corresponding pseudoternary phase diagram monitored Microemulsion formation field of different lecithin/alcohol. Solid lipid nanoparticles (SLN) were prepared by dispersing warm microemulsion in cold water under magnetic stirring. Then appropriate microemulsions that can contain more water phase and suitable oil phase were selected to prepare SLN. The influence of formulation, process variables on the preparation and quality of SLN were studied. Based on the investigation of single factors, orthogonal design was used to optimize SLN formulation and preparation process, and more, the reproducibility of the optimized results were studied.

RESULTSThe results showed that the device temperature (Ti), water temperature (Tw), and delivery rate (Rd) were the key factors that influence the preparation process of SLN, and Tw was extremely important. The ratio of microemulsion formulation, the ratio of microemulsion and distilled water had also influence on its quality.

CONCLUSIONMicroemulsion technique can be used to prepare solid lipid nanoparticles.

Alcohols ; Drug Carriers ; Emulsions ; Lipids ; chemical synthesis ; chemistry ; Nanotechnology ; Particle Size ; Phosphatidylcholines ; Solubility ; Technology, Pharmaceutical ; methods

With the diversion rate of ginkgolide A, B, K as comprehensive evaluation indexes, the amount of activated carbon, ad- sorption time, mix rate, and adsorption temperature were selected as factors, orthogonal design which based on the evaluation method of information entropy was used to optimize activated carbon adsorption technology of ginkgo diterpene lactones meglumine injection. Opti- mized adsorption conditions were as follows: adsorbed 30 min with 0.2% activated carbon in 25 °C, 40 r ·min⁻¹, validation test re- sult display. The optimum extraction condition was stable and feasible, it will provide a basis for ginkgo diterpene lactone meglumine injection' activated carbon adsorption process.
Adsorption ; Charcoal ; chemistry ; Chemistry, Pharmaceutical ; instrumentation ; methods ; Diterpenes ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Ginkgo biloba ; chemistry ; Lactones ; chemistry ; isolation & purification

AIMTo detect the hepatotoxic pyrrolizidine alkaloids (HPA) in the genus Ligularia Cass..

METHODSThe alkaloid extracts of Ligularia plant materials were detected and analyzed by the method of combination of TLC, and LC/MSn.

RESULTSAmong 22 species of Ligularia Cass., HPA were detected in 18 species with LC/MSn, and no HPA was detected in the remaining 4 species.

CONCLUSIONHPA was first detected with LC/MSn in L. tongelensis and other 15 species of Ligularia Cass.; HPA from these plants should be isolated, separated and identified and it is necessary to study the activities and toxicities of the HPA. The types and kinds of HPA from different species and sources are different, they should be detected separately.

Asteraceae ; chemistry ; classification ; Chromatography, Thin Layer ; Molecular Structure ; Plants, Medicinal ; chemistry ; Pyrrolizidine Alkaloids ; analysis ; chemistry ; Species Specificity ; Spectrometry, Mass, Electrospray Ionization

Objective To evaluate the accuracy of automated quantification of myocardial perfusion imaging (MPI) using a method based on a Western normal database for the detection of coronary artery disease (CAD) in a group of Chinese patients. Methods Seventy-two Chinese patients who underwent coronary angiography (CAG) and MPI within 3 months were recruited into this study. Eighty selected from 140 Chinese patients with low probability of CAD ( ＜ 5% ) were enrolled into local normal database of 99Tcm-methoxyisobutylisonitrile (MIBI) MPI using Cedars quantitative perfusion SPECT (QPS) database. Two Western MPI normal databases (CSMC MibiMbiAuto and Mibimibi) were used for processing the Chinese CAD patients recruited in this study, and the results were compared with those using local normal database and visual interpretation. T-test and z-test were used for statistical analysis. Results The extent (EXT)measurement obtained from Mibimibi and local database was ( 10.73 ± 14.54)% and ( 14.22 ± 16.51 )%,respectively ( t = 7.87, P ＜ 0.001 ); the severity (SEV) was 1.07 ± 0.93 and 1.34 ± 1.20, respectively ( t =7.45, P＜0.001). The area under curve(AUC) by using EXT measurement for local database (0.85 ±0.05) was larger than that for CSMC MibiMbiAuto ( AUC = 0.72 ± 0.06, z = 2.50, P ＜ 0.01 ) and Mibimibi ( AUC = 0.77 ± 0.06, z = 2.47, P = 0.014). The AUC of local database showed no significant difference from that of visual interpretation (AUC=0.83 ±0.05, z=0.05, P＞0.05). Conclusion Quantification of MPI of our Chinese patients using Western normal database would decrease the accuracy for the detection of CAD.

BACKGROUNDSentinel lymph node (SLN) biopsy has become a common procedure for early breast cancer patients. The GeneSearch(TM) Breast Lymph Node (BLN) Assay is a real-time RT-PCR assay for the detecting nodal metastases larger than 0.2 mm. China Breast Cancer Clinical Study Group (CBCSG)-001a is a prospective multi-center clinical trial that was conducted to validate the GeneSearch(TM) BLN Assay in China.

METHODSThe SLNs from 90 consecutive patients were identified and dissected, and then sectioned along the short axis into multiple blocks. Intra-operatively, the odd blocks were tested by BLN assay and the even ones were used for frozen section, while all the blocks were evaluated by touch imprint cytology. Post-operatively, the remaining tissues were assessed by histological evaluation.

RESULTSA total of 189 SLNs was tested by BLN assay. The sensitivity, specificity, positive predictive value, and negative predictive value were 88.9%, 97.4%, 88.9% and 97.4%, respectively, for BLN assay, 75.0%, 100%, 100% and 94.4%, respectively, for frozen section, and 63.9%, 100%, 100% and 92.2%, respectively, for touch imprint cytology. The sensitivity of BLN assay was higher than that of touch imprint cytology (P = 0.01) and frozen section (P = 0.13). When assessing the nodes with micro-metastases, BLN assay had a significant higher sensitivity than frozen section (P = 0.023) and touch imprint cytology (P = 0.005).

CONCLUSIONThe GeneSearch(TM) BLN Assay is an accurate and rapid intra-operative assay for breast SLNs and it is suitable for application in general medical practice.

Adult ; Aged ; Breast Neoplasms ; complications ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; diagnosis ; Middle Aged ; Sentinel Lymph Node Biopsy ; methods

OBJECTIVEAlpha-thalassemia is one of the most common monogene disorders in the world. Most frequently, it is caused by deletions of alpha-globin gene (-alpha or --), and less commonly resulted from the non-deletional mutation (alpha(T)alpha). Hemoglobin H (HbH) disease is the most severe type among survivors of alpha-thalassemia. The clinical presentation of children with the disease was highly heterogeneous. The aim of this study was to investigate the effect of alpha-globin genotypes in the children with HbH disease on predicting the phenotypic severity and to define the factors involved in the disease progress.

METHODSForty-three children with the disease in Zhuhai area of Guangdong, China were examined by using established techniques to detect genotypes of alpha-globin and to determine all hematological parameters. All detailed clinical data of the cases were recorded. Then clinical and hematological findings, and the correlation with genotypes were evaluated.

RESULTSSix alpha-thalassemia mutations were detected and interacted to produce 5 HbH disease genotypes. Of these genotypes, -alpha(3.7)/--(SEA)(60%), -alpha(4.2)/--(SEA) (19%) and alpha(CS)alpha/--(SEA) (12%) HbH diseases were prevalent in the area. Compared with -alpha(3.7)/--(SEA) HbH disease, significantly lower red blood cell (RBC) count, hemoglobin (Hb), mean corpuscular hemoglobin (MCHC) and HbA(2) (P < 0.05, 0.01, 0.01 and 0.01, respectively), and significantly higher mean corpuscular hemoglobin volume (MCV) and HbH levels (both P < 0.01), and more severe clinical phenotypes were found in the HbH disease with alpha(T)alpha/--(SEA) genotype. While the differences were much more significant when compared with -alpha(3.7)/--(SEA) then compared with -alpha(4.2)/--(SEA) not only in the hematological parameters, but also in the severity of clinical phenotypes. In addition, HbH levels showed anegatively correlation with the RBC count (r = -0.39, P < 0.01).

CONCLUSIONThe phenotypes of HbH disease may be mainly related to the underlying genotypes. The children with alpha(T)alpha/--(SEA) genotype presented with more severe hematological and clinical phenotypes followed by the -alpha(4.2)/--(SEA) and then -alpha(3.7)/--(SEA) genotypes. But phenotypic severity was not simply related to the degree of alpha-globin deficiency. HbH levels were found to exacerbate anemia. These data might provide comprehensive and very valuable and basic information for the management of HbH disease, genetic counseling and prenatal diagnosis.

Child ; China ; Disease Progression ; Genotype ; Hemoglobin H ; genetics ; Humans ; Phenotype ; alpha-Globins ; genetics

OBJECTIVETo investigate cell apoptosis induced by survivin ASODN and clarify the precise mechanism of anti-apoptotic action of survivin.

METHODSCells of lung cancer cell line NCI-H446 were treated with survivin ASODN at different concentrations. The changes of survivin mRNA and protein expression were assessed by RT-PCR and Western blot assay. The apoptosis index (AI) and proliferation index (PI) were determined by flow cytometry (FCM). After 500 mmol/L survivin ASODN treatment, cells were stained with Rh123 to detect changes of mitochondrial membrane potential (deltapsim) by FCM. The concentration of cytoplasmic cytochrome c (cyt-c) was continuously determined by ELISA. Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay. The expression of caspase-8 protein was measured by Western blot assay. The apoptotic rates of lung cancer cells induced by survivin ASODN with or without mitochondrial permeability transition pole (MPTP) inhibitor CsA treatment were assessed by FCM.

RESULTSDown-regulated survivin mRNA was shown to be in dose-dependent and time-dependent manners. Its maximal effect was achieved at a concentration of 500 nmol/L for 72 h, at which mRNA was down-regulated by 62.7%, the expression of survivin protein in NCI-H446 cells was also obviously decreased. After treatment with survivin ASODN at concentration of 500 mmol/L for 72 h, AI was 48.35%, higher than that of control, lipofectin, NSODN, survivin ASODN 100 mmol/L and 300 mmol/L groups (3.75%, 3.41%, 4.69%, 19.85% and 34.39%, respectively). PI was 24.38%, lower than that of control, lipofectin, NSODN, survivin ASODN100 and 300 mmol/L groups (75.74%, 73.12%, 71.76%, 51.03% and 38.94%, respectively). Deltapsim was decreased in 9.54% of NCI-H446 cells treated with survivin ASODN for 3 h and 97.06% for 24 h. Following it, release of cyt-c from mitochondria to cytosol and activation of caspase-9 and caspase-3 increased significantly. The above mentioned indicators changed with a time-dependent and time diversity relationship. In the presence of CsA, the apoptotic rate of lung cancer cells induced by survivin ASODN was decreased significantly. No up-regrulation and activation in caspase-8 protein was observed.

CONCLUSIONSurvivin inhibits apoptosis via regulation of mitochondrial-dependent pathway. survivin ASODN can not only induce apoptosis but also inhibit cell proliferation through blocking the expression of survivin mRNA and protein.

Apoptosis ; drug effects ; genetics ; physiology ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; metabolism ; Cytosol ; drug effects ; enzymology ; metabolism ; Down-Regulation ; Humans ; Immunosuppressive Agents ; pharmacology ; Inhibitor of Apoptosis Proteins ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Microtubule-Associated Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

OBJECTIVETo study the clinical problems about posterior atlanto-axial internal-fixation and fusion for atlanto-axial instability or dislocation.

METHODSSurgical treatments of 138 cases with atlanto-axial instability or dislocation were reviewed. There were 62 cases of odentoid malformation, 54 cases of odentoid fracture or rupture of transverse ligament, 22 cases of subluxation and rotation. All cases were treated using Gallie's technique. Six cases were also fixed with transarticular screws, and protected with Philadelphia collar. Other patients were fixed with plaster paris brackets. The followed-up period was 1 to 12 years with an average of 3 year and 5 months.

RESULTSAccording to Sumi's criteria, excellent 70 cases (50.7%), good 40 cases (29.0%), fair 15 cases (10.9%), poor 13 cases (9.4%). 9 cases with bone graft postponed fusion were cured by enhance external-fixation. 2 cases with nonunion were treated with revision surgery. Complication of cord injury happened in 1 case.

CONCLUSIONGallie's fusion technique is an effective method to manage the atlanto-axial instability or dislocation. Skull distraction before operation and reliable external-fixation post operative are important assistant measures. Key points for successful operation are careful wiring or cable traversing, decortication of posterior arc of C1, and maintaining the physiological height between C1 and C2 posterior arc. Indications and objectives should be conformed before revision surgery for failure cases.

Adolescent ; Adult ; Atlanto-Axial Joint ; surgery ; Bone Transplantation ; Child ; Female ; Humans ; Joint Dislocations ; surgery ; Joint Instability ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Spinal Fusion ; adverse effects ; methods ; Transplantation, Autologous