Objective To explore the association of Toll-like receptor 4 TLR4 and lipopolysaccharide receptor CD14 gene polymorphisms with Kawasaki disease (KD) susceptibility.Methods Three-color fluorescent staining flow-cytometry was used to detect the expression of TLR4 in peripheral blood white blood cell of 76 KD children and 118 healthy control group.The gene of TLR4 (-896A/G), (-1196C/T) and CD14 (-260C/T) polymorphisms was identified by polymerase chain reaction-restriction fragment length polymorphisms; and the relationship between genotype and KD was analyzed.Results 1.The values of mean fluorescence intensity (MFI) of TLR4 in peripheral blood white blood cell of the KD groups and the healthy control groups were 2.87?0.96, 10.55?4.87, 23.36?8.28 and 3.26?0.65, 7.55?1.21, 25.41?6.97, respectively; There was a gradual increase of these values on lymphocyte, neutrophilic leukocyte and mononuclear cell in both groups.2.(-896A/G), (-1196C/T) polymorphisms of TLR4 gene were not found in both groups.3.The frequency of each genotype of CD14 gene (-260C/T) was 35.5%CC, 30.3%CT, 34.2%TT in KD group and 38.1%CC, 47.5%CT, 14.4%TT in healthy control group.The frequency of each genotype was significantly different in 2 groups(?2=11.62 P

AIM To investigate the equilibrium solubilities,oil-water partition coefficients and in vitro skin permeation features of brucine and strychnine in total alkaloids from Strychni Semen.METHODS Saturated dissolution method was applied to determining the equilibrium solubilities of two constituents in ethanol (10％,20％,30％,60％,90％,anhydrous ethanol),trichloromethane,n-octanol and surfactants (0.5％ tween,0.5％ sodium deoxycholate,0.5％ oleic acid).Shake-flask method was adopted in detecting their oil-water partition coefficients in PBS (pH 2.5,4.0,5.0,5.8,6.8,7.0,7.4,9.0).Modified Franz diffusion cell method was used for evaluating their in vitro skin permeation features in PBS,20％ ethanol and anhydrous ethanol.RESULTS Both brucine and strychnine showed the highest equilibrium solubilities in trichloromethane and the lowest equilibrium solubilities in surfactants.The equilibrium solubility of strychnine was higher than that of brucine in ethanol (＞ 20％) or PBS (pH ＜ 8.0),which reached the highest in 60％ ethanol and pH 2.5 PBS,respectively.The similar oil-water partition coefficients of two constituents,proportional to pH value,reached the highest at pH9.0.And they exhibited the highest accumulated transdermal absorptivities in anhydrous ethanol and pH 9.0 PBS,respectively.CONCLUSION Solvent type has obvious effects on the equilibrium solubilities,oil-water partition coefficients and in vitro skin permeation features of both brucine and strychnine.This study can provide a reference for the bioavailability improvement of transdermal drug delivery and development of related preparations.

Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.

Background Adeno-associated virus-based vector is one of most efficient vehicles.It presents with a long-term and efficient transfer and expression of therapeutic genes with minimal toxicity.But its delayed-and low-efficient transgene expression limits the application of AAV vector.To explore an improving method of AAV infecting RPE cells is the hot spot. Objeetive Present study was to investigate whether adeno-associated virus (AAV)combined with low dose non-replieable adenovirus(Ad-null)can enhance its infection efficieney on RPE ceils in vitro. Methods Human RPE cells were isolated from the donate eyeballs under the approval of the Ethic Committee of this hospital.The cells were cultured in DMEM containing 10%fetal bovine serum.AAV particles with enhanced green fluorescence protein(EGFP)were added into the medium alone or in combination with different amount of adenovirus for 30 days.The cells were detected under the fluorescence microscope,and the protein expression levels of report gene EGFP in RPE cells were analyzed with Western blotting assay. Results Melanin granules could be found in cultured RPE cells.EGFP was expressed in RPE cells at 2 days after AAV-EGFP infection and peaked at 12 days and remained for about 3-week duration,showing the green influorescence under the influorescence mwroscope.After the cells were infected by AAV2-EGFP with 0.01 to 1000 MOI Ad-null respectively,the number of cells with green influorescence was obviously increased with the enhanced infiuorescence intensity.The enhance of the infection efficiency began in the 0.1 MOI Ad-null group and peaked in 10 MOI Ad-null group.Dead cells were exhibited in the 100 or more MOI Ad-nulor group.Western blotting assay demonstrated that the protein expression level of EGFP in RPE cells enhanced significantly in 1 and 10 MOI Ad-null groups compared with only AAV infection group. Conclusion These finding suggested that the infection efficiency of AAV can be improved significantly when it is used with low dose Ad-null in vitro.This offers a basis for further study of gene therapy.

OBJECTIVETo evaluate the effect of different preparation methods on the encapsulation efficiency (EE) and drug loading (DL) of paclitaxel-loaded polybutylcyanoacrylate nanoparticles (PTX-PBCA-NPs) and optimize the preparation of PTX-PBCA-NPs.

METHODSWith DL and EE as the major indexes, the qualities of PTX-PBCA-NPs produced by the interfacial polymerization and emulsion polymerization method were compared. The optimized prescription was obtained by orthogonal design.

RESULTSThe ranges of EE of PTX-PBCA-NPs with the two methods were both 94.39%-99.23%. The highest DL with interfacial polymerization was (1.07-/+0.03)%, as compared to (0.86-/+0.01)% with emulsion polymerization. The optimized preparation conditions resulted in the mean size of PTX-PBCA-NPs of 235.6 nm, DL of 0.80%, and EE of 95.71%.

CONCLUSIONThe EE of PTX-PBCA-NPs prepared by the above two methods is consistent with the requirement of the Pharmacopoeia of China, and PTX-PBCA-NPs containing higher DL can be obtained via interfacial polymerization.

Delayed-Action Preparations ; chemical synthesis ; Drug Carriers ; chemistry ; Drug Delivery Systems ; Enbucrilate ; chemistry ; Nanoparticles ; chemistry ; Paclitaxel ; administration & dosage ; Polymerization

OBJECTIVETo examine serum 25-hydroxyvitamin D levels in children with autism spectrum disorders (ASD) and to explore the relationship between vitamin D level and ASD.

METHODSSerum levels of 25-hydroxyvitamin D levels were determined by the HPLC-MS/MS method in 117 children with newly diagnosed ASD and 109 healthy controls. Vitamin D status were classified into normal (>30 ng/mL), insufficiency (10-30ng/mL) and deficiency (<10 ng/mL) according to 25-hydroxyvitamin D levels.

RESULTSSerum level of 25-hydroxyvitamin D (19±9 ng/mL)in children with ASD was significantly lower than that in healthy controls (36±13 ng/mL; P<0.01). The rate of vitamin D insufficiency plus deficiency in the ASD group was significantly higher than in the control group (89.7% vs 52.3%; P<0.01).

CONCLUSIONSVitamin D insufficiency or deficiency is common in children with ASD and might be as one of the environmental or genetic factors for ASD.

Child ; Child Development Disorders, Pervasive ; blood ; Child, Preschool ; Chromatography, High Pressure Liquid ; Female ; Humans ; Infant ; Male ; Vitamin D ; analogs & derivatives ; blood ; Vitamin D Deficiency ; epidemiology

OBJECTIVETo further study the mechanism of the inhibitory effect of interferon beta-1a (IFN beta-1a) on the activation of human hepatic stellate cell (HSC) LX-2, and to analyze the differences on the protein expression in LX-2 induced by I IFN beta-1a.

METHODSCultured LX-2 cells were treated with 2000 U/ml IFN beta-1a for 48 h. Two-dimensional gel electrophoresis (2-DE) was performed to compare protein patterns of the control (untreated) and IFN beta-1a treated LX-2 and for quantitative and qualitative analyses of protein expression. A rat liver fibrosis model was established and the rats were sacrificed and their various tissues were obtained for the same analyses. Western blotting and RT-PCR were used to validate the expression of the changed proteins after treatment of IFN beta-1a in LX-2 cells and of various tissues of the rats.

RESULTS708 +/- 25 spots were detected in control LX-2 cells and 804 +/- 32 spots in IFN beta-1a-treated LX-2 cells. A match rate of 73%-82% was achieved. The results also showed that 31 protein spots displayed quantitative changes in expression after IFN beta-1a treatment. Of the 31 spots, 21 proteins were identified, of which, one was newly found, two were enhanced in abundance and 18 showed lower expressions. The newly found protein was glia maturation factor beta (GMF beta). The treatment of LX-2 with IFN beta-1a increased the production of GMF beta(GMF beta) protein in comparison with the untreated cells (t=1.81, P < 0.01). The expression of GMF beta protein (1.81 vs 0.10) and mRNA (0.85 vs 0.12) were more in the normal liver tissues than in the cirrhotic liver tissues (t=2.53, 2.13 respectively, P < 0.01). The expressions of GMF beta protein and mRNA were weak in rat heart and lung tissues, however, they were strong in rat liver, kidney, spleen and brain tissues (t=1.91, 1.94 respectively, P < 0.01).

CONCLUSIONThere is a significant difference of protein expression levels between IFN beta-1a untreated and treated LX-2 cells. These proteins, especially GMF beta, may be involved in an inhibition process of IFN beta-1a on activation and apoptosis of LX-2 cells. This proteome study may be useful in further studies of the relationship of IFN beta-1a treatment and human liver diseases.

Animals ; Cell Line ; Female ; Glia Maturation Factor ; metabolism ; Hepatic Stellate Cells ; metabolism ; Humans ; Interferon beta-1a ; Interferon-beta ; pharmacology ; Liver ; cytology ; Liver Cirrhosis ; metabolism ; Proteome ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the clinical signification of c-kit gene mutation in gastrointestinal stromal tumor (GIST) and examine whether the presence of mutation of c-kit gene is important as a prognostic factor.

METHODSThe c-kit mutation had been detected by PCR-SSCP, DNA sequence, statistical comparison were used for the relationship of c-kit gene mutation and clinical pathology, clinical behavior, recurrence, et al.

RESULTSThe presence of c-kit mutation correlated with tumor size, proliferating cell nuclear antigen index, mitotic cell number, presence of necrosis, microscopic invasion to adjacent tissues, recurrence and distant metastasis. The age, sex, location of tumor, cell type, the presence of hemorrhage, and c-kit expression were independently related to the presence of c-kit mutation.

CONCLUSIONSThe c-kit gene mutation is an important prognostic factor for GIST.

Adult ; Age Factors ; Base Sequence ; DNA Mutational Analysis ; Female ; Gastrointestinal Neoplasms ; genetics ; pathology ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Prognosis ; Proto-Oncogene Proteins c-kit ; genetics ; Sex Factors ; Stromal Cells ; pathology

Purpose: This study aimed to evaluate the effectiveness of an interactive virtual reality (VR) play intervention including instructional play and emotional catharsis play sessions in reducing children's pain and fear during intravenous placement. Methods: A randomized controlled trial with parallel groups was conducted. The sample consisted of 134 hospitalized children aged 6–12 years (intervention group: n = 69; comparison group: n = 65). The intervention involved one immersive intravenous scene in VR before the actual intravenous placement and one emotional catharsis VR play after injection. The comparison group received an educational photo book about intravenous placement before receiving intravenous placement. The children and their caregivers rated their pain and fear by using the Wong–Baker FACES Pain Rating Scale and the Children's Fear Scale. The time required for successful intravenous insertion was also compared between the two groups. Results: Children's pain (p = .028) and fear scores (p = .004) were significantly lower in the intervention group than in the comparison group. Their caregivers' pain and fear scores (both p < .001) were significantly lower in the intervention group. The time required for successful intravenous insertion did not differ significantly between the intervention and comparison groups. Conclusions The interactive play intervention with VR effectively reduced children's levels of pain and fear during the intravenous placement procedure. The results of this study can serve as a reference for the implementation of a feasible, child-friendly care practice for clinical intravenous placement in school-aged children.

Objective To investigate the factors associated with the occurrence of pulmonary complication in patients with cervical spine fiactures and concurrent cervical cord injury in Wenchuan earthquake. Methods A retrospective analysis was conducted among the 9 patients with cervical spine fractures and cervical cord injuries treated in our department between May 12 to August 6, 2008. Results All the patients received surgical treatment for cervical spine fractures and cervical cord injuries. Six of the patients developed pulmonary complications 5 days after the injury, including 3 patients with pneumonia, 2 with ventilation disorder, and 1 with lung edema and hemopnenmothorax. Aggressive respiratory management was administered in these patients, and the pulmonary complications were effectively controlled. Conclusion Patients with cervical spine fractures and concurrent cervical cord injury often experience severe pulmonary complications during the acute phase (<5 days), which can be more likely in patients with high level injury, chest trauma, old age, preexisting pulmonary illnesses or smoking history. Early detection of the complications results in better therapeutic effect with conservative therapy.