Objective To explore continuous renal replacement therapy(CRRT) machine for plasma exchange in critical disease in children.Methods Retrospective study of 8 patients(8 month to 14 years,mean 5.7 years) and 32 plasma exchange treatments,after(adowble) lumen catheter inserted into the subclayian venous,using the Baxter BM25 machine with commercially available plasma filters.Results Five patients(3 ABO-incompatibility in bone marrow transplantation,1 thrombotic thrombocytopaenic purpura TTP,1 sepsis) gained full recovery.One systemic lupus erythematosus(SLE) and 1 sepsis experienced moderate improvement while 1 case of acute disseminated encephalomyelitis failed PE treatment.The average total exchange volume was 80-100 mL/kg,achieved at a blood flow rate of 5-10 mL/(kg?min) and a turnover rate of 60-120 mL/(kg?h) over a 3-hours duration.Thirty-one PE treatments were finished smoothly,one of which experienced the serious complication involving plasma filter.Conclusion Plasma exchange therapy is a safe and effective procedure for severe autoimmune abnormalities and pathogen removal in children.

AIM: To screen TIGR/myocilin gene (MYOC) mutation in high myopic Chinese children with family history.METHODS: Gene sequencing was performed in exon 3 of the TIGR gene in high myopic Chinese Children. The coding sequence of TIGR exon 3 was screened by capillary electrophoresis sequencing. The sequence alterations were analyzed by bioinformatics.RESULTS: TIGR gene mutation was not found in high myopic patients and normal controls group.CONCLUSION: No identified gene mutation is found in TIGR gene in high myopic Chinese children.

Objective To study the expression and phosphorylation of focal adhesion kinase(FAK) in rat's au- tologous vein graft and the olmesartan modulating effect.Methods Autologous external jugular veins were grafted to common carotid arteries in 40 male Sprague Dawley rats.After surgery,rats were randomly assigned to the fol- lowing groups:sham;control;olmesartan treatment(10mg/kg.d by gavage);or physiological saline.The intimal thickness,the I/M in vein grafts was quantitated by HE stain.The expression and phosphorylation of focal adhe- sion kinase were assessed by Western-blotting,PCNA and ?-smooth muscle actin were measured by immunohisto- chemistry.Results Neointimal hyperplasia in control group was characterized by significantly increased intimal thickeness I/M(P

OBJECTIVETo explore the differential proteomic expression in human liver cells L-02 induced by different dosages of trichloroethylene (TCE).

METHODSHuman liver cells L-02 were treated with different concentrations of TCE and the solvent control (dimethylsulfoxide). The total cellular proteins were separated using 2DE and visualized with silver staining after TCE treatment. The images were analyzed with Image Master 2D Platinum 5.0 analysis software. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS).

RESULTSFifteen protein spots with significant difference were found, and went upward or downward or disappeared after the stimulation of TCE with different dosages, which indicated that TCE induced the change of the proteomic expression in the liver cells. The mass spectrum identification and the IPI human database retrieval were used for identifying 9 proteins related to the L-02 Liver cells induced by TCE.

CONCLUSIONThe result provides an insight to TCE-related molecular mechanism and which might be useful for further study of the TCE-associated proteins and molecular markers.

Cell Line ; Dose-Response Relationship, Drug ; Electrophoresis, Gel, Two-Dimensional ; Hepatocytes ; drug effects ; metabolism ; Humans ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Trichloroethylene ; toxicity

Objective To analyze the clinical characteristics,muscle pathological features and pathogenic gene mutation in 4 cases with LMNA-related congenital muscular dystrophy (L-CMD).Methods Clinical data of the probands and the parents were collected.Skeletal muscle specimens were biopsied from the probands for pathological analysis.Genomic DNA and RNA were extracted from peripheral blood leukocytes,and PCR,reverse transcription(RT)-PCR and DNA direct sequencing were employed to analyze the LMNA gene to determine the gene mutation and confirm the pathogenicity.Results Four patients had symptoms from fetal period to several months after birth.They presented with motor retardation,muscle weakness with prominent the proximal upper limbs,distal lower limbs and neck extensor,hypotonia,contractures,with mild to moderate elevation of CK level.The muscle biopsies showed muscular dystrophic and with inflammatory changes,and the abnormal nuclear morphology was observed with transmission electron microscopy.Genetic analysis of them detected 4 dominant de novo mutations.Three of them had unreported pathogenic mutations.The same sites of the LMNA gene were wild type in their parents.Conclusions Four cases of L-CMD are genetically identified.Genetic counseling of the family can be possible.The patients should be considered LMNA gene mutation of they present themselves with muscle weakness with the proximal upper limbs,distal lower limbs and neck extensor,hypotonia,contractures,mild to moderate elevation of CK level,and if the biopsies show muscular dystrophic changes but also with inflammatory changes should be considered LMNA gene mutation.Genetic analysis is the most reliable method for diagnosing L-CMD.

OBJECTIVETo study the clinical feature of a Chinese family with muscle-eye-brain disease (MEB) and the mutation of protein O-linked-mannose beta-1, 2-N-acetylglucosaminyltransferase 1 gene (POMGNT1).

METHODSClinical data of the proband and his family members were collected. Genomic DNA from the patient and his parents was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze all of the exons to determine the mutation, and the relationship between genotype and phenotype was analyzed.

RESULTSThe proband was diagnosed as floppy baby, presented with delayed psychomotor development and myopathic face. His serum creatine kinase (CK) level elevated moderately and brain MRI showed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, cerebellar cysts and cerebellar and brain stem hypoplasia, consistent with congenital muscular dystrophy with eye brain disorder. Further test with DNA detected a compound heterozygous mutation of c.1896 1 G to C before exon 22 which may induce splicing error, and missense mutation c.1319T to G, p.L440R in exon 16. Both parents had a heterozygous mutation at the mutation sites.

CONCLUSIONAccording to our study, the family is diagnosed as MEB. The proband carried compound heterozygous mutations in the POMGNT1 gene, and his parents are heterozygous carriers, which is consistent with autosomal recessive inheritance. The child is definitely diagnosed as having muscle eye brain disease.

Adult ; Amino Acid Sequence ; Asian Continental Ancestry Group ; Base Sequence ; Brain ; pathology ; Child, Preschool ; Exons ; genetics ; Female ; Heterozygote ; Humans ; Magnetic Resonance Imaging ; Male ; Molecular Sequence Data ; Mutation ; genetics ; N-Acetylglucosaminyltransferases ; genetics ; Phenotype ; Sequence Alignment ; Walker-Warburg Syndrome ; diagnosis ; genetics

OBJECTIVETo study the protocol of construction of a KCNQ2-c.812G>T mutant and it's eukaryotic expression vector, the c.812G>T (p.G271V) mutation which was detected in a Chinese pedigree of benign familial infantile convulsions, and to examine the expression of mutant protein in human embyonic kidney (HEK) 293 cells.

METHODSA KCNQ2 mutation c.812G>T was engineered on KCNQ2 cDNAs cloned into pcDNA3.0 by sequence overlap extension PCR and restriction enzymes. HEK293 cells were co-transfected with pRK5-GFP and KCNQ2 plasmid (the wild type or mutant) using lipofectamine and then subjected to confocal microscopy. The transfected cells were immunostained to visualize the intracellular expression of the mutant molecules.

RESULTSDirect sequence analysis revealed a G to T transition at position 812. The c.812G>T mutation was correctly combined to eukaryotic expressive vector pcDNA3.0 and expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both the wild type and mutant molecules on the plasma membrane, which suggested that the c.812G>T mutation at the pore forming region of KCNQ2 channel did not impair normal protein expression in HEK293 cells.

CONCLUSIONSSuccessful construction of mutant KCNQ2 eukaryotic expression vector and expression of KCNQ2 protein in HEK293 cells provide a basis for further study on the functional effects of convulsion-causing KCNQ2 mutations and for understanding the molecular pathogenesis of epilepsy.

Epilepsy, Benign Neonatal ; genetics ; Fluorescent Antibody Technique ; Genetic Vectors ; HEK293 Cells ; Humans ; Infant, Newborn ; KCNQ2 Potassium Channel ; analysis ; genetics ; physiology ; Mutagenesis, Site-Directed ; Polymerase Chain Reaction

Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

OBJECTIVETo clarify the key morphological signs for the survival of adult rat cardiomyocytes in primary culture.

METHODSThe adult rat hearts were retrogradely superfused by Langendorff apparatus. Cardiomyocytes were digested by collagenase I and cultured in three groups: (1) Serum free medium + BA (Bongkrekic acid, apoptotic inhibitor), (2) 5% serum medium, and (3) 5% serum medium + BA. The morphological alterations were observed and the percentage of rod-shaped cardiomyocytes, the apoptotic rate of cells, the rate of pseudopodium formation and the nuclear distances of cardiomyocytes were detected during culture.

RESULTS(1) The percentage of rod-shaped cardiomyocytes decreased gradually in the first 3 days of cell culture. The percentage of rod-shaped cardiomyocytes cultured without fetal bovine serum (FBS) decreased more rapidly than those cultured with FBS. No differences were noticed between with and without the addition of apoptotic inhibitor BA. The apoptotic rate of cardiomyocytes increased in the first 3 days of cell culture, and the apoptotic rate of cells cultured without FBS increased more than that cultured with FBS. Also BA had no effect on apoptotic rate. (2) Cardiomyocytes cultured with FBS spread from the intercalated disk and extended pseudopodium on the second or third day of cell culture. Cardiomyocytes with thin membranous pseudopodium developed would survive and spread laterally at the 6th day of culture. Cells with the elongated morphology gradually spread extensively and took on a spheroidal shape. Myofibrils gradually lost their parallel. Cells cultured without FBS had no pseudopodium formation. The intercalated disk of cells gradually changed blunt. There was no effect on the rate of pseudopodium formation when added with apoptotic inhibitor BA. (3) Cytoskeletal remodeling occurred in survived cardiomyocytes. After 6 days of culture, cardiomyocytes exhibited characteristic of redifferentiation. (4) The distance between nuclei decreased in a single cardiomyocyte cultured with FBS for the cytoskeletal reconstruction, whereas it remained unchanged in cardiomyocytes cultured without FBS.

CONCLUSIONWe clarify the pseudopodium developed on the second or third day of cell culture will be the critical morphological signs of survival cultured adult rat cardiomyocytes. It is necessary to add FBS for the formation of pseudopodium.

Animals ; Cell Culture Techniques ; Cell Surface Extensions ; physiology ; Cell Survival ; Cells, Cultured ; Male ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley

This study was aimed to discover the novel HLA recombination haplotypes and investigate the distribution of haplotypes in Chinese Han population. Based on the HLA-A, B, DRB1 typing results of 179 family members, 791 haplotypes were assigned by the mode of inheritance. The results showed that a total of 4 novel recombinant haplotypes in HLA-DRB1 locus region were observed in 4 families, which ratio of paternal to maternal chromosomes was 3:1. The recombination ratio between HLA-DRB1 and HLA-A or B loci was 0.92% (4/433). There were a total of 362 kinds of HLA-A, -B, -DRB1 haplotypes to be confirmed in Chinese Han partial population. A33-B58-DR17, A2-B46-DR9, A30-B13-DR7, A11-B13-DR15, A11-B75-DR12 and A2-B46-DR14 were the most common haplotypes that was consistent with the distribution of HLA alleles in unrelated donors. There were A1-B63-DR12, A29-B46-DR15, A1-B61-DR10, A34-B35-DR9, A29-B54-DR4, A23-B13-DR16 and A34-B62-DR15 haplotypes and so on, which were rare haplotypes not yet reported in Chinese. It is concluded that the HLA-A-B-DRB1 haplotypes would be confirmed by analysis of their family pedigree. The results obtained in this study are basic data for study of Chinese anthropology, organ transplantation and disease correlation analysis.
Alleles ; Asian Continental Ancestry Group ; genetics ; Gene Frequency ; HLA Antigens ; classification ; genetics ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Haplotypes ; Humans ; Pedigree ; Recombination, Genetic