Objective To identify mutations site and clinical characteristics of a familial hypercholesterolemia(FH) proband diagnosed clinically through DNA sequencing and family analysis in the proband and his family members of 3 generations.Methods Blood samples and clinical data of the kindred of total 29 from 3 generations members were collected.Proband had a physical examination electrocar-diogrom and vascular ultrasound.The proband and his family members took routine clinical exams,and genomic DNA was isolated.The promoter region and the 18 exons of low density liporotein receptor(LDLR) gene were screened by Touch down polymerase chain reaction -single strand conformation polymorphism(PCR-SSCP) and DNA sequencing.The result of sequencing were matched gene sequence published in the BLAST database.Results 1.Increased intima-media thickness and plaque were detected in the common carotid artery,right subclavian artery of the proband.Aortic valve regurgitation was found by echocardiography.2.No mutation R3500Q of ApoB100 was observed.3.Two heterozygous mutations in exon 10 and 13 of LDLR gene (W462X and A606T) were identified.The proband and 5 members of paternal relatives showed W462X heterozygosis mutation in exon 10 of LDLR gene which introduced the change from tryptophone to a new stop codon.The proband's mother and grandmother harboured A606T heterozygous mutation in exon 13 of LDLR gene due to a single base pair substitution of G for A in the codon for residue 1 879.Conclusions Disease causing mutations of proband are W462X and A606T compound heterozygosis mutation in exon 10 and 13 of LDLR gene inherited from mother and father.Proband shows homozyous phenotype though the genotype analysis indicates heterozygous mutations.

OBJECTIVESTry to induce liver-derived dendritic cells proliferation by plasmid-IL-3 and CD40L genes in mice in vivo.

METHODSRapid tail-vein injection of large amounts of plasmid-carrying genes was performed to transfect genes in mice livers. Liver nonparenchymal cells were isolated by Percoll gradient centrifugation. Dendritic cell markers were detected and dendritic cells were sorted out by flow cytometry. Morphology of dendritic cells was studied microscopically (with Giemsa staining) and under scanning electron microscopy.

RESULTSLiver nonparenchymal cells dramatically increased in the liver lobules, portal and periportal areas in the treated group, but not in the control group. B220+/DEC205+ dendritic cells were detected and sorted by flow cytometry. There were more B220+/DEC205+ dendritic cells (16.0%) in the experiment group than those in the control group (1.1%). Morphologically, the sorted B220+/DEC205+ cells showed irregular shaped nuclei, paucity of cytoplasmic granules and extensive cytoplasmic processes.

CONCLUSIONB220+/DEC205+ dendritic cells were expanded in vivo in mice livers by rapid tail-vein injection of plasmid-carrying genes encoding IL-3 and CD40L in a large amount. Inducing liver dendritic cell proliferation in vivo provides a more convenient way for studying the biology of these cells.

Animals ; CD40 Ligand ; genetics ; Dendritic Cells ; cytology ; Flow Cytometry ; Hepatocytes ; cytology ; Interleukin-3 ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Transfection

Objective To explore the molecular basis of familial hypercholesteraemia(FH)by analyzing the phenotype and genotype relationship through identify the low density liporotein receptor(LDL-r)gene mutation in a FH kindred.Methods A male patient of 15 years old was selected to examine the electrocardiogram,lipid.Color Doppler was used to examine heart and great vessels.The promoter region and the 18 exons of the LDL-r gene were screened by touch-down polymerase chain reaction(PCR)and DNA sequencing.Results The caro-tid intima-media thickness(IMT)was increased to 0.23 cm,while coronary flow velocity reserve(CFVR)was decreased to 1.57,and mode-rate mitral regurgitation was found in the proband.The genetic alteration G→A change at 1 448 of exon 10 causing premature stop codon(W462X).The same heterozygous nonsense mutation was also found in his father.The mutation had been reported in other Chinese patients.In vitro experiments showed that W462X mutation leads to low LDL binding and internalization ability.Conclusions The homozygous mutation(W462X)in exon 10 of the LDL-r gene were identified in the clinically heterozygous FH proband.The W462X mutation is the underl-ying cause of hypercholesterolaemia and clinical AS manifestations.W462X is recurrent mutation among Chinese FH patients.It might be a hot spot mutation in LDL-r in Chinese FH.J Appl Clin Pediatr,2009,24(1):18-20

OBJECTIVETo investigate the effect of local application of bFGF combined with sucralfate on the cell proliferation during continuous tissue expansion (CTE).

METHODSNine white pigs were selected to undergo the continuous tissue expansion in this study and treated with bFGF and sucralfate, respectively as the following groups: group 1 with both bFGF and sucralfate, group 2 only with bFGF, group 3 with only sucralfate, and group 4 with saline as control. Fifteen samples were taken in each pig for immunohistochemistry analysis 1-14 days and 6 weeks after the operation.

RESULTSIn the group with both bFGF and sucralfate, the epidermic basal cells proliferated significantly after the operation and reached top level in 3 days, which was statistical higher than the control group, but the multiplication of basal cell was the lowest 14 days after the operation, still more than the control group. In dermal layer, proliferation of fibroblasts, vessel endothelial cells, hair follicles epidermic cells and sweat gland epicytes was also significant higher in the group with both bFGF and sucralfate than that the control group and reached top level 7 day after the operation, but the proliferation of cells decreased obviously 14 days after the operation, still higher than the control group. The mitotic activity of cells returned to the basal level in 42 days. There were no significant differences among the group 2, group 3 and group 4.

CONCLUSIONLocal application of both bFGF and sucralfate could be more effect to induce cells multiplication during early skin expansion to facilitate the growth of neoformed skin soft tissue.

Animals ; Cell Proliferation ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Fibroblast Growth Factors ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Sucralfate ; pharmacology ; Swine ; Time Factors ; Tissue Expansion ; Tissue Expansion Devices

AIMTo observe the expression of Presenilin-1 (PS-1) and production of amyloid beta-protein (Abeta) in hippocampus of female senile rats and to investigate the effect of vitamin E(VE) on preventing Alzheimer's disease after menopause.

METHODSThe animal model was established using female senile rats. Experimental groups (n=8) were respectively given different doses of VE(5 mg/kg, 15 mg/kg, 60 mg/kg) per day. The expression of PS-1 in hippocampus was detected by immunohistochemistry, the level of Abeta in hippocampus was measured by Radioimmunoassay, and neuronal ultrastructure in hippocampal DG area was observed using transmission electron microscope.

RESULTSThe expression of PS-1 in rat hippocampus of senile control group was stronger than that of adult control group. PS-1 expressed weakly in three medication groups along with augmentation of dosage. The levels of Abeta were found to correlate statistically with the expression of PS-1. The content of Abeta in VE groups was significantly decreased compared to that in senile control group (P < 0.01). There were some changes in the neuronal ultrastructure of senile rats. Neurons were gradually recovered in VE groups.

CONCLUSIONVE may depress the production of Abeta by regulating the expression of PS-1, reducing neuronal injuries. VE may play a role in neuronal protection.

Aging ; Alzheimer Disease ; metabolism ; Amyloid beta-Peptides ; metabolism ; Animals ; Female ; Hippocampus ; drug effects ; metabolism ; Presenilin-1 ; metabolism ; Rats ; Rats, Wistar ; Vitamin E ; pharmacology

OBJECTIVETo analyze the correlation between myocardial ischemia and carotid atherosclerosis in hypertensive patients.

METHODSThe clinical data were collected from 85 hospitalized hypertensive patients admitted between May 2005 and September 2008 without the complication of coronary artery disease as confirmed by cardiac computed tomographic angiography (CTA). According to the results of treadmill exercise test, the patients were divided myocardial ischemia group and ischemia-free group. Univariate and multivariate analyses were used to screen the risk factors of myocardial ischemia. The correlations were analyzed between myocardial ischemia, common carotid artery intima-media thickness (IMT), Crouse score of the carotid plaque, thickness of the intraventricular septum and left artrium. The receiver operating characteristic (ROC) curves were used to evaluate the sensitivity and specificity of IMT and Crouse score in predicting the presence of myocardial ischemia in hypertensive patients.

RESULTSCarotid plaque formation was identified as the major risk factor of myocardial ischemia in hypertensive patients (OR=4.982, P=0.004). The incidence of myocardial ischemia in the hypertensive patients with carotid plaques was significantly higher than that in the patients without the plaque (Chi2=9.317, P=0.002). Myocardial ischemia in hypertensive patients was positively correlated to the thickness of the intraventricular septum (r=0.362, P=0.001) and left artrium (r=0.298, P=0.009), and the IMT of the common carotid artery was positively correlated to the thickness of the intraventricular septum (r=0.231, P=0.045). The area under cure (AUC) of the ROC curve of Crouse score was 0.726-/+0.061 in predicting the presence of myocardial ischemia in the hypertensive patients (P=0.001), and that of IMT was 0.682-/+0.061 (P=0.006).

CONCLUSIONCarotid plaque formation is the major risk factor of myocardial ischemia in hypertensive patients and shows a positive correlation to the onset of myocardial ischemia, but both the common carotid artery IMT and the Crouse score of the carotid plaque are not accurate markers for predicting myocardial ischemia in patients with hypertension.

Adult ; Aged ; Aged, 80 and over ; Atherosclerosis ; complications ; pathology ; Carotid Artery, Common ; diagnostic imaging ; pathology ; Coronary Angiography ; methods ; Female ; Humans ; Hypertension ; complications ; pathology ; Male ; Middle Aged ; Multivariate Analysis ; Myocardial Ischemia ; etiology ; pathology ; Risk Factors ; Tomography, X-Ray Computed ; Ultrasonography

OBJECTIVETo investigate the initial changes in the gut microenvironment that accompany intestinal endotoxemia related to alcoholic fatty liver disease (ALD) in order to explore the potential initiating factors and to observe the effect of probiotic therapy on these factors.

METHODSFifty Sprague-Dawley male rats were randomly divided into an ALD model group (alcoholic intragastric administration), an intervention group (ALD with probiotic intragastric administration), and a control group (physiological saline intragastric administration). Histological changes of the liver were evaluated using hematoxylin-eosin staining and light microscopy. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglycerides (TG), and plasma endotoxin and coli bacillus were determined. The structural integrity of intestinal mucosa and tight junctions were observed by transmission electron microscopy. Occludin protein expression in intestinal epithelial cells was detected by immunohistochemistry.

RESULTSAfter four weeks, the three groups showed significant differences in the plasma endotoxin levels [control: (0.67+/-0.14) pg/ml, model: (4.42+/-1.28) pg/ml, and intervention: (2.88+/-0.83) pg/ml; F = 27.288, P = 0.000] and numbers of Escherichia coli [control: (2.31+/-0.39) lg3/ml, model: (3.23+/-0.41) lg3/ml, and intervention: (2.24+/-0.44) lg3/ml; F = 10.692, P = 0.001]. The plasma endotoxin level and E. coli number were significantly higher in the model group than in the control group and the intervention group (all P less than 0.05). The three groups showed no significant differences in the levels of ALT, AST, and TG at four weeks. After eight weeks, however, all three serum markers were significantly different between the three groups [ALT: control: (62.33+/-7.12) U/L, model: (95.50+/-8.73) U/L, and intervention: (81.33+/-6.19) U/L; F = 18.051, P = 0.000]; [AST: control: (90.50+/-10.67) U/L, model: (130.00+/-14.91) U/L, and intervention: (110.33+/-7.26) U/L; F = 30.170, P = 0.000]; [TG: control: (0.84+/-0.84) mmol/L, model: (1.40+/-0.17) mmol/L, and intervention: (1.10+/-0.17) mmol/L; F = 10.592, P = 0.001]. In addition, the three groups showed significant differences in E. coli number [control: (2.23+/-0.46) lg3/ml, model: (4.81+/-0.29) lg3/ml, and intervention: (3.61+/-0.50) lg3/ml; F = 23.579, P = 0.000] and plasma endotoxin level [control: (0.52+/-0.21) pg/ml, model: (12.46+/-2.61) pg/ml, intervention: (6.83+/-1.74) pg/ml; F = 30.731, P = 0.000]. The levels of ALT, AST, TG and endotoxin, and the number of E. coli were all significantly higher in the model group than in the control group and the intervention group (all P less than 0.05). Small intestinal epithelial cell structural failure was more apparent and intercellular gaps more broad after eight weeks than after four weeks for all three groups. However, the intervention group showed clearer cell connection structures and less extensive cell gap broadening than the model group at eight weeks. After eight weeks, the occludin protein had become significantly down-regulated and distributed in a non-continuous pattern in the model group, as compared with the control group. However, the occludin protein expression was higher in intervention group than in the model group.

CONCLUSIONIntestinal endotoxemia related to perturbations in the microenvironment occurs in the early phase of ALD, and the increased intestinal permeability appears to be the initial factor of elevated plasma endotoxin, which may lead to liver damage. Probiotic therapy can reduced plasma endotoxin levels and postpone ALD progression by altering the composition of the gut microbiota and up-regulating expression of the occludin protein in intestinal epithelial cells.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Endotoxins ; blood ; Escherichia coli ; isolation & purification ; Fatty Liver, Alcoholic ; microbiology ; therapy ; Intestinal Mucosa ; metabolism ; microbiology ; Intestine, Small ; metabolism ; microbiology ; Male ; Occludin ; metabolism ; Probiotics ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Triglycerides ; blood

OBJECTIVETo determine the value of fluorescence in situ hybridization (FISH) to the diagnosis of chromosome abnormality in genetic diseases and prenatal diagnosis.

METHODSFISH was performed using appropriate probes, including alpha-satellite DNA probe, chromosome sequence specific probe and whole chromosome painting probe, to examine the blood samples from 36 patients who were suspected of having chromosome abnormality by conventional cytogenetics, and to examine the amniocytes from 45 pregnant women who were in need of prenatal diagnosis.

RESULTSAmong 36 patients, the following karyotypes 45, X; 45, X/46, XX; 45, X/46, Xr(X); 46, X, i(Xq); 47, XXY; 46, XX, t(4;7); 47, XYY; 47, XXX; 47, XXY, inv(7); 46, XY, inv(7); 47, XX, +21 were detected by FISH. Of the fetuses of the 45 pregnant women, two fetuses with chromosomal abnormalities were diagnosed by FISH; the karyotypes were 47, XX, +18 and 46, XY, der(15) t(Y;15) respectively.

CONCLUSIONFISH can precisely and rapidly detect the chromosome abnormalities. It is a complement to the conventional cytogenetics and can be widely used in the diagnosis of genetic diseases and prenatal diagnosis.

Adult ; Amniocentesis ; Chromosome Aberrations ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Male ; Prenatal Diagnosis ; Sex Chromosome Aberrations ; Turner Syndrome ; diagnosis

AIMTo establish chromatographic fingerprint of Carthamus tinctorius L. by RP-HPLC in order to control the quality of Carthamus tinctorius L.

METHODSThe gradient elution mode was applied in chromatographic separation, and data were analysed by "Computer Aided Similarity Evaluation" software to compare the quality of Carthamus tinctorius L. samples from different habitats.

RESULTSSamples from different habitats were of high similarity, though a few samples showed evident difference in fingerprint graphics.

CONCLUSIONThe RP-HPLC fingerprint method is repeatable, feasible in analysis of Carthamus tinctorius L. and can be used in quality assessment of Carthamus tinctorius L. Chemical components in Carthamus tinctorius L. samples from various habitats are similar, and their ratios between each other are stable.

Carthamus tinctorius ; chemistry ; Chalcone ; analogs & derivatives ; chemistry ; isolation & purification ; China ; Chromatography, High Pressure Liquid ; methods ; Ecosystem ; Flowers ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Quinones ; chemistry ; isolation & purification

AIMTo observe the effect of vitamin E (VE) on ovarian apoptosis-related protein Bcl-2 and Bax and its impact on antioxidant capacity in aged female rats and to study the senility-delaying effect and mechanism of VE on ovary.

METHODSNatural aging female rats were given different doses of exogenous VE. Then apoptosis regulatory protein Bcl-2, Bax expression in ovarian grandlose cells were detected by using immunohistochemical methods and Western blot. The contents of serum total superoxide dismutase (SOD) activity and malondialdehyde (MDA) were detected by using biochemical methods.

RESULTSContrasted with adult control group, the level of Bcl-2 expression in Senile control group was lower and the level of Bax expression was higher (P < 0.01), Serum SOD activity decreased and the level of MDA significantly increased (P < 0.01). Contrasted with senile control group, the level of Bcl-2 expression increased in VE group, the level of Bax expression decreased (P < 0.05), the level of MDA expression significantly decreased (P < 0.01).

CONCLUSIONVE can regulate apoptosis-related protein Bcl-2, Bax expression and confront free radical damage which contribute to a protective effect for ovarian grandiose cells.

Aging ; Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Female ; Granulosa Cells ; cytology ; Ovary ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Vitamin E ; pharmacology ; bcl-2-Associated X Protein ; metabolism