Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.

Adult ; Aneurysm, Dissecting ; etiology ; Coronary Aneurysm ; etiology ; Humans ; Male ; Myocardial Infarction ; etiology ; Wounds and Injuries ; complications

Acute Disease ; Coronary Aneurysm ; diagnostic imaging ; etiology ; Female ; Humans ; Infant ; Intracranial Hemorrhages ; diagnosis ; diagnostic imaging ; etiology ; Male ; Mucocutaneous Lymph Node Syndrome ; complications ; diagnosis ; diagnostic imaging ; Tomography, X-Ray Computed

Objective To establish a stable in vitro model of blood-brain barrier (BBB) simulating in vivo state using the primary-cultured rat brain microvascular endothelial cells (BMVECs) and pericytes.Methods The primary rat BMVECs and pericytes were isolated,purified and cultured.The isolated cells were identified by immunocytochemical staining method.An in vitro model of BBB was constructed using Transwell inserts (pore size 0.4 μm) coculture.Its barrier function was evaluated by the 4-hour leakage test,tight junction protein identification,transendothelial resistance detection,and permeability test.The difference between the cocultured model and simple BMVEC model across the membrane resistance values,and the permeability difference of the small molecule sodium fluorescein (Na-F) were compared.Results Confluent BMVEC monolayers demonstrated a typical cobblestone appearance and the pericytes displayed irregular shape and overlapping growth.Immunodouble labeling technique identification showed that the pericytes positively expressed α-srmooth muscle actin (α-SMA) and neuron-glial antigen 2 (NG2); after the fusion of cocultured model endothelial cells,the surface leakage test became positive; immnocytochemical staining shows that a continuous and dense tight junction formed between the endothelial cells; compared to the BMVEC model,the transendothelial electrical resistance of the cocultured model increased significantly (190.762 ± 10.326 Ω/cm2 vs.96.503 ± 8.012 Ω/cm2; t=- 24.489,P ＜0.01),and the permeability decreased significantly (56.149％ ± 3.572％ of the single endothelial model; t =19.330,P ＜ 0.01 ).Conclusions The primary isolated rat BMVECs and pericytes cocultured the morphology,structure and barrier function of in vitro model have the basic characteristics of BBB,and they have provided a useful tool for the research of BBB.

OBJECTIVETo discuss the diagnosis of adrenoleukodystrophy(ALD) and analyse the causes of its misdiagnosis.

METHODSThe clinical and laboratory data of six cases with ALD were analyzed.

RESULTSAmong the six cases of ALD, 4 cases were of childhood cerebral ALD, 1 case of Addison only, and 1 case of adolescent cerebral ALD. Pigmentation of skin was the first symptom in 3 cases. The delay from the diagnosis of Addison's disease to that of ALD ranged from 1 to 6 years. Another 2 cases was misdiagnosed as multiple sclerosis in early stage.

CONCLUSIONThe most important reason of delay is that the physician is unfamiliar with the ALD clinical features. Assay of very long chain fatty acid (VLCFA) is useful for the early diagnosis of ALD.

Addison Disease ; diagnosis ; Adolescent ; Adrenoleukodystrophy ; diagnosis ; Child ; Child, Preschool ; Diagnosis, Differential ; Diagnostic Errors ; Humans ; Male

Adolescent ; Adult ; Aged ; Endoscopy ; Female ; Humans ; Male ; Maxillary Sinus ; surgery ; Middle Aged ; Otorhinolaryngologic Surgical Procedures ; methods ; Young Adult

Cause of Death ; Cerebrospinal Fluid Rhinorrhea ; etiology ; mortality ; Head Injuries, Penetrating ; etiology ; mortality ; Humans ; Male ; Middle Aged ; Postoperative Complications ; Sphenoid Sinus ; surgery

Objective To explore the mutation of androgen receptor(AR)gene in a patient with 46,XY disorder of sex development(DSD)and to improve the diagnostic level and understanding of androgen insensitivity syndrome(AIS).Methods The clinical data of the child was analyzed,including physical examination,relevant laboratory examination,karyotype,pelvic B ultrasound,pelvic magnetic resonance imaging(MRI)and AR gene mutation.The peripheral blood of the child and his parents were drawn,and peripheral blood DNA was extracted.The polymerase chain reaction(PCR)-DNA sequencing method was used to amplify all exons of the AR gene in the child and his parents.Then,they were directly sequenced.Results A 7-years and 2-months old child who suffered from DSD,revealed physical examination that the child had normal female external genitalia,as the clitoris length was 2.0 cm×0.8 cm,with visible vaginal opening,and there were masses at bilateral inguinal region,with a size of 1.5 cm×0.8 cm.The results of human chorionic gonadotropin(HCG)stimulation test:testosterone was 0 nmol/L,androstenedione was 1.78 nmol/L,dihydrotestosterone was 0.07 nmol/L before HCG was injected;but testosterone was 4.69 nmol/L,androstenedione was 2.10 nmol/L,dihydrotestosterone was 0.33 nmol/L after HCG was injection.Sex chromosome analysis reported 46,XY karyotype.Pelvic B ultrasound revealed the absence of a uterus and ovaries and the presence of bilateral testes like gonad at each side of internal inguinal ring,with a size of 1.4 cm×1.0 cm×0.8 cm in the left,1.5 cm×0.7 cm×0.8 cm in the right;but the kidney,ureter,urinary bladder,adrenal gland and retroperitoneal for B ultrasound revealed no abnormality.Pelvic MRI(non-enhanced and enhanced)showed the presence of a blind ending vagina between rectum and urinary bladder(40 mm in depth)and the absence of uterus and ovarian tissue.DNA sequencing found one c.1685T>C heterozygous mutation(p.Ile562Thr)on exon 2 of AR gene in the child.But retrieving and summarzing documents of the domestic and foreign information databases and websites,the locus mutation of AR gene had never been reported.The structure prediction of the mutated protein(Polyohen2 and SIFT software)was significantly changed.By verifying the locus site of the parents of this child,it was found that his mother carried the same mutation,but his father was found to be normal.Conclusions A c.1685 T>C mutation(p.Ile562Thr)on exon 2 of AR gene is a novel mutation.Combined with the patient's clinical manifestations and computer prediction results,it may suggest that the novel mutation of AR gene can lead to the occurrence of AIS.

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.

Objective To explore the effects of thiamine and riboflavin on H2O2-induced DNA oxidative damage in human umbilical vein endothelial cell line ECV304.Methods ECV304 cells were incubated with 10,100,500,1000 mg/L of thiamine or 20,100,300,500 nmol/L of riboflavin for 24 h,and then oxidative damage of cells were induced by 25 mol/L H2O2 for 30 min.DNA damage was detected with single cell gel electrophoresis(SCGE)assay.ECV304 cells incubated without H2O2,thiamine and riboflavin were served as negative controls,and those incubated with H2O2 and without thiamine and riboflavin were served as positive controls.Results H2O2 induced DNA damage,and the indices of percent of DNA damage cells,percent of tail DNA,tail length and Olive tail moment were increased.The indices of cells pretreated with 10,100,500 mg/L of thiamine or 20,100,300 nmol/L riboflavin were significantly decreased(P0.05).Conclusion Proper supplementation of thiamine and riboflavin may decrease H2O2-induced DNA oxidative damage,while excess thiamine and riboflavin supplementation may be harmful to DNA and enhance the susceptibility to H2O2 potentially.