Animals ; Extinction, Psychological ; physiology ; Fear ; physiology ; Male ; Mental Recall ; physiology ; Rats ; Rats, Sprague-Dawley ; Sleep Deprivation ; complications ; physiopathology ; Sleep, REM ; physiology

Objective To study the antigenicity of human thyrotropin receptor(hTSHR)amino terminus (amino acid 29～280)and its association with Graves' disease.Methods Total thyroid RNA was prepared from human normal thyroid tissue.RNA was then reversely transcripted and cDNA was subjected to PCR amplification.PCR product was cloned into pcDNA3.1 and the recombinant plasmid was named pcDNA3.1/hTSHR188～940bp. Balb/c mice were immunized with peDNA3.1/hTSHR188～940bp. The levels of serum thyroxin,anti-TSHR antibody(TRAb)and thyroid stimulating antibody(TSAb)were measured,and the pathological changes of thyroid tissue were also observed.Results A 753 bp fragment encoding hTSHR ectodomain amino end was obtained after PCR amplification.Confirmed by Hind Ⅲ restriction enzyme digestion and DNA sequencing,pcDNA3.1/hTSHR188～940bphad been constructed successfully,with the correct sequence and direction of hTSHR188～940bp.In the Balb/c mice treated with pcDNA3.1/hTSHR188～940bp,elevated TRAb in week 6(0.148±0.018)were observed compared with those at week o(0.106±0.006,P＜0.01),and kept a higher level till week 10(0.134±0.011,P＜0.01).T4 and TSAb index values were significantly increased in week 10.Serum T4 concentration increased from(41.02±7.97)μg/L in week 0 to(62.20±12.77)μg/L in week 10(P＜0.01);TSAb index values rose from 0.864±0.076 at week 0 to 1.392±0.615(P＜0.01).Thyroid pathological examination showed that proliferated thyroid follicular epithelial cells and foll icular eapacity increased.Inflammatory cells were occasionally found.Conclusions There are antigen epitopes in hTSHR ectodomain amino acid 29～280,which can stimulate the production of TSAb.And the latter induces hyperthyroidism and Graves' disease like manifestations.It suggests that hTSHR ectodomain amino acid 29～280 is closely associated with Graves' disease,and maybe one of important etiological factors leading to the disease.

Abstract?AlM:To analyze of the clinical features of acute retinal pigment epitheltis ( ARPE) .?METHODS: The clinical data of 36 ARPE patients ( 40 eyes) attending this center from January 2008 to January 2014 were reviewed retrospectively. Of them, 21 patients (58.3%) were male (male :female=1:0. 71). The mean age was 40. 92±7. 13 years old (range:17~60y). The mean best-corrected visual acuity (BCVA) was 0. 50±0. 26 with a range of 0. 3 ~ 1. 0. Thirty-two patients were unilateral cases. All the patients were examined for BCVA, funds photography, fluorescein fundus angiography ( FFA ) , optical coherence tomography ( OCT) . FFA was shown as three types: type ▏ to multiple black light or grape variety fluorescent spot; Type II for l lesions visible fluorescence leakage; Type Ⅲ lesions with choroid neovascularization ( CNV ) . OCT was the following three forms: multiple RPE lesions layer reflection intermittent, proliferation ( type ▏); pigment epithelial detachment with limitations neural epithelium ( typeII);types l and ll with CNV ( type Ⅲ) .?RESULTS: Ocular fundus showed that the lesions were multiple dark-gray spots with a dark circumscribed area at the macular or nearby in all 40 eyes. FFA showed:21 eyes were type ▏, 17 eyes were type II and 2 eyes were typeⅢ, BCVA between type ▏ and type II was statistically significant (P<0. 05), the same was between type 芋. BCVA between different cases in the same type and between type II, Ⅲ, was no statistical difference ( P>0. 05). OCT showed 21 eyes wwere type ▏, 17 eyes were type II and type Ⅲ 2 eyes. BCVA average between type▏ andIIwas statistically significant (P<0. 05). The mean BCVA was no statistically significant difference between type II and Ⅲ patients (P>0. 05).?CONCLUSlON:ARPE fundus demonstrated the multiple dark gray discrete lesions, the degree of visual impairment related with the presence of pigment epithelial barrier and lesion location. OCT and FFA characterized three types. FFA is shown asblack light orgrape variety fluorescent spot, and is the basis of diagnosis. OCT can display the lesions organization form of each layer clearly. lt plays a more and more important role in the diagnosis and differential diagnosis of ARPE.

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

OBJECTIVETo retrospectively analyze the effects of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on childhood chronic myelogenous leukemia (CML).

METHODOf the 24 consecutive cases, 16 were boys and 8 were girls. The median age of patients was 12 (3 - 16) years old; 16 cases were in chronic phase (CP) of CML, 1 case in accelerated phase (AP) and 5 cases in blastic phase (BP). Allo-HSCT from HLA identical siblings were performed for 5 cases, HLA haplotype was performed for 14 cases and unrelated allo-HSCT for 5 cases. Twenty-four cases underwent allo-HSCT with conditioning regimen of BUCY. Prophylaxis of graft versus host disease (GVHD) included CsA + MTX plus MMF. The average follow-up was 36 months.

RESULTAll of patients were successfully engrafted. The 5-year overall survival (OS) of the 24 cases was 81%. Four patients died after allo-HSCT including 3 cases in BP from haploidentical donors and 1 case in CP from HLA identical sibling. The 5 cases who received unrelated allo-HSCT have been alive. Among the 10 cases who survived over 5 years, 3 had chronic GVHD.

CONCLUSIONChildren with CML could be treated effectively with allo-HSCT. There were no significant differences among different donors. Transplantation to children with CML should be performed as early as possible. Preparative regimen adjustment before transplantation, the transplantation of associated comorbidities and effective prevention and treatment for CML patients after prolonged graft survival of high quality have important significance.

Adolescent ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Child, Preschool ; Cyclophosphamide ; administration & dosage ; Female ; Graft vs Host Disease ; mortality ; prevention & control ; Hematopoietic Stem Cell Transplantation ; adverse effects ; methods ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; mortality ; therapy ; Male ; Methotrexate ; administration & dosage ; Retrospective Studies ; Survival Analysis ; Transplantation Conditioning ; methods ; Transplantation, Homologous ; Treatment Outcome

OBJECTIVETo investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.

METHODSIGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.

RESULTSIGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).

CONCLUSIONIGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Podophyllotoxin ; analogs & derivatives ; pharmacology ; Receptor, IGF Type 1 ; metabolism

OBJECTIVETo investigate the expressions of chemokine receptors and interleukin (IL) receptors on the peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and their correlations with clinical features as well as SLE disease activity index (SLEDAI).

METHODSThe mRNA expressions of chemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CXCR3, CXCRS, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis.

RESULTSThe level of CCR5 mRNA in SLE patients (including active and inactive SLE) was significantly higher than that in healthy controls (P < 0.05), and there was no significant difference between active and inactive patients in this respect (P > 0.05). CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others (except for CCR8, CXCR3, and IL-10R) in active SLE patients were significantly higher than those in both inactive SLE patients and healthy controls (all P < 0.05). There were positive correlations between SLEDAI and CCR2 (r = 0.424, t = 4.313, P < 0.001), CCR3 (r = 0.518, t = 5.410, P < 0.001), CCR4 (r = 0.376, t = 3.851, P < 0.001), CCR6 (r = 0.457, t = 4.513, P < 0.001), CXCR5 (r = 0.455, t = 4.629, P < 0.001), CX3CR1 (r = 0.445, t = 4.523, P < 0.001), as well as XCR1 (r = 0.540, t = 5.445, P < 0.001). And CCR5 mRNA expression level was positively correlated with IL-4R mRNA (r = 0.313, t = 2.353, P < 0.05). The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CR1, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity (r = 0.426, t = -2.155, P < 0.05).

CONCLUSIONIncreased expressions of CCR5 and CX3CR1 on PBMCs may be indicators in clinical survey for SLE.

Adolescent ; Adult ; CX3C Chemokine Receptor 1 ; Child ; Female ; Humans ; Leukocytes, Mononuclear ; immunology ; Lupus Erythematosus, Systemic ; etiology ; immunology ; Male ; Middle Aged ; RNA, Messenger ; blood ; Receptors, CCR5 ; genetics ; Receptors, Chemokine ; genetics ; Receptors, Interleukin-10 ; genetics ; Receptors, Interleukin-4 ; genetics

OBJECTIVESTo investigate the effects of FAK-related non-kinase (FRNK) on expressions of type I collagen and matrix metalloproteinase-2 (MMP-2) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in rat hepatic stellate cells (HSC).

METHODSUsing in vitro cell culture technique, FRNK plasmids were transfected into HSC mediated by cationic liposome. Type I collagen synthesis capability in HSC was examined by 3H-Pro incorporation assay. The levels of FRNK in HSC were assayed by Western blot, and the expressions of MMP-2 and TIMP-2 were assayed by RT-PCR on mRNA levels.

RESULTSThe exposure of HSC to FRNK caused the expression of FRNK protein to be up-regulated, and the FRNK protein contents reached the highest point at 48 h after the transfection, P less than 0.05. The expressions of MMP-2 mRNA were up-regulated by FRNK; the expressions of TIMP-2 mRNA were down-regulated by FRNK; the ratios of MMP-2 mRNA/TIMP-2 mRNA were enhanced by FRNK.

CONCLUSIONAfter FRNK was transfected, the capability of type I collagen synthesis in HSC was inhibited, which may be related to the up-regulation of MMP-2 mRNA/TIMP-2 mRNA.

Animals ; Cells, Cultured ; Collagen Type I ; metabolism ; Hepatic Stellate Cells ; cytology ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Plasmids ; Protein-Tyrosine Kinases ; genetics ; RNA, Messenger ; genetics ; Rats ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transfection

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.
Animals ; Blood-Brain Barrier ; metabolism ; Brain ; metabolism ; Cell Nucleus ; metabolism ; Cerebral Cortex ; metabolism ; Female ; Fibroblast Growth Factor 1 ; administration & dosage ; pharmacokinetics ; Gene Products, tat ; administration & dosage ; pharmacokinetics ; Hippocampus ; metabolism ; Injections, Intravenous ; Male ; Mice ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; administration & dosage ; pharmacokinetics

Objective To analyze the relationship between prevalence of metabolic syndrome (MS) and behavior habits such as smoking,alcohol intake,physical activity,sleeping hours.Methods A multi-stage stratified cluster sampling was conducted in 31 provinces,autonomous regions,and municipalities in China according to the program of National Nutrition and Health Survey.Questionnaire survey,interview,physical examination,measurement of biochemical indices,and dietary investigation were done.In total,4937 men aged 18 to 45 years old were selected.Results The MS prevalence was 6.9%(329/4937).The rate of drinking was 49.4% and smoking rate was 54.4%.The percentage of sleeping was hours from 7 to 8 was 70.5%.The percentage of spending time on physical activity over 420 minutes/week was as high as 41.9%.Data from single logistic regression showed volume of smoking more than 600 packs and alcohol intake were associated with high risk of MS and no significantly associations were found between MS and the duration of physical activity and the sleeping time.Multivariate logistic regression showed that the risk of MS in smokers with the volume more than 600 packs age increased significantly as compared to nonsmokers with the odds ratio as 1.443 (95%CI:1.044-1.993 ) and 1.765 (95%CI:1.150-2.708) in smokers with volume from 600 to 899 packs age,and more than 900 packs age respectively.Compared to the nondrinkers,the odds ratios were 1.525 (95%CI:1.135-2.048),2.322(95%CI:1.671-3.255) and 2.033 (95%CI:1.478-2.796) in subjects volume of alcohol dranking as 1 to 2 times per week,3 to 4 times per week and more than 5 times per week respectively.Conclusion Tobacco and alcohol were associated with high risks of MS.