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Objective To investigate the association of polymorphisms in the filaggrin (FLG)gene with the occurrence and clinical phenotypes of atopic dermatitis (AD).Methods A questionnaire survey was carried out to collect data from 261 patients with AD,including the diagnosis of allergic rhinitis and asthma,and the severity of AD.Mixed food allergen screening test and mixed inhaled allergen screening test were performed in a part of patients,so was the detection of total serum IgE and eosinophil cationic protein (ECP).Among the above AD patients and 276 healthy controls,17 polymorphic sites in exon 3 of the FLG gene,including R444G,T454A,P478S,H519N,D836D,S1482Y,A1805V,R1891Q,1961Q,S2166S,Y2194H,H2330H,D2339N,S2366T,E2398Q,K2444E and E2652D,were genotyped by overlapping PCR and DNA sequencing.Results Binary logistic regression analysis and chi-square test showed no correlations between the 17 polymorphic sites in the FLG gene and the occurrence of AD (all P ＞ 0.05).However,the H519N polymorphic site was associated with AD complicated by asthma (x2 =8.680,P =0.011),and the AA genotype of H519N could increase the risk of asthma in the AD patients (P =0.004,OR =1.061,95％ CI:1.016-1.109).The S2366T and K2444E polymorphic sites were associated with food sensitization in the AD patients (x2 =6.520,6.121,P =0.038,0.047,respectively),and the GG + CG genotype of S2366T (P =0.012,OR =1.396,95％ CI:1.054-1.849)and its G allele (P =0.037,OR =1.350,95％ CI:1.008-1.807) both could increase the risk of food sensitization in the AD patients.Similarly,the AA + GA genotype of K2444E (P =0.013,OR =1.393,95％ CI:1.049-1.850)and its G allele (P =0.028,OR =1.380,95％ CI:1.025-1.857) could increase the risk of food sensitization in the AD patients.Conclusions The FLG polymorphisms may be predisposing factors for some AD-related clinical phenotypes in Chinese Han population.The H519N gene may be associated with AD complicated by asthma,and the S2366T and K2444E genes may be related to food sensitization in AD patients.
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Objective To investigate the association between high-risk types of human papillomavirus (HR-HPV) load and the expression of nestin in different cervical lesions.Methods The hybridization capture Ⅱ (HC-Ⅱ) assay was used to test the HR-HPV load from 60 patients who were the first time to do the cervical cancer screening in Xiangya Hospital,and immunohistochemistry was used to detect the expression of nestin in those biopsy tissue samples.Results (1) The lgHPV level (logarithm of HR-HPV load) in the high level lesion group was higher than the low level one (P ＜0.05),and HR-HPV load was positively associated with the degree of cervical lesions (rs =0.269,P =0.037).(2) The expression of nestin in A group was weaker than groups B and C (H =7.271,22.843,P ＜0.01),and the expression of nestin in C group was stronger than B group (H =7.270,P ＜0.01),and the expression of nestin was positively associated with the degree of cervical lesion (rs =0.646,P =0.000).(3) The HR-HPV load was positively associated with the expression of nestin (P ＜ 0.05).Conclusions The HR-HPV load and the expression of nestin are closely related to the cervical lesions,and the joint detection has a referential value for early prevention of cervical lesions and prediction of progress of cervical precancerous lesion.It might guide the early prevention and treatment of cervical cancers.
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BACKGROUND:Some scholars believe that anesthesia can improve the quality of cord blood separation and colection, and subarachnoid block is commonly used in cesarean section, but there is no research on the influence of subarachnoid block on differentiation potential of mononuclear cels into neural stem cels and astrocytes. OBJECTIVE: To study the influence of subarachnoid block on differentiation potential of mononuclear cels in the cord blood of neonates into neural stem cels and astrocytes. METHODS:Mononuclear cels isolated from 20 neonates delivered spontaneously and 20 neonates born by cesarean delivery undergoing subarachnoid block were culturedin vitro, acting as control group and study group. After delivery, cord blood samples were taken to isolate and culture mononuclear celsin vitro. After 3 days of routine culture, the cels were subject to non-induced culture and induced differentiation into neural stem cels. The cultured cels were identified by the cellmakers Nestin and glial fibrilary acidic protein with immunohistochemistry identification. RESULTS AND CONCLUSION:The Nestin and glial fibrilary acidic protein showed no difference between the two groups after induced differentiation (P > 0.05), indicating subarachnoid block has no impact on the differentiation of mononuclear cels in the cord blood from neonates into neural stem cels and astrocytes.
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ObjectiveTo investigate whether neuronal intermediate filament protein(NF-66) could be used as a molecular marker for the determination of malignancy of insulinoma.MethodsThe expression of NF-66 protein was detected in insulinoma and pana - cancerous tissues and 3 insulinoma cell lines by immunohistochemistry staining. The relationship between the expression of NF-66 protein and the clinicopathological characteristics,survival was analyzed by univariate and multivariate statistical analysis.ResultsExpression of NF-66 was found in 102(77％ ) out of 132 insulinomas and none of 98 paired control (P =4.86 × 10-31 ).NF-66 was highly expressed in 3 insulinoma cell lines.The expression of NF-66 was found in 96 (81％) out of 118 benign tumors (P =0.003),while out of 14 malignant insulinomas,6(43％ ) were found to express NF-66 ( P =0.003 ).The expression of NF-66 was significantly associated with tumor size (3 cm as the cut-off point),distant metastasis (38％ vs 81％,P =0.013) and distant plus lymph node metastasis (46％ vs 81％,P =0.009),respectively.The expression of NF-66 was not correlated with age,gender,recurrence and overall survival.ConclusionsDown-regulation of NF-66 was significantly associated with tumor malignancy,suggesting that NF-66 could be a potentially novel molecular bionarker to distinguish malignancy from benign insulinoma.
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Objective: To study the influence of morphine on the expression of nestin in the ependymal epithelia, central gray and hippocampal formations in mice. Methods: Twenty health mice were evenly randomized into control group and experiment group. Mice in the control group were injected with normal saline (0.1 ml daily) and those in the experimental group were injected with morphine (0.1 ml, 1 mg daily). Thirty days later, the mice brain samples were harvested and made into paraffin sections. Immumohistochemical ABC technique was used to observe the expression of nestin under light microscope. The images were analyzed with the image analytical system. Results: In the control group, the ependymal epithelia, the central gray, the periventricular gray substances and the hippocampal formations had weak expression of the nestin, with a mean gray scale of 150.98±13.31; there were 5 kinds of nestin-positive cells: (1) the basal cells of ependymal epithelium, (2) cells distributed in the periventricular gray substance and the deep lamella of central gray, (3) cells distributed in the superficial lamella of central gray, the subiculum, the parahippocampal gyrus and the cortex in II, III layers of the entorhinal area, (4) cells frequently seen in the tectum of rnidbrain and the subiculum, and (5) cells distributed in the tectum of midbrain, the hippocampus, gyrus dentatus, parahippocampal gyrus and the cortex in V layer of the entorhinal area; the density of nestin in the subiculum and entorlimal area was (7.20 ± 1.23) mm2. In the experiment group, the ependymal epithelia, the central gray, the periventricular gray substances and the hippocampal formations had positive expression of the nestin, with the mean gray scale being 133.03 ± 22.28; the density of the above-mentioned 5 kinds of cells increased; the density of nestin in the subiculum and entorhinal area was (10.50±1.43) mm2. The mean value of gray scale and nestin-positive neurons were significantly different between the 2 groups (P<0.05). The cellular proliferation was seen in the ependymal epithelia. The second kind of cells appeared in the superficial lamella of central gray, parahippocampal gyrus and the cortex in I, II and III layers of the entorhinal area. The third kind of cells increased in the hippocampal pyrarnidal layer and the fourth kind of cells increased in the tectum of midbrain and the subiculum. Conclusion: Morphine can promote nestin expression in the ependymocyte, the central gray, the periventricular gray substance and the hippocampal formation; it can also promote the proliferation, differentiation and migration of the ependymocytes and neurocytes.
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Objective To construct the recombinant plasmid containing human filaggrin gene,purify and identify the immunoreactivity of the recombinant protein,and establish the indirect ELISA to detect AFA for diagnosis of RA.Methods The constructed plasmids were transformed into E. Coli Rosettagami(DE3).This fusion protein was purified by NAT chromatography.ELISA coated with the fusion protein Was established to detect the AFA in serum of patients,which included 114 cases of RA,56 cases of SLE,32 cases of OA and 40 cases of normal controls. The correlation between the results of AFA and anti-CCP in RA group were compared. Results 321 bp fragment of filaggrin gene was amplified and the recombinant expression vector pET-28a( + )-filaggrin was constructed. The sequence of filaggrin gene was the same as the sequence reported in the literatures. The Rosetta-gami (DE3) strains of E. Coli with recombinant vector showed high level of filaggrin protein after induction. The SDS-PAGE showed that the plasmid expressed the filaggrin fusion protein with molecule weight of 14 000 Da. The expression protein could be purified by Ni-NAT with activity. The absorbance value of AFA in RA group was 0.473 ±0. 248 while they were 0. 160 0. 088, 0. 121±0. 070, 0.050 0. ±018 in SLE, OA and normal groups respectively. There were significant differences of absorbance values of AFA between RA and SLE, OA, control group (t = 12.004, 14. 464, 18.078, P<0. 01, respectively). The positivities of anti-filaggrin in RA, SLE and OA were 48.2%, 5.4% and 3. 1% respectively. The positivities of AFA were significantly different between RA, OA and normal control groups (x~2 = 67. 088, P < 0. 01). There was positive correlation of results between AFA and anti-CCP antibody (r = 0.42, P < 0. 05 ) . The consistency rate of results between AFA and anti-CCP was 70. 1%. Anti-CCP was negative in 10 out of 114 patients with AFA positive. AFA can be used to diagnose RA with sensitivity of 48. 2% , specificity of 96.9% , positive predictive value of 93. 2% and negative predictive values of 67. 9% . Conclusions The purified human filaggrin fusion protein is successfully purified. The indirect ELISA method based on the recombinant protein shows good sensitivity and specificity. Joint detection with AFA and anti-CCP can improve the positive rate of detection.
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Objective To explore the expression and significance of nestin in renal tubular epithelial cells in hypercholesterolemic rats. Methods Dietary-induced hyperlipidemia were induced in female SD rats by given 4% cholesterol and 1% cholic acid diet for 16 weeks. Changes of serum lipid, urinary albumin, serum creatinine and renal interstitial pathological changes were assessed. The expression of nestin and a-smooth muscle actin (α-SMA) were detected by immunohistochemical stain. Results The serum levels of total cholesterol, low density lipoprotein, urinary albumin and serum creatinine were significantly increased in hyperlipidemia group, accompanied with renal interstitial injury and fibrosis. As time extended, the expression of nestin and a-SMA in renal tubular epithelial cells were increased significantly. There was positive correlation among the expression of nestin and total cholesterol, low density lipoprotein, urinary albumin and serum creatinine( r =0.963,0.830,0.944,0.706, P <0.01). Nestin also had a positive correlation with tubular-interstitial index ( r = 0. 974, P < 0. 01) and α-SMA ( r = 0. 804, P < 0. 01). Conclusion The increased expression of nestin may be associated with renal tubular-interstitial fibrosis and tubular epithelial myofibroblast transdifferentiation in hypercholesterolemic rats.
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Objective To explore effects and mechanism of inactivation of vitamin D receptor (VDR) in intestinal tumor growth of APCmin/+ mice. Methods To generate APCmin/+VDR -/- mice through breeding, the number and size of small intestinal and colonic tumors were assessed and compared between APCmin/+ and APCmin/+ VDR -/- mice. The intestinal tumors were diagnosed with HE stain. The expressions of BCL-2,vimentin-1,Stat-1 and MSH-2 proteins of tumors were determined by immanohistocbemistry and compared be-tween APCmin/+ and APCmin/+VDR -/- mice. Results A comparison between APCmin/+ and APCmin/+ VDR -/- mice revealed that the number of the tumors, which were larger than 3mm, was significantly increased in APCmin/+ VDR -/- mice at 4 months of age (P< 0.01). HE staining indicated fistulous adenomas from small intestine and colon of two groups, Immunostaining showed Stat - 1 level in APC-min/+ tumors were increased and MSH-2 and vimentin-1 levels in APCmin/+ VDR-/- mice were increased, compared to APCmin/+tumors. Conclusion These observations suggested that inactivation of VDR promoted the intestinal tumor growth of APCmin/+ mice.
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Objective To investigate the expression of nestin, a type Ⅵ intermediate filament protein in the glomeruli with foot process effacement and the potential relationship between nestin expression in the kidney and the degree of proteinuria. Method Immunohistochemistry was used to determine the localization of nestin in the kidney samples obtained from needle biopsies of normal human and patients with minimal change disease (MCD). Puromycin aminonucleoside (PAN) nephrosis rat models were established by a single intraperitoneal injection of PAN. Both real time quatitative reverse PCR and Western blot methods were applied to evaluate the levels of nestin expression at day 1, 4, 10 and 20 after PAN injection. Results Immunohistochemistry showed that the expression of nestin in glomeruli of MCD patients was significantly reduced compared with normal samples (0.93±0.08 vs 1.65±0.12, P<0.05) . The mRNA and protein expressions of nestin in the rat kidney were transitorily increased by 1.23 folds and 1.48 folds of control group (NC) after 1 day of PAN injection (P<0.05), then decreased quickly in the following days. The mRNA levels of nestin in the kidney were 35.8% and 12.1% of NC after 4 days and 10 days of PAN injection, respectively, (P<0.01) as determined by real time PCR. After 20 days of PAN injury, nestin mRNA expression partly recovered to 65.8% of NC (P< 0.05 ). The protein levels of nestin detected by Western blot presented the similar trend, which were 77.0%, 58.0% and 83.4% of NC after 4 days, 10 days and 20 days of PAN injection, respectively (P<0.05). The degree of proteinuria in puromycin aminonucleoside nephrosis rats was negatively correlated with both mRNA and protein levels of nestin in the kidney(r=-0.667,P<0.05 and r=-0.621 ,P<0.05, respectively). Conclusions The expression of intermediate filament protein nestin is down-regnlated in the kidney characterized with foot process effacement and negatively correlated with the degree of proteinuria in puromycin aminonucleoside nephrosis rats. Nestin may play a potential role in modulating the structure and function of podocyte.
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Objective:To study the influence of morphine on the expression of nestin in the ependymal epithelia,central gray and hippocampal formations in mice.Methods:Twenty health mice were evenly randomized into control group and experiment group.Mice in the control group were injected with normal saline(0.1 ml daily)and those in the experimental group were injected with morphine (0.1 ml,1 mg daily).Thirty days later,the mice brain samples were harvested and made into paraffin sections.Immumohistochemical ABC technique was used to observe the expression of nestin under light microscope.The images were analyzed with the image analytical system.Results:In the control group,the ependymal epithelia,the central gray,the periventricular gray substances and the hippocampal formations had weak expression of the nestin,with a mean gray scale of 150.98?13.31;there were 5 kinds of nestin-positive cells:(1) the basal cells of ependymal epithelium,(2)cells distributed in the periventricular gray substance and the deep lamella of central gray, (3)cells distributed in the superficial lamella of central gray,the subiculum,the parahippocampal gyrus and the cortex inⅡ,Ⅲlayers of the entorhinal area,(4)cells frequently seen in the rectum of midbrain and the subiculum,and(5)cells distributed in the tectum of midhrain,the hippocampus,gyrus dentatus,parahippocampal gyrus and the cortex in V layer of the entorhinal area;the density of nestin in the subiculum and entorhinal area was(7.20?1.23)mm~2.In the experiment group,the ependymal epithelia,the central gray,the periventricular gray substances and the hippocampal formations had positive expression of the nestin,with the mean gray scale being 133.03?22.28;the density of the above-mentioned 5 kinds of cells increased;the density of nestin in the subiculum and entorhinal area was(10.50?1.43)mm~2.The mean value of gray scale and nestin-positive neurons were significantly different between the 2 groups (P