Objective:To study the trinucleotide repeats of GCN (GCA, GCT, GCC, GCG) encoding Alanine in exon 3 of the PHOX2B gene among healthy individuals from southwest China and two patients with Congenital central hypoventilation syndrome (CCHS). Methods:The number and sequence of the GCN repeats of the PHOX2B gene were analyzed by capillary electrophoresis, Sanger sequencing and cloning sequencing of 518 healthy individuals and two newborns with CCHS, respectively. Results:Among the 1036 alleles of the 518 healthy individuals, five alleles were identified, including (GCN) 7, (GCN) 13, (GCN) 14, (GCN) 15 and (GCN) 20. The frequency of the (GCN) 20 allele was the highest (94.79%). And five genotypes were identified, which included (GCN) 7/(GCN) 20, (GCN) 13/(GCN) 20, (GCN) 14/(GCN) 20, (GCN) 15/(GCN) 20, (GCN) 20/(GCN) 20. The homozygous genotypes were all (GCN) 20/(GCN) 20, and the carrier rate was 89.58%. Four GCN sequences of the (GCN) 20 homozygous genotypes were identified among the 464 healthy individuals. The GCN repeat numbers in the exon 3 of the PHOX2B gene showed no significant difference between the expected and observed values, and had fulfilled the, Hardy-Weinberg equilibrium. The genotypes of the two CCHS patients were (GCN) 20/(GCN) 25 and (GCN) 20/(GCN) 30, respectively. Conclusion:It is important to determine the GCN repeats and genotypic data of the exon 3 of the PHOX2B gene among the healthy individuals. The number of GCN repeats in 518 healthy individuals was all below 20. The selection of appropriate methods can accurately detect the polyalanine repeat mutations (PARMs) of the PHOX2B gene, which is conducive to the early diagnosis, intervention and treatment of CCHS.

Objective To explore the role of lung dendritic cells (DCs) and Th17/regulatory T cells (Treg) pathway in the pathogenesis of chronic obstructive pulmonary disease(COPD).Methods COPD patients who received lobectomy from Sep.2015 to Mar.2016 in our hospital were enrolled and classified into non-smoking non-COPD group,smoking without COPD group and COPD group.The expression of CD80,chemokine recepter-6 (CCR6),interleukin-17A (IL-17A) and fork-head transcription factor P3 (FoxP3) were detected by immunohistochemistry (IHC) in lung tissue.Mature DCs (mDCs),immature DCs (imDCs),Th17 cells and Treg cells in lung tissue were detected by flow cytometry (FCM) and the correlation between Th17/ Treg cells with lung function was analyzed.Results (1) The expression of CD80 and FoxP3 in COPD group was decreased,while the expression of CCR6 and IL-17A was increased (P＜0.05).(2) The percentage of mDCs and Treg in lung tissue of COPD group was significantly decreased.In contrast,the proportion of imDCs and Th17 cells in COPD group was significantly increased (P＜0.05).(3) The imbalance of Th17/Treg ratio in lung tissue was seen in patients with COPD,suggesting the potential mechanism of Th17 cell-mediated proinflammatory response.(4) The percentage of Th17 cells and Th17/Treg ratio in COPD patients was negatively correlated with forced expiratory volume in the first second (FEV1) as a percentage of predicted value (FEV1％ pred),forced vital capacity(FVC) as a percentage of predicted value (FVC％ pred),FEV1/FVC.On the other hand,the percentage of Treg cells was positively correlated with FEV1％ pred,FVC％ pred,FEV1/FVC.Conclusions The data in this study demonstrate the maturation disorder of dendritic cells in lung tissue of COPD patients.The imbalance of Th17/Treg ratio suggests that Th17 cell-mediated proinflammatory response may be involved in the pathogenesis of COPD.

OBJECTIVETo assess the accuracy of quantitative fluorescence PCR(QF-PCR) for the detection of fetal chromosomal aneuploidies and its values for prenatal diagnosis.

METHODSQF-PCR and chromosomal karyotyping were used to analyze 6066 amniotic fluid samples derived from 6034 pregnant women.

RESULTSBoth QF-PCR and karyotyping analysis have detected 135 cases of fetal aneuploidies involving chromosomes 21, 18, 13, X, and Y. The QF-PCR assay was also successful in 67 cases for which amniotic fluid culture has failed. Furthermore, it has identified maternal cell contamination in 7 cases. By determining the consistency of short tandem repeat (STR) sites, the QF-PCR assay has identified 22 dizygotic twins among 32 twins with double chorions and double amniotic sacs. In 12 cases, it has signaled numerical chromosomal aberration by critical or partial abnormal values for the fluorescence peak area ratio, which were verified by karyotyping analysis as mosaicisms of chromosome aneuploidies.

CONCLUSIONThe QF-PCR can provide an useful supplement for chromosomal karyotyping and has an important role in rapid prenatal diagnosis.

Adolescent ; Adult ; Aneuploidy ; Female ; Fluorescence ; Humans ; Karyotyping ; Microsatellite Repeats ; Middle Aged ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult

BACKGROUND:Primates are considered to be the most appropriate animal model of lumbar intervertebraldisc degeneration, but the disc degenerated characteristics of monkeys were rarely reported. OBJECTIVE:To verify the degenerated regularity and characteristics of lumbar intervertebral disks in rhesus monkeys with magnetic resonance T2 mapping and T1ρimaging technology. METHODS:The sagittal lumbar intervertebral disc magnetic resonance T2 weighted imaging,T2 weighted mapping imaging and T1ρweighted imaging of 63 adult rhesus monkeys were acquired on 1.5T magnetic resonance equipment. The T2-map value and T1ρvalue of lumbar intervertebral disc regions of interest were calculated on the post-processing workstation. RESULTS AND CONCLUSION:(1) This study obtained 425 better magnetic resonance images of lumbar intervertebral disks in adult rhesus monkeys. T2-map value and T1ρvalue of nucleus pulposus were most consistent by different persons, and the Kappa coefficient was more than 0.93. (2) The T2-map value and T1ρvalue of nucleus pulposus were both negatively correlated significantly with Pfirrmann grades (r=-0.842, P<0.01;r=-0.896, P<0.01). The T1ρvalue and T2-map value of nucleus pulposus were significantly statistical y different between Pfirrmann grades I-IV (P<0.001, P<0.001). The T1ρvalue of nucleus pulposus was negatively correlated significantly with Pfirrmann grade II-III (r=-0.517, P<0.01) and Pfirrmann grade IV-V (r=-0.499, P<0.01). The T2-map value of nucleus pulposus was also negatively correlated significantly with Pfirrmann grade II-III (r=-0.617, P<0.01) and Pfirrmann grade IV-V (r=-0.652, P<0.01). (3) The T2-map value of L1-2 and L2-3 segments nucleus pulposus were significantly lower than that in L6-7 and L7-S1 segments (P<0.05). (4) There were significant differences in age among the T1ρvalue and T2-map value of nucleus pulposus (r=-0.702, P<0.001, r=-0.730, P<0.001). (5) It is concluded that magnetic resonance T2 mapping and T1ρimaging technology can objectively and sensitively assess the degenerated process of nucleus pulposus in rhesus monkeys. The degeneration in upper lumbar segments (L1-2 and L2-3) was earlier and more severe than that in lower lumbar segments (L6-7 and L7-S1) in rhesus monkeys. Age is one of the most important factors in lumbar intervertebral disc degeneration of adult rhesus monkeys.

Objective: To investigate the effects of PRX-2 gene on phenotype changes in epidermal stem cells differentiating into sweat gland cells. Methods: Epidermal stem cells and sweat gland cells separated and cultured from healthy foreskin and adult full-thick skin respectively, were identified by immunofluorescence staining. Lentiviral vector-mediated overexpression and knockdown of PRX-2 gene in epidermal stem cells were performed respectively, with empty vector-mediated epidermal stem cells as a control group. Overexpression、blank control and knowdown group′s PRX-2 expressions in gene and protein levels were detected using RT-PCR and Western blot technology. The ESCs of each group were co-cultured with sweat gland cells through transwell plate, and the expressions of CEA and β1 integrin in epidermal stem cells were determined by flow cytometry before and after co-culturing. Results: Epidermal stem cells and sweat gland cells were in line with their respective specific antigens. Before co-cultured, epidermal stem cells highly expressed β1 integrin (98.69±0.67)%, hardly expressed CEA (6.20±3.15)%. After co-cultured, β1 integrin expression levels were showed as knockdown group (19.30±0.53)% blank control group (51.20±0.79)%> overexpress group (45.91±0.93)%. There had significant differences between those of each two groups. Conclusions PRX-2 gene can inhibit the phenotypic change of Epidermal Stem Cells differentiating into Sweat Gland Cells and improve the ability to maintain their own specific antigens.

As a model organism, zebrafish have many advantages over other animal models and is suitable for studies on establishment of human disease model and mechanism.In zebrafish, there are two phases of endocrine formation during early development, which are directed by concomitant activity of many signaling pathways.Zebrafish pancreas possess similar cell structure with that of other animals, which can express various endocrine hormones including insulin.The main organs required for metabolic control, such as the pancreas, islet, and insulin sensitive tissue (muscle, liver) are conserved in zebrafish, and the mechanisms of glucose regulation in zebrafish is similar to that seen in mammalian models.These render it an excellent model to study glucose metabolism.Hyperglycemia in zebrafish model can be induced by administration of the diabetogenic drug, streptozotocin (STZ), alternatively immersion of the fish in glucose solution and water, or disturbing of signaling pathways associated with glucose metabolism.Glucose levels in adult zebrafish blood or embryo tissue and phenotype of retinal cell layers or retinal vasculature are the commonly used measurement organs in zebrafish diabetic models.

Objective To observe the changes in the lung aquaporin 1 (AQP1) expression in adrenaline-induced pulmonary edema(PE),and the effect of Methylprednisolone (MP) on its expression.Methods Fifty Wister rats of 1-month old were randomly divided into 5 groups (10 rats in each group):control,adrenaline PE,MP A,MP B,and MP C groups,respectively.Control group animals were treated with 0.27 mL 9 g/L saline;PE group was given 2.7 mg/kg adrenaline (1 ∶ 1 000) by intraperitoneal injection;MP A,MP B and MP C groups rats were intraperitoneally injected 10 mg/kg,20 mg/kg,and 30 mg/kg MP intraperitoneally immediately after intraperitoneal injection of adrenaline,respectively.The morphology changes in the lungs were observed with HE staining,and lung wet/dry weight (W/D) was measured.The levels of AQP1 mRNA,AQP1 protein,and AQP1 distribution in the lung tissues were detected by using real time-polymerase chain reaction,Western blot,and immunohistochemical method.Results (1)PE group exhibited a faster breathing rate,and double lung volume increased significantly;there was a visible hemorrhagic distribution in the lung surface and cross section,endotracheal filled with white or pink foam liquid.(2) The W/D of rats in PE group was higher than that of the control group (6.50 ± 0.53 vs.4.59 ± 0.36,P ＜ 0.05).(3) Pathological grading of PE group (3.80 ± 0.42) increased significantly compared with that of the MP A group (3.30 ± 0.48),MP B group (2.30 ± 0.68) and MP C group (1.20 ± 0.42),and there were significant differences (all P ＜ 0.05).(4) Immunohistochemistry showed that the expression of AQP1 in PE group (1.20 ± 0.79) was reduced compared with that of the control group (4.20 ± 1.03),and there were significant differences (all P ＜ 0.05).(5) The levels of AQP1 mRNA and AQP1 protein (0.12 ± 0.43 and 0.20 ± 0.04) were significantly lower than those of the control group (0.90 ± 0.32 and 0.60 ± 0.15),and there were significant differences (all P ＜ 0.05);compared with PE group,AQP1 mRNA and AQP1 protein of each group with MP treatment showed the highest values (MP A group:0.17 ±0.06 and 0.32 ±0.04,MP B group:0.39 ±0.13 and 0.37-±0.09,MP C group:0.61 ±0.21 and 0.44 ± 0.07) (all P ＜ 0.05).Conclusion The expression of AQP1 reduced in adrenaline-induced PE rats.MP could improve the expression of AQP1,and significantly ameliorate the PE and bleeding.

Objective To evaluate the short-term prediction of high-sensitivity cardiac troponin T (hs-cTnT) and other cardiovascular risk biomarkers in patients undergoing maintenance hemodialysis (MHD).Methods We conducted a cohort survey in 296 consecutive MHD patients whose clinical data were retrospectively analyzed.Before MHD,hs-cTnT and other relative cardiovascular biomarkers were detected.The end point (all-cause death) and time of occurring were recorded in the next 13 months.The differences between survival and all-cause death were analyzed by t-test,Mann-Whitney test and x2 test.The best two percentile cutoff point was calculated by X-tile and the survival rate was calculated by Kaplan-Meier Logistic regression analysis was applied to analyze the odd ratio between high risk and non-high risk hs-cTnT group.Non-high risk group was divided into intermediate risk and low risk group based on the 99th percentile of hs-cTnT in healthy population,to further evaluate its short-term prediction value for MHD patients.The short-term significance of hs-cTnT was proved to be independently associated with all-cause death by Logistic regression analysis.Results The mean value of serum hs-cTnT in survival group was 0.05 (0.03～0.07) ng/mL,while in the death group it was 0.07 (0.04～0.14) ng/mL,which had statistical significance (P =0.027).The best two percentile cutoff of hs-cTnT in MHD patients was 0.1 ng/mL.The survival rate in high risk group (hs-cTnT＞0.1 ng/mL) is lower than it in non-high risk group (hs-cTnT≤0.1 ng/mL) (76.67％ vs.96.62％,P ＜0.05).The odd ratios for high risk group and non-high risk group was 7.288 (P＜ 0.001).Moreover,further grouping the non-high risk group by hs-cTnT =0.014 ng/mL,intermediate risk group (hs-cTnT＞0.014 ng/mL) group has lower survival rate than low risk group (hs-cTnT≤0.014ng/mL),while there wasn't any death case occurred in the low risk group.Conclusions Hs-cTnT is an independent risk factor to all-cause death.Thus hs-cTnT can be a strong indicator of short-term prediction and prognostic evaluation.

Aim To study the effect of rhein on renal damage induced by aristolochic acid. Methods Ze-brafish model of aristolochic acid nephropathy, genera-ted by treating zebrafish larvae with aristolochic acid for 24 h, was treated with rhein simultaneously . Mor-pholigical changes were observed and the creatinine level in larvae tissue was measured. And mRNA ex-pression levels of inflammatory factor cox2 a and fibrosis factor TGF-β1 in larvae tissue were detected using qPCR. Results Some larvae show periocular edema and circulation system defection e. g. weak heart beat, narrow cardiac vesicle, decreased blood flow and even blockage , with a dose-response relationship after expo-sure to aristolochic acid for 24 h. The creatinine level in larvae tissue of the treated group was significantly higher than that of the control larvae. And the expres-sion levels of cox2 a and TGF-β1 in larvae tissue of the treated group were also significantly increased. Per-centage of abnormal larvae and creatinine level in lar-vae tissue were decreased when treated with rhein sim-ultaneously. And the expression levels of cox2a was down-regulated by rhein compared with the aristolochic acid treated group. But rhein had no effect on TGF-β1 expression. Conclusion To some extent rhein can protect renal from damage induced by aristolochic acid.

Aim To investigate the modeling of breast cancer in zebrafish embryos and its related protein expression. Methods 48hpf wild type AB/ TG(Transgenic) zebrafishs were micro-in-jected with breast cancer cell line: MCF-7,T-47D, MDA-MB-231 respectively, the relationship between the number of tumor and model application was investigated, and the number of sub-intestinal veins(SIVs) was detected under confocal microscope, as well as the metastasis of tumor cells in embryos; then the ze-brafish xenografts of MB-231 were co-cultured with tofacitinib/ptk787 for 48 h, optical density(OD) of the cell survival and subintestinal veins(SIVs) were evaluated under confocal micro-scope, and Western blot(WB) analysis was used to test micro-circumstances related protein. Results When the number of in-oculated cells was more than 200 per embryo, xenograft model rate woule be more than 0. 90;MB-231 xenografts showed metas-tasis feature in zebrafish, which could be inhibited by tofacitinib (P < 0. 01), while the number of xenograft MB-231 cells was reduced significantly(P < 0. 01); in another zebrafish xenografts SIVs assay, the tumor could promote the proliferation of SIVs, and 4 mg·L - 1 PTK787 showed inhibiton effect( P < 0. 01). Western blot showed 4d T-47D xenograft zebrafish got more HER2 expression than AB embryos; VEGFa expression in ze-brafish MB-231 model group was higher, and model zebrafish P53 expressi was higher after treated by tofacitinib. Conclusion A zebrafish xenograft model of human brest cancer can be es-tablished, which demonstrates applicability for screening com-pounds in drug discovery studies.