OBJECTIVE: To observe the effect of manual acupuncture stimulation (MAS) of "Baihui"(GV 20)-"Shenmen"(HT 7)or GV 20-"Sanyinjiao"(SP 6) on sleep and expression of circadian rhythm genes (Clock and Bmal 1) in the hypothalamus in insomnia rats, so as to select a better acupoint group for insomnia. METHODS: A total of 53 SD rats were randomly divided into normal control (n=12),insomnia model (n=8), GV 20-HT 7(n=12), GV 20-SP 6(n=11),and GV 20-non-acupoint (n=10) groups. The insomnia model was established by intraperitoneal injection of p-chlorophenylalanine (PCPA,500 mg/kg,100 mg/mL) once daily for 2 days. The MAS (uniform reinforcing-reducing needling) was applied to GV 20-HT 7, GV 20-SP 6 or GV 20-non-acupoint for 30 min,once daily for 7 days. The sleep onset latency and sleep duration were gauged after intraperitoneal injection of pentobarbital sodium (35 mg/kg). The expression levels of Clock mRNA and Bmal 1 mRNA in the hypothalamic tissues containing ventrolateral preoptic area (VLPO) and suprachiasmatic nucleus (SCN) region were detected by fluorescence quantitative real-time PCR. RESULTS: Following administration of pentobarbital sodium,the sleep latency was significantly prolonged and the sleep duration was considerably shortened in rats of the model group(P<0.05). After the treatment, the increased sleep latencies in the GV 20-HT 7, GV 20-SP 6 and GV 20-non-acupoints were all significantly down-regulated (P<0.05), and the decreased sleep duration was significantly increased only in the GV 20-HT 7 group relevant to the model group (P<0.05), but not in the GV 20-SP 6 and GV 20-non-acupoint groups (P<0.05). There were no significant differences in the sleep latency among the 3 treatment groups (P<0.05). The sleep duration was obviously prolonged in the GV 20-HT 7 group than in the GV 20-SP 6 and GV 20-non-acupoint groups (P<0.05). After modeling, the expression levels of Clock mRNA and Bmal 1 mRNA in hypothalamic VLPO and SCN regions were significantly down-regulated relevant to the normal control group (P<0.01). Following the treatment, the expression levels of Clock mRNA in the VLPO and SCN regions of the GV 20-SP 6 and GV 20-HT 7 groups, and those of Bmal 1 mRNA in the VLPO and SCN regions of the 3 treatment groups were considerably increased relevant to the model group (P<0.05, P<0.01). The effects of GV 20-HT 7 were significantly superior to those of GV 20-SP 6 and GV 20-non-acupoint (and also the action of GV 20-SP 6 was evidently superior to that of GV 20-non-acupoint) in up-regulating the expression of Clock mRNA and Bmal 1 mRNA in both VLPO and SCN regions (P<0.05, P<0.01). CONCLUSIONS: Manual acupuncture stimulation of GV 20-HT 7 can improve the sleep latency and duration in insomnia rats,which may be associated with its effects in up-regulating the expression levels of circadian Clock mRNA and Bmal 1 mRNA in hypothalamic VLPO and SCN regions, and the efficacy of GV 20-HT 7 is obviously better than that of GV 20-SP 6 and GV 20-non-acupoint.

Objective To investigate the effect of continuous heated humidification on critical patients with artificial airway. Methods 60 patients with artificial airway were randomly divided into experimental group and control group, 30 cases each group. Experimental group received continuous heated humidification and control group used artificial nose to humidify airway. To compare sputum viscidity, sputum scab, amyctic cough, respiration mucosa bleeding of two groups. Results There were significant differences between the two groups in sputum viscidity, sputum scab, amyctic cough, respiration mucosa bleeding (P ＜ 0. 05). Conclusions Continuous heated humidification can bring much better effects to patients than hygroscopic condenser humidifier.

Animals ; Fatty Liver, Alcoholic ; drug therapy ; Female ; Male ; Phytotherapy ; Rats ; Rats, Wistar ; Saponins ; therapeutic use ; Trigonella

Objective To detect serum level of long noncoding RNA ( lncRNA) BC200 in gastric cancer(GC) patients, and investigate its relationship with clinical features , and evaluate its diagnostic value for GC.Methods A case-control study was performed.From November 2014 to July 2015, serum levels of lncRNA BC200 were detected by real-time quantitative polymerase chain reaction in 124 patients with GC , 41 patients with atrophic gastritis and 59 normal controls who were hospitalized in Qilu Hospital of Shandong University.Meanwhile , serum carcinoembryonic antigen ( CEA ) and carbohydrate antigen 72-4 ( CA72-4 ) were detected by electrochemical luminescence immunoassay .Serum levels of lncRNA BC200, before and 3, 7, 10, 30, 100 days after radical operation in another 31 patients with GC were determined.The sensitivity and specificity of serum lncRNA BC200, CEA and CA72-4 were analyzed by using of the receiver operating characteristic ( ROC) curve.The comparison between two groups was performed with Mann-Whitney U test and the comparison among many groups was conducted with Kruskal-Wallis H test.Results Serum levels of lncRNA BC200 in GC patients with stage Ⅰ and Ⅱ[1.041(0.794,1.462)] and stage Ⅲ and Ⅳ[1.290 (0.978,1.794)]were significantly higher than those in patients with precancerous lesion [0.969(0.699, 1.219)]and normal controls[0.801(0.556,1.599)](H =54.68,P<0.000 1).Compared with pre-operation[1.120 (0.859,1.663)], the serum BC200 levels decreased significantly in 10 days [0.903 (0.724,1.182)](U=55.0,P<0.000 1), 30 days[0.759(0.671,1.037)](U=299.0,P=0.026 1), and 100 days[0.478(0.378,0.635)](U=41.0,P<0.000 1) after surgery.The area under the receiver operating characteristics curve ( AUC) of serum lncRNA BC200 was 0.865 for GC diagnosis, which was significantly higher than that of serum CA 72-4 ( AUC =0.699 ) or CEA ( AUC =0.807 ) .The AUC of combined detection of three tests was 0.934.Conclusion Serum lncRNA BC200 levels are significantly increased in GC patients , which may be used as a potential biomarker in GC diagnosis and monitoring .

The polysomnogram (PSG) analysis is considered the golden standard for sleep staging under the clinical environment. The electroencephalogram (EEG) signal is the most important signal for classification of sleep stages. However, in-vivo signal recording and analysis of EEG signal presents us with a few technical challenges. Electrocardiogram signals on the other hand, are easier to record, and can provide an attractive alternative for home sleep monitoring. In this paper we describe a method based on deep neural network (DNN), which can be used for the classification of the sleep stages into Wake (W), rapid-eye-movement (REM) and non-rapid-eye-movement (NREM) sleep stage. We apply the sleep stage stacked autoencoder to constitute a 4-layer DNN model. In order to test the accuracy of our method, eighteen PSGs from the MIT-BIH Polysomnographic Database were used. A total of 11 features were extracted from each electrocardiogram recording The experimental design employs cross-validation across subjects, ensuring the independence of the training and the test data. We obtained an accuracy of 77% and a Cohen's kappa coefficient of about 0.56 for the classification of Wake, REM and NREM.
Classification ; Electrocardiography ; Electroencephalography ; Hand ; Methods ; Polysomnography ; Research Design ; Sleep Stages

Objective：This study was designed to express chimeric anti-VEGF (vascular endothelial growth factor) antibody in dihydrofolate reductase-deficient Chinese hamster ovary (CHO-dhfr-)cells at high-level, and explore an optimum method to obtain high-level expression cells clone. Methods：The light chain and heavy chain genes of chimeric anti-VEGF antibody were induced into CHO-dhfr-cells using a novel eukaryotic high-level expression vectors system for genetic engineering antibodies. High-level expression was achieved after subcloning and several rounds of co-amplification of methotrexate (MTX). Biological features and productive amount of chimeric antibody was charactered by ELISA. Result：The cells strain that secret anti-VEGF chimeric antibody at the highest level of 28 μ g/ml was established. The cells were subcloned following each round of co-amplification of MTX, while greatly different results were obtained using three methods. The chimeric antibody contained constant regions of human immunoglobin and had the specificity against VEGF by ELISA. Conclusion：The anti-VEGF mouse-human chimeric antibody was expressed at high-level successfully in CHO cells. This may be an optimum method to obtain high-level expression cells clone for the eukaryotic high-level expression vectors system.

Objective To explore the effect of polyethylene glycol-polyethyleneimine (PEG-PEI) serving as a non-viral vector in delivering small interfering RNA (siRNA) into C17.2 neural stem cells (NSCs) in vitro. Methods Complexes of PEG-PEI and siRNA targeting Nogo receptor were prepared, and their characterizations were estimated by measurements of particle size and zeta potential,and the complex abilities of PEG-PEI/siRNA complexes were observed by gel retardation assay. In addition, with liposome complex system (Lipofectamine 2000/siRNA) as positive control, the transfection efficiency of PEI-PEG/siRNA complexes at different N/P ratios (cationic nitrogen/siRNA phosphate molar ratio) was detected by flow cytometry. Results The siRNA molecules were condensed by PEG-PEI to form nanoseale complexes. As the proportion of N/P ratio enhancing, the surface potential of nanoparticles gradually increased and the particle sizes of PEI-PEG/siRNA complexes showed a decreasing trend. Gel retardation electrophoresis suggested that siRNA could be fully composited with PEG-PEI as a result of the coulombic foree between them. Meanwhile, flow cytometry experiments revealed that the transfection efficiency of PEG-PEI mainly depended on N/P ratios of the nanoparticles,and the highest one was obtained at N/P=15 ([78.72±8.18)]％). Conclusion PEG-PEI might be a prospective candidate for siRNA delivery system, which enjoys its value in NSC gene therapy.

Aim To explore the optimal way of breeding and genotype identification of Arrb2 knockout mice, and to find a simple and quick polymerase chain reaction ( PCR) method for the genotyping of Arrb2 knockout mice. Methods Breeding homozygote genotype of Arrb2 gene knockout mice were copula-ted with wild-type C57BL/6J mice, and then the heterozygous mating were used for mating. The growth and development of off-spring were observed. The genomic DNA was extracted from the tail of two-week-old mice. PCR was employed to amplify the Arrb2 gene fragment, and electrophoresis was used to present the gene type. Results The breeding and reproducing were successful and three genotype offspring, including wild-type,heterozygous and homozygous knockout mice were obtained. Agarose gel electrophoresis results showed the size of PCR prod-ucts was about 186 bp and 224 bp, which was consistent with the expected target gene fragment, and identified Arrb2 gene knockout mice of different genotypes successfully. Western blot analysis demonstrated the lack ofβ-arrestin2 protein in the major organs from Arrb2 -/ - mice compared with Arrb2 +/ + and Arrb2 +/ - mice. Conclusions It is feasible to obtain the homo-zygous Arrb2 knockout mice by inbreeding heterozygotes. It is simple, rapid and reliable to identify mouse genetype by PCR.

Wuzhuyu Tang is a classical formula for treating migraine, but its' pharmacological ingredients is unclear yet. Present study employed the everted intestinal sac model to collect the absorption samples of 10 kinds of Wuzhuyu decoction, and then analyzed the contents of 9 ingredients in Wuzhuyu Tang and absorption samples quantitatively or semi-quantitatively by HPLC-DAD method. Reserpine was used to establish the mice model of migraine, and then the contents and activities of 5-hydroxytryptamine, noradrenaline, dopamine, nitric oxide and nitricoxide synthase in brain tissues and serums were determined respectively after oral administration of Wuzhuyu Tang. Using the partial least squares regression method to correlate the total absorption quantity of 9 ingredients and pharmacodynamics. The result shows that limocitrin-3-O-beta-D-glucoside, ginsenoside Rg1 and Rb1, rutaevine, limonin, evodiamine and rutaecarpine are the main ingredients influenced the effects in absorption samples in everted intestinal sacs, especially ginsenoside Rg1, rutaevine, evodiamine and rutaecarpine among them have obvious improving effects to most pharmacodynamics index, might be the pharmacological ingredients influenced the therapeutical effects of Wuzhuyu Tang treating migraine.
Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Intestinal Absorption ; drug effects ; Intestines ; drug effects ; Male ; Mice ; Mice, Inbred ICR ; Migraine Disorders ; drug therapy ; Rats ; Rats, Wistar

OBJECTIVETo investigate the large germline deletion of the VHL gene in Chinese families with von Hippel-Lindau disease (VHL).

METHODSThe large deletion of the VHL gene in 20 unrelated Chinese VHL families was analyzed by using universal primer quantitative fluorescent multiplex polymerase chain reaction (UPQFM-PCR) and GeneScan analysis.

RESULTSPartial and complete VHL gene deletions were detected in 6 probands, including 3 exon 1 deletions, 1 exon 3 and 2 complete deletions. Of the 2 families with the complete deletions, patients developed multi-centric hemangioblastoma in the retina and central nervous system (CNS), and none developed renal cell carcinoma (RCC).

CONCLUSIONPartial and complete VHL gene deletions could be detected in Chinese kindreds with von Hippel-Lindau disease and the test for large deletion of the VHL gene should be implemented in routine DNA diagnosis for VHL disease. Further investigations are required to confirm that entire VHL deletions may be associated with a high risk of hemangioblastomas in the retina and central nervous system.

Asian Continental Ancestry Group ; genetics ; Exons ; Female ; Gene Deletion ; Germ-Line Mutation ; Humans ; Male ; Pedigree ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; genetics