OBJECTIVETo observe the intervention effects of electroacupuncture at "Yishu" (EX-B 3) on rats with type-2 diabetes mellitus (T2DM), so as to provide experiment references for acupuncture to treat T2DM.

METHODSAmong seventy male Wistar clean-grade rats, 8 rats were randomly selected into a control group; the rest rats were made T2DM model. Fifty-two rats which were successfully made T2DM model, according to randomized block method, were divided into a model group (10 rats), a medication group (10 rats), an electroacupuncture at "Shenshu" (BL 23) group (11 rats), an electroacupuncture at "Pishu" (BL 20) group (10 rats) and an electroacupuncture at "Yishu" (EX-B 3) group (11 rats). Seven days after successful establishment of model, the rats in the model group were fixed in the self-made rat bag without receiving any treatment; the rats in the medication group, according to body mass (10 mL/kg), were treated with intragastric administration of glimepiride; the rats in all the electroacupuncture groups were treated with electroacupuncture at "Shenshu" (BL 23), "Pishu" (BL 20) and "Yishu" (EX-B 3), respectively. The continuous wave was selected with a frequency of 15 Hz and a current intensity of 4 to 6 mA. The treatment was given 20 min per treatment, once a day, 5 treatments per week for continuous 4 weeks. Before the establishment of model and continuous 4 weeks after the intervention, blood samples were collected from rats' caudal vein, and fasting blood glucose (FBG) was measured with FBG device each week. After the last intervention, the rats were killed and hypothalamus, pituitary and adrenal gland were collected. The colorimetric method was applied to measure the contents of triglyceride (TG), cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C); radioimmunoassay was used to test the contents of glycated serum protein (GSP), fasting insulin (FINS), corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortin (CORT).

RESULTSFour weeks after the intervention, except that the rat's body mass in the normal group continued to increase, body mass in the model group, medication group and each electroacupuncture group were significantly reduced (P<0.01, P<0.05). Compared with the model group, the FBG in the electroacupuncture at "Pishu" (BL 20) group and electroacupuncture at "Yishu" (EX-B 3) group were obviously reduced (P<0.05, P<0.01); FBG in the electroacupuncture at "Yishu" (EX-B 3) group was lower than that in the medication group and electroacupuncture at "Shenshu" (BL 23) group (both P<0.05). The contents of TG, HDL-C and LDL-C in the electroacupuncture at "Yishu" (EX-B 3) group were reduced (P<0.05, P<0.01), the content of TG was significantly lower than that in the medication group and electroacupuncture at "Shenshu" (BL 23) group (both P<0.05), the content of LDL-C was significantly lower than that in electroacupuncture at "Shenshu" (BL 23) group (P<0.05). Insulin sensitivity index (ISI) in the medication group, electroacupuncture at "Pishu" (BL 20) group and electroacupuncture at "Yishu (EX-B 3)" group were evidently increased (P<0.05, P<0.01); ISI in the medication group was lower than that in the electroacupuncture at "Yishu" (EX-B 3) group (P<0.05). The content of CRH in the electroacupuncture at "Yishu" (EX-B 3) group was lower than that in the medication group and electroacupuncture at "Shenshu" (BL 23) group (P<0.05, P<0.01); the content of CORT in the electroacupuncture at "Yishu" (EX-B 3) group was lower than that in the medication group and electroacupuncture at "Pishu" (BL 20) group (both P<0.05).

CONCLUSIONElectroacupuncture at "Yishu" (EX-B 3) could reduce the level of CORT to improve the insulin resistance in rats with T2DM, improve insulin sensitivity index, regulate blood lipid metabolism and relieve the hyperactivity of the HPA axis.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; therapy ; Electroacupuncture ; Hormones ; metabolism ; Humans ; Hypothalamus ; metabolism ; Male ; Pituitary-Adrenal System ; metabolism ; Rats ; Rats, Wistar

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

BACKGROUNDThe low-temperature resistance aging performance of Yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) is the key effective factor that influences the long-term success rate of prosthesis. The objective of this study was to test and compare the aging performances for resisting low temperature of Lava Frame, Cercon Smart, and Upcera Yttria-stabilized zirconia core materials, via analyzing the micro and the crystal phases of the materials, and measure the three-point bending strength and the fracture toughness.

METHODSThe three zirconia green bodies were prepared as 60 test samples for three-point bending strength and as 60 test samples for fracture toughness. The test samples for three-point bending strength and fracture toughness were assigned to five groups and were treated respectively for 0, 5, 10, 15, and 20 hours to observe the micro and the crystal phases of the test samples. Then the three-point bending strength and fracture toughness were tested by X-ray diffraction (XRD).

RESULTSThe m phase content of Lava Frame was raised from 7.70% to 13.01%; the m phase content of Cercon Smart was raised from 4.95% to 8.53%; and Lava Frame is raised from 10.84% to 35.18%. The three-point bending strengths of the three zirconia core materials were higher than 1100 MPa and the fracture toughness was higher than 3 MPa·m(1/2). The three-point bending strength and the fracture toughness of Upcra zirconia decreased the most, followed by Lava Frame, and then by Cercon Smart.

CONCLUSIONThe aging resistance sequences of the three zirconia core materials are, from strong to weak, Cercon Smart, Lava Frame, and Upcera.

Ceramics ; chemistry ; Dental Porcelain ; chemistry ; Microscopy, Electron, Scanning ; Temperature ; X-Ray Diffraction ; Yttrium ; chemistry ; Zirconium ; chemistry

Objective To explore the relationship between immature platelet fraction(IPF) with severity of sepsis and prognosis in patients with septic shock.Methods A total of 40 patients admitted to intensive care units of Tianjin First Central Hospital from June 2016 to June 2017 were enrolled.Of them,10 patients contracted non-sepsis infected,13 patietns with septic shock,and 17 patients with non-complicated sepsis.Ten healthy subjects were recruited as control groups from Tianjin Medical University.IPF and immature reticulocyte fraction (IRF) were detected,and SOFA and APACHE Ⅱ scores were calculated,and clinical findings of all groups were recorded.The differences in IPF and IRF between the groups were analyzed.The relationship between the IPF and SOFA score was studied,and the role of IPF in the diagnosis of septic shock was evaluated.Statistical methods include t test,MarmWhitney test,Spearman correlation analysis,and ROC procedure,and P＜0.05 was considered significant.Results Significantly higher IPF level was observed in patients with sepsis than that in patients with nonsepsis infection.(6.25 + 2.92) vs.(2.49 ± 1.03),P＜0.01.Significantly higher IPF level was observed in patients with septic shock than that in patients with non-complicated sepsis(4.71 ± 1.79) vs.(8.25 ± 2.94),P＜0.01.IPF correlated with sepsis severity scores (7.41 ± 3.51) vs.(4.5 ± 1.7),P=0.005;r=0.58,P=0.001.This study presented the highest diagnostic accuracy for the presence of sepsis by all studied clinical and laboratory parameters (AUC=0.78,P=0.01).Conclusion IPF levels could be used as a biomarker for diagnosis and severity of sepsis.

There are many E. coli rare codons in the gene of porcine interferon alpha-1. In order to obtain high expression of poIFN-alpha1 in E. coli, the cDNA encoded poIFN-alpha1 mature protein was synthesized using biased codons of E. coli without changing the original amino acid sequence and the terminator was changed as TAA. At the same time, Adenine and Thymine were used to the largest extent near the 5' terminus of poIFN-alpha1 mature protein gene. The synthesized gene was inserted into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-alpha1. The poIFN-alpha1 is highly expressed in E. coli DH5alpha when the induction was carried out at 42 degrees C . The expressed poIFN-alpha1 account for 24.5% of the total cellular proteins and existed as inclusion body. The poIFN-alpha1 inclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subsequently the denatured poIFN-alpha1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured poIFN-alpha1 was most efficiently re-natured in refolding buffer containing 1 mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natured poIFN-alpha1 was purified by Sephacryl S-200 chromatography. As a result, the purified poIFN-alpha1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 6.4 x 10(6) u/mg. This study paved the way for large-scale production of recombinant poIFN-alpha1 and its usage in virus disease control of pigs.
Animals ; Codon ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transduction, Genetic

Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .

Objective To study the relation between aquaporin 4 (AQP4) expression in the perihematomal tissue and changes of MRI indicators after intracerebral hemorrhage of rats, and explore the relationship between AQP4 expression and formation of hemorrhagic brain edema. Methods Forty-five male SD rats were randomized into sham-operated group (n=15) and hemorrhage group (n=30). The rats of these groups were equally subdivided into the 1', 2nd, 3rd, 5th and 7th d measurement groups, respectively. The models of intracerebral hemorrhage were established by infusing collagenase into globus pallidus of the rats. MRI was performed 1, 2, 3, 5 and 7 d after the success of model making;edema volume around the hematoma and signal intensity ratio of T1WI, T2WI, and FLAIR sequences in the edema zone were measured and calculated. The rats were sacrificed at the corresponding time point after imaging. Immunohistochemistry staining was performed to observe the expression of AQP4 at each time point. Results The AQP4 expression level of perihematomal tissue in the hemorrhage group was obviously higher than that in the sham-operated group (P＜0.05). Liner positive correlation between the AQP4 expression level and the volume of cerebral edema around the hematoma was noted (r=0.687,P＜0.05). Liner positive correlations between the AQP4 expression level and both signal intensity ratio of T2WI and FLAIR sequences in the cerebral edema region were also found (r=0.640, 0.662; P＜0.05).Conclusion AQP4 has a close relation with the formation and expansion of hemorrhagic cerebral edema; over-expression of AQP4 may promote the formation of edema after intracerebral hemorrhage.

OBJECTIVETo evaluate the efficacy and safety of Capsule metadoxine in the treatment of alcoholic liver disease.

METHODSA randomized double blind multicenter placebo-controlled clinical study was performed to evaluate the therapeutic effectiveness and safety of capsule metadoxine. Patients in metadoxine group received capsule metadoxine 500mg tid po. Patients in placebo group received placebo 2 pillows tid po. The treatment duration was 6 weeks. Patients were followed up 2 weeks after the treatment. Patients were visited once every 3 weeks during the treatment period. Clinical symptoms and liver function were evaluated in all the patients before treatment, at week 3, week 6 and 2 weeks after therapy. CT scan was done in some patients before treatment and at the end point of therapy.

RESULTS254 patients were recruited in the study, 126 in metadoxine group and 128 in placebo group. Median ALT, AST, GGT level in metadoxine group were decreased from 80.0 U/L, 59.2 U/L, 123.0 U/L (before treatment) to 41.1 U/L, 36.0 U/L, 57.0 U/L (after 6 weeks therapy). The improvement in liver function was more significant in metadoxine group than in placebo group (P less than 0.05). For the patients who stopped drinking during the study, the total effective rate of improvement in liver function was 82.8% in metadoxine group, much higher than that in placebo group (55.7% , P=0.0000). For the patients who did not stop drinking during the study, the total effective rate of improvement in liver function was 65.4% in metadoxine group, which is not significantly higher than that in placebo group (44.8%, P=0.1767). The CT value ratio of liver to spleen was significantly improved in metadoxine group (P=0.0023), and there was no significant difference between the two groups (P=0.6293). The rate of adverse was 1.6% in both of groups.

CONCLUSIONCapsule metadoxine is an effective and safe treatment for alcoholic liver disease.

Administration, Oral ; Adult ; Aged ; Alanine Transaminase ; blood ; Alcohol Deterrents ; administration & dosage ; therapeutic use ; Analysis of Variance ; Aspartate Aminotransferases ; blood ; Capsules ; Double-Blind Method ; Drug Combinations ; Fatty Liver, Alcoholic ; blood ; drug therapy ; pathology ; Female ; Follow-Up Studies ; Humans ; Liver ; diagnostic imaging ; pathology ; Liver Diseases, Alcoholic ; blood ; drug therapy ; pathology ; Liver Function Tests ; Male ; Middle Aged ; Pyridoxine ; administration & dosage ; therapeutic use ; Pyrrolidonecarboxylic Acid ; administration & dosage ; therapeutic use ; Treatment Outcome ; Ultrasonography ; Young Adult ; gamma-Glutamyltransferase ; blood

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; blood ; immunology ; Binding Sites ; genetics ; Blotting, Western ; CHO Cells ; Cell Proliferation ; Cricetinae ; Cricetulus ; Female ; Glycosylation ; Mice ; Mice, Inbred BALB C ; Mutation ; Open Reading Frames ; genetics ; Porcine Reproductive and Respiratory Syndrome ; immunology ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; metabolism ; Swine ; virology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Vaccines, DNA ; administration & dosage ; immunology ; Viral Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; administration & dosage ; immunology

Vp1 gene of O type foot-and-mouth diseases virus and M. tuberculosis HSP70 were expressed in methylotrophic yeast Pichia pastoris expression system. The results of cellular immune responses and humoral immune response were examined after BALB/c mice were immunized with fusion protein expressed in methylotrophic yeast Pichia pastoris. The genes was cloned into the vector pPICZalpha-A by routine molecular technique. The plasmid fusion (pPICZalphaA-vp1-HSP70) was created that HSP70 located downstream of VP1 gene of O type foot-and-mouth disease virus. Vp1 was expressed by fusing to the amino terminus of M. tuberculosis hsp70 in yeast Pichia pastoris. The recombined fusion plasmid was transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24h. The expressived product analyzed by SDS-PAGE and Western blotting. The result indicated that the fusion protein(vp1-HSP70) has specific antigenicity. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein, PBS and conventional inactivated vaccines. To evaluate the prophylaxtic efficacy of fusion protein, Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited slightly lower FMDV antibody level but stronger T cell proliferation.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease Virus ; immunology ; HSP70 Heat-Shock Proteins ; genetics ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Pichia ; genetics ; Recombinant Fusion Proteins ; immunology ; Vaccines, Synthetic ; immunology ; Viral Vaccines ; immunology