Background and purpose: Itch protein is an established regulator of T cell immune response thresholds, belong to a class of E3 ubiquitin-transferring enzymes, widely involve in the ubiquitination of several key signaling molecules, such as ZAP70, P85, VAV, PLC-γ, PKC-θ, etc, plays a critical role in tumor induced immu-nosuppression. Itch ligase activity regulate T-cell anergy and development of regulatory T cells in the periphery by modulating key components of T-cell receptor and transforming growth factor-βsignaling. Therefore, manipulation of Itch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmunity and cancer. speciifc small interfering RNA(siRNA) was utilized to silence the expression of Itch gene of T-lymphocytes and investigate the cytotoxicity activity of transfected T lymphocytes against MFC stomach neoplasms cells in vitro. Methods:T lymphocytes were isolated from the spleen of 615 mice and transfected by speciifc siRNA to silence the expression of Itch gene, The expression of Itch protein were examined by Western bolt in each group;72 hours after transfection, The secretion level of IL-2, INF-γwere measured by enzyme-linked immunosorbent assay (ELISA). At the end, the cytotoxicity activity changes against MFC stomach neoplasms cells was compared between transfected T lym-phocytes, negative control and blank control in vitro. Results:Compared with control group, the expression rate of Itch protein of transfected T-lymphocytes was decreased to 16%after transfection 48 hours;72 hours after transfection, the secretion level of IL-2 in transfection group, negative control and blank control respectively were (1 891.96±141.91)pg/mL, (1 241.69±91.67)pg/mL and (1 175.03±89.14)pg/mL (P<0.001), the secretion level of INF-γin transfection group, negative control and blank control respectively were (958.33±75.46)pg/mL, (683.33±66.67)pg/mL and (691.72±68.72) pg/mL (P<0.05). Transfected T lymphocyte also showed more efifcient killing ability against MFC stomach neoplasms cells than negative control and blank control in vitro, the highest killing rate has reached (54.18±2.96)%. Conclusion:Silencing Itch gene can signiifcantly promoted the secretion level of IL-2, INF-γof mice T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against MFC stomach neoplasms cells in vitro.

To establish a method for determination contents of schizandrin, tanshinone I, cryptotanshinone, tanshinone II (A) and schizandrin B in rongxin pills. The HPLC method was performed on an Agilent C18. The mobile phase was composed of methnol and water wish gradient elution. The flow rate was 1.0 mL x min(-1). The column temperature was 30 degrees C and the detection wavelength wash 240 nm. The linear of schizandrin, tanshinone I, cryptotanshinone, Tanshinone II (A) and schizandrin B were 3.000-48.00 (r = 1.000), 3.985-63.76 (r = 0.999 9), 6.370-101.9 (r = 1.000), 8.690-139.0 (r = 0.999 9), 1.700-27.20 mg x L(-1) (r = 0.999 9), respectively. The average recoveries were 98.44%, 100.3%, 99.29%, 99.07%, 98.42%, and RSDs were 0.61%, 1.1%, 0.52%, 0.72%, 0.97%. The method is convenient, accurate and has good precision. It can be used for determination of the preparation.
Chromatography, High Pressure Liquid ; Cyclooctanes ; analysis ; Diterpenes, Abietane ; analysis ; Drugs, Chinese Herbal ; chemistry ; Lignans ; analysis ; Linear Models ; Organic Chemicals ; analysis ; Phenanthrenes ; analysis ; Polycyclic Compounds ; analysis ; Quality Control ; Reproducibility of Results

Objective To explore the association of Gly71Arg mutation in gene of bilirubin uridine 5'-diphosphate-glucuronosyltransferase(UGT1A1)and neonatal jaundice in Beijing city Han population.Methods The genotypes and alleles of the Gly71 Arg polymorphism for UGT1A1 gene were identified by polymerase chain reaction-restricted fragment length polymorphism assay in infants of Beijing city Han population of China,including 96 infants with neonatal jaundice[serum bilirubin(307.06?38.5)?mol/L,indirect bilirubin(292.9?35.9)?mol/L] and 101 healthy control infants [serum bilirubin(131.2?42.1)?mol/L,indirect bilirubin(126.3?39.7)?mol/L].The genotypes and allele frequencies of the polymorphism were compared between infants with neonatal jaundice group and healthy infant group(control group).The effect of polymorphism in infants with neonatal jaundice group on serum bilirubin level were analyzed.Results There were significant differences in genotypes distribution in Gly71Arg polymorphism for UGT1A1 gene between the 2 groups(?2=9.47 P=0.002).Compared with control group,neonatal jaundice group had significantly higher Arg allele frequency in the polymorphism for UGT1A1 gene(?2=10.34 P=0.001).There were independent effects of Gly71Arg mutation in the gene on serum bilirubin level in neonatal jaundice group,at the carriers of homozygote of the Arg allele of Gly71Arg polymorphism had higher serum bilirubin levels compared to carriers of heterozygote of the Arg allele of the polymorphism and non-carriers of the Arg allele of the polymorphism(Pa

Objective To observe the levels of serum leptin in Gaves disease(GD)and thyroiditis(HT)Datients and to discuss the immunological role of leptin in the pathogenesis of autoimmune thyroid disease(AITD).Methods 102 newly diagnosed female AITD patients were divided into 3 groups:GD hyperthyroid group,HT hypothyroid group and subclinical hypothyroid group.Age,sex and BMI-matched 27 euthyroid,healthy subjects served as controis.The levels of FT3,FT4 and sTSH were determined by immunofluorometrie assay.ELISA kit was aDplied to measure the levels of serum leptin.Results Serum FT3 and FT4[(19.74±15.39),(78.25±58.68)pmol/L]levels of GD hyperthyroid patients were obviously higher than those of the controls[(4.87±0.25),(15.96±3.15)pmol/L,P＜0.01],but serum sTSH and leptin levels[(0.15±0.08)mU/L,(8.73±1.92)μg/L]were obviously lower than those of the controls[(3.81±0.19)mU/L,(12.38±3.51)μg/L,P＜0.01or＜0.05].Serum FT3 and FT4[(3.36±0.26),(6.95±3.29)pmol/L]levels of HT hypothyroid patients were obviously lower than those of the controls(P＜0.05),but serum sTSH and leptin levels[(45.48±35.83)mU/L,(17.17±3.82)μg/L]were obviously higher than those of the controls(P＜0.01 or＜0.05).Serum FT3 and FT4[(4.67±0.60),(14.87±2.14)pmol/L]levels of subclinical hypothyroid patients had not statistical difference comparing with those of the controls(P＞0.05),but serum sTSH and leptin levels[(13.67±8.66)mU/L,(16.25±3.67)μg/L]were obviously higher than those of the controls(P＜0.01 or＜0.05).Conclusions Leptin might have an immuoregulation role in the pathogenesis of AITD.In addition,serum levels of leptin in AITD is also influenced by many other related hormones.

OBJECTIVETo observe the effect of Cangfu Congxian Decoction (CCD) on oxidative stress in granulosa cells of polycystic ovary syndrome (PCOS) patients.

METHODSForty PCOS patients underwent in vitro fertilization-embryo transfer (IVF-ET) were assigned to the treatment group and the control group 1 according to random digit table, 20 in each group. Patients in the treatment group took CCD (200 mL, once in the morning and once in the afternoon) 2 months before IVF-ET, while those in the control group 1 took no Chinese medical decoction. Recruited were another 20 patients undergoing IVF-ET for tubal factors (as the control group 2). The clinical effect of IVF-ET were observed, including oocyte retrieval number, 2 pronuclear (2PN) fertilization rate, good quality embryo rate, clinical pregnancy rate, and ovarian hyperstimulation syndrome (OHSS) induced transplantation cancel rate. The expression of relative oxygen species (ROS) in granulosa cells was detected using cell immunofluorescence combined with confocal microscopy and FCM.

RESULTSCompared with the control group 1, occyte retrieval number, 2PN fertilization rate, and good quality embryo rate increased in the control group 2 and the treatment group (P <0. 05). OHSS induced transplantation cancel rate decreased in the control group 2 (P < 0.05). Fluorescence intensity of ROS decreased in the treatment group and the control group 2, as compared with the control group 1 (P < 0.01).

CONCLUSIONCCD increased good quality embryo rate by down-regulating the expression of ROS protein in ovarian granulosa cells, and correcting in vivo oxidative stress.

Drugs, Chinese Herbal ; therapeutic use ; Embryo Transfer ; Female ; Fertilization in Vitro ; Granulosa Cells ; drug effects ; metabolism ; Humans ; Oocyte Retrieval ; Ovarian Hyperstimulation Syndrome ; prevention & control ; Oxidative Stress ; drug effects ; Polycystic Ovary Syndrome ; drug therapy ; Pregnancy ; Pregnancy Rate ; Reactive Oxygen Species ; metabolism

Objectiye To optimize the depth of the microchannel and the time point for sperm collection,and improve the efficiency of sperm screening on a microfluidic device. Methods Microchannels with four different depths of 25, 50, 100 and 200 μm were tested. Mice sperm were added to the inlet of the microchannel. The relative quantity and motility of sperm in the outlet were recorded at different collection times, i.e. ,5, 15, 30 and 60 min. Statistical method one-way ANOVA and appropriate post-hoc testing were applied to analyze differences between different groups, and further to select the best-fit depth of the microchannel and the time point for collection. Results In microchannels with depths of 25, 50, 100 and 200 μm, the sperm motilities measured in each outlet were (85.4 ± 2.3)%, (85.8 ± 5.8)%,( 87. 2 ± 2. 8 ) %, (76. 5 ± 2. 8 ) % respectively with statistical significance ( F = 5.8, P ＜ 0. 05 ). No obvious differences were found among 25-100 μm channels, however the motility dramatically decreased in the 200 μm group. The relative sperm quantities were (5.2 ±2.0)%, (7.2 ±2.5)%,(12.3 ±2.0)%,(7. 7 ± 1.1 ) % respectively with statistical significance ( F = 6. 9, P ＜ 0. 05), which increased with channel depth from 25 to 100 μm,while it decreased in the 200 μm channel Taking 2 indexes into account, 100 μmwas the most fit channel depth for sperm motility screening. The sperm motility in the outlet gradually decreased with time. At the time points of 5, 15, 30 and 60 min after adding sperm, the sperm motilities were (99. 6 ±0. 7)%, (87.2 ±2. 8)%, (79. 3 ±2. 2)% and (62. 6 ±8.0)% respectively with statistical significance ( F = 37. 3, P ＜ 0. 01 ). Yet the relative quantities of sperm in the outlet increased almost three times in this process. At the time points mentioned above, the relative quantities of sperm were (5.8±1.1)%, (10.6 ± 0.9)%, (12.1 ± 1.7)%, (17.9 ± 3.4)% respectively with statistical significance ( F = 17.8, P ＜ 0. 01 ). Thus 15-30 min was the ideal screening time. Conclusion An effective microdevice for sperm screening with optimized depth and collection time period is developed,which may contribute significantly for the screening of healthy sperm on microfluidic chips.

Binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3. 1/ myc-his(-) B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection (900 microg x mL(-1)) for subsequent bioassay analysis. K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were obtained in competition with [125I]-NDP-MSH for binding studies. The cyclic AMP level was tested by using [3H]-cyclic AMP kit. It is showed that K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were (0.159 +/- 0.040), (35.430 +/- 6.743), (19.293 +/- 2.780) and (2.230 +/- 0.670) nmol L(-1), respectively. Its EC50 values for MC1, MC3, MC4, and MC5 receptors were (0.45 +/- 0.07), (7.80 +/- 0.65), (2.55 +/- 0.23) and (0.33 +/- 0.09) nmol L(-1), respectively. In these tests, the novel alpha-MSH analogue is a MC1R and MC5R selective agonist.
Amino Acid Sequence ; Binding, Competitive ; Cell Line ; Cell Line, Tumor ; Cyclic AMP ; metabolism ; Genetic Vectors ; Humans ; Iodine Radioisotopes ; Kinetics ; Molecular Sequence Data ; Plasmids ; genetics ; Radioligand Assay ; Receptor, Melanocortin, Type 1 ; agonists ; genetics ; metabolism ; Receptors, Corticotropin ; agonists ; genetics ; metabolism ; Receptors, Melanocortin ; agonists ; genetics ; metabolism ; Transfection ; Tritium ; alpha-MSH ; analogs & derivatives ; chemistry ; metabolism ; pharmacology

Objective To study the effects of five different kinds of Chinese medicine materials on dissolution of pills; To evaluate their in vitro - in vivo correlation.Methods Five different types of traditional Chinese medicine materials, such as starch, fiber, protein, grease and polysaccharide materials, were selected by uniform design to the proposed 8 model formulation and preparation of pills, and the cumulative release rate and in vivo plasma concentration of berberine hydrochloride were determined by HPLC. The effects of Chinese medicine materials on the drug release behavior of pills in vitro - in vivo were investigated. And the in vitro - in vivo correlation of the pills was evaluated.Results Starch and fiber materials could promote the release of the pills, and protein and grease materials has a blocking effect on the pills dissolution. Polysaccharide materials have no significant effect on the dissolution of the pills. Pills in vitro - in vivo correlation was significant.Conclusion Chinese medicine materials have the characteristics of medicine-assisted unity, which can control the dissolution and bioavailability of pills by adjusting the proportion of powder in the prescription. And the pills have good correlation in vitro - in vivo.

Objective To explore the effect of iodine on the pathogenesis of postpartum thymiditis.Methods Forty-four female C57BL/6J mice,8-week old,fed by low iodine dietary(the concentration of iodine≤35 μg/kg),were randomly divided into 4 groups:non-pregnancy experimental autoimmune thymiditis(non-pregnancy EAT)group with 8 mice,EAT of mice was induced by immunization with pig's thyroglobulin(Tg)in the presence of complete Freund's adjuvant.Six mice in non-pregnancy EAT group survived at the end of experiment;normal iodine-PPT(NI-PPT)group,10-fold high iodine-PPT(10HI-PPT)group and 50-fold high iodine-PPT(50HI-PPT)group with 12 mice in each group.The last 3 groups mice,who received the same immunization schedule as the above,were mated with adult male mice followed by induction of EAT.In the end,7,6 and 6 mice were noticed to be pregnant in each group.All animals were killed 4 weeks after postpartum.Histological severity of thyroid specimens was evaluated.The serum level of thyroglobulin antibody(Tg-Ab),thyroid pomxidase antibody(TPO-Ab),TT3 and TT4 were measured by radioimmunoassay(RIA).The expression level of IFN-γ/IL-4 mRNA in spleenwere assayed by RT-PCR.Results Pathological examination showed the infiltration of inflammatory cells.epithelial cell applanation,follicle atrophy or destruction.The severity of inflammation in non-pregnancy EAT bgroup.NI-PPT group and 10HI-PPT group was less serious than that in the 50HI-PPT group,the difference has bstatistical significance(P＜0.05).The level of TPO-Ab in non-pregnancy EAT group,NI-PPI-group,10HI-PPTgroup and 50HI-PPT group wag(14.32±8.85)%,(64.45±10.52)%,(38.46±5.57)%and(90.09±9.98)%.respectively the difference being statistically significant between any two groups(P＜0.05).There was no statisticaldifference(F＝0.484,P＞0.05)of Tg-Ab among non-pregnancy EAT group[(33.74±3.71)%],NI-PPT group [(29.65±2.06)%],10HI-PPT group[(37.21±3.87)%]and 50HI-PPT group[(33.87±4.17)%].There was no statistical difference(F＝1.596,P＞0.05)of TT3 among non-pregnancy EAT group (2.47±0.69)%,NI-PPT group(1.57±0.25)%,10HI-PPT group[(1.60±0.28)%]and 50HI-PPT group[(1.82±0.75)%].The level of TT4 in 50HI-PPT group[(66.68±5.47)%]was lower than that in non-pregnancy group,NI-PPT group and 10HI-PPTgroup[(99.87±5.97)%,(89.13±7.64)%and(91.05±5.82)%],the difference being statistically significant(P＜0.05).The expression level of IFN-γ mRNA was increasing,being 1.02±0.10,1.37±0.10,1.39±0.12 and 1.68±0.06 in non-pregnancy EAT group,NI-PPT group,10HI-PPT group and 50HI-PPT group.The difference had a statistical significance between any two groups except for NI-PPT group and 10HI-PPT group(P＜0.05).The expression level of IL-4 mRNA in 10HI-PPT group(0.49±0.04)and 50HI-PPT group(0.53±0.06)were all higher than non-pregnancy EAT group(0.24±0.05)and NI-PPT group(0.35±O.05),the differences being statistically significant (P＜0.05).Conclusions Adequate iodine supplementation during pregnancy and postpartum period is necessary,but iodine excess could induce postpartum thyroiditis.So iodine supplementation during pregnancy and postpartum should be adequate and reasonable.

Obecfive To study the clinical significance of detecting serum proeollagen type Ⅲ(PCⅢ) and hyaluronie acid(HA)in patients with autoimmune thyroid diseases(AITD).Methods According to the thyroid function,the 114 patients with AITD were divided into hyperthyroidism group(38),hypothyroidism group(35),and sub-hypothyroidism group(41).In addition,40 healthy persons were served as controls.The level of serum PCⅢ was determined with ELISA and that of serum HA with RIA.The level of FT3,FT4 and sTSH were detected by immumnofluorometric assay.Results Serum FT3(18.35±6.19)pmol/L]and FT4[(76.28±23.49)pmol/L]level of patients with hyperthyroidism were obviously higher than those of the controls[(4.75±0.31),(16.12±3.27) pmol/L],but serum sTSH[(0.15±0.07)mU/L]was obviously lower than that of the control[(3.78±0.15)mU/L],the differences were statically significant(P＜0.01).Serum FT3[(3.36±0.26)pmol/L]and FT4 [(6.37±2.19) pmol/L]level of patients with hypothyroidism were both lower than those of the controls(P＜0.05).but serum sTSH[(44.58±13.29)mU/L]was obviously higher than that of the control(P＜0.01).Serum FT3 [(4.86±0.45)pmol/L]and FT4[(15.26±2.78)pmol/L]level of patients with sub-hypothyroidism had no statistical difference compared with those of the controls(P＞0.05),but serum sTSH[(14.26±4.73)mU/L] was obviously higher than that of the controls(P＜0.01).The level of sernm PCⅢ[(4.63±1.22)μg/L]in pafients with hyperthyroidism was significantly higher than that of any other group(P＜0.05).There waB no statistical significant difference in PCⅢ among the patients with hypothyroidism,the patients with sub-hypothyroidism and controls [(3.64±1.12),(3.54±1.17)and(3.56±1.07)μg/L],respectively(P＞0.05).The level of serum HA [(31.13±10.28)μg/L]in patients with hypothyroidism was significantly higher than that of any other group(P＜0.05).There was no statistical significant difference in HA among the patients with hyperthyroidism,the patients with sub-hypothyroidism and controls[(22.24±7.22),(22.43±7.99)and(23.09±9.19)μg/L,respectively,P＞0.05].Conclusions It is very significant to understand myocardial fibrosis early through detecting sernm PCⅢ in patients with hyperthyroidism.Measurement of serum PCⅢ and HA will be useful to discovery hepatic fibrosisearly in patients with a long course of hyperthyroidism.