Objective To investigate the changes of iodide uptake and the expression of thyroidspecific genes in poorly differentiated follicular thyroid carcinoma (FTC) cells after transfection of human TSH receptor (hTSHR) gene in vitro. Methods The recombinant eukaryotic expression plasmid PcDNA3. 1/hTSHR-cDNA was transformed into DH5a bacterial for amplification and then the recombinant plasmid was extracted. The recombinant was identified with PCR amplifying, restriction enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1/hTSHR was transfected into FTC-133 cell line by lipofectin methodin vitro. Immunofluorescence, iodide uptake studies and real time-PCR were applied to detect target protein expression. Statistical analysis was performed with t-test using SPSS 13. 0 software. Results Kpn Ⅰ and Xba Ⅰ restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3. 1/hTSHR was successfully constructed. After transfection of the recombinant plasmid pcDNA3. 1/hTSHR-cDNA and the stimulation of hTSH, the tumor cells displayed the expression of hTSHR protein at cell surface and cytoplasm. The iodine uptake in pcDNA3. 1/hTSHR transfected cells was 2. 9 times higher than that of control(pcDNA3.1(+) transfected cells) group(t = 28.63, P ＜0. 01). The expression of TSHR,NIS, TPO and Tg (mRNA levels) in pcDNA3. 1/hTSHR transfected cells were also significantly elevated by 1.74 (t =5.959, P＜0.01), 7.2 (t =3.807,P＜0.05), 2.88 (t=4.769,P＜0. 01) and 2.67 times (t=6.388,P ＜0.01) respectively compared to those of the control group. Conclusion The study demonstrates that iodide uptake may be reactivated by hTSHR receptor gene transfection in poorly differentiated FTC cell.

OBJECTIVETo investigate the ability of human dendritic cells (DCs) inducing specific T lymphocyte response and inhibit the expression of HBeAg and HBsAg in 2.2.15 cell culture supernatant.

METHODSDCs were prepared from peripheral blood mononuclear cells induced with granulocyte macrophage colony-stimulating factor(GM-CSF) and interleukin 4. DCs was impulsed with pure HBsAg before DCs maturation and cocultured with self-blood T lymphocyte, while DCs without pure HBsAg stimulated group, T lymphocyte group and only T lymphocyte group were prepared as control group. The culture supernatant of 2.2.15 cell with stimulated T lymphocytes was collected on day 1, day 3, day 5 and day 7, respectively. The expressed levels of HBeAg and HBsAg were detected by ELISA method.

RESULTSDCs after antigen stimulation had a strong ability to present antigen and induce immune activation, DCs after loading with antigen in normal control and chronic hepatitis patients group had stronger stimulative ability for T lymphocytes proliferation than that of DCs without loading with antigen and only T lymphocyte group(P less than 0.01). The stimulating ability of DCs had a positive correlation to the dosage of loaded antigen; CTLs produced as a result of DCs stimulation had a specific inhibitive effect on the expression of HBeAg in 2.2.15 cell supernatant,but not on the expression of HbsAg.

CONCLUSIONHuman dendritic cells stimulated with HBsAg in vitro can efficiently present antigen and stimulate the production of specific CTLs to HBV, which can efficiently inhibit the expression of HBeAg in 2.2.15 cell supernatant- DC vaccine may become an antiviral therapy strategy for chronic hepatitis B virus infected patients in future.

Adolescent ; Adult ; Antigen Presentation ; Cells, Cultured ; Child ; Dendritic Cells ; drug effects ; immunology ; Female ; Hepatitis B Surface Antigens ; pharmacology ; Hepatitis B, Chronic ; immunology ; Humans ; Lymphocyte Activation ; Male ; Middle Aged

To evaluate the adjuvant effect of recombinant enterovirus 71 (EV71) subunit vaccine formulated with chitosan, rabbits were orally immunized with recombinant VP1 (rVP1) or rVP1 mixed with chitosan adjuvant. Levels of virus-specific IgG and IgA antibodies in sera, mucosal wash buffer (intestine, nasal cavity, and lung), and feces were determined by indirect enzyme-linked immunosorbent assay (ELISA). The titers of neutralizing antibodies against EV71 were determined using cytopathic effect-based neutralizing assay, and levels of cytokines (IFN-gamma and IL-4) secreted from in vitro-cultured rabbit splenic lymphocytes under antigen stimulation were also determined by ELISA. Results showed that immunization with rVP1 alone could only induce low levels of serum IgG and mucosal IgA, while rVP1 combined with chitosan adjuvant were able to induce significantly higher levels of antibodies, rVP1 can only induce neutralizing antibodies when used in combination with chitosan. Levels of IFN-gamma and IL-4 in the group immunized with rVP1 plus chitosan were significantly higher than those in the group immunized with rVP1 only or those in the control groups. Our study lays the foundation for development of oral VP1 vaccine against EV71 infection.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; Chitosan ; administration & dosage ; immunology ; Enterovirus A, Human ; genetics ; immunology ; Enterovirus Infections ; immunology ; prevention & control ; virology ; Female ; Humans ; Rabbits ; Vaccination ; Vaccines, Subunit ; administration & dosage ; genetics ; immunology ; Viral Proteins ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Objective To analyze the application of multi‐slice spiral CT and 3D airway reconstruction technique on obstruc‐tive sleep apnea syndrome (OSAS) of pharyngeal soft tissue and upper airway stenosis .Methods Selected 92 patients with OSAS in our hospital as the observation group and another 92 healthy persons were selected as control group ,all patients achieved multi‐slice CT and 3D airway reconstruction technical inspections ,counted and compared their pharyngeal soft tissue and upper airway stenosis .Results Airway stenosis of tongue back ,and hypopharynx retro‐palatal of control group were more narrow compared with the control group(P<0 .05);the observation group′s pharyngeal soft tissue was more thick than the control group ,so as the length and width(P< 0 .05) .Conclusion Multi‐slice spiral CT and 3D airway reconstruction technique can effectively share pharyngeal soft tissue and upper airway stenosis of patients with OSAS .

OBJECTIVETo develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA.

METHODSA double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method.

RESULTSThe results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949).

CONCLUSIONThe novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.

Bunyaviridae ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Fever ; virology ; Fluorescent Antibody Technique ; Humans ; Thrombocytopenia ; virology

Background Since the measurement method establishment of serum ferritin abroad in early period of theseventies, the iron deficiency had been divided into two types: the non-anemia and anemia types. In orderto go step further studies, we must ertablish the bemoglobin targets of the two types. Methods One hurdred and fifty-two children in experimontal group, from 6 to 7 years old, and allcome from Qinghai province. There are 29 children in Xining city, 24 in Guide, 26 in Gongbe, 40 in Gui-nan and 33 in Maduo countics. There are 36 health children aged from 6 to 7 years old in the controlgroup, and all comes from Beijing. The Hb, RBC, HCT, HCTW and FEP wcre determined. Results The three targets correlating with Hb (Hb, MCH and MCHC); correlating with RBC (RBC,HCT and MCV); the two targets correlating with RBC_weight (HCTW and CMCW) and correlating withFEP of RBC(FEP and MCEP) have very significant difference between experimental group and control group. Conclusion The determination values of the 10 targets are not same in children in different districts,and the values of all the target: are increased on different degree along with the increase in altitude of ele-vation. There is very important significance on the studies of iron deficiency and altitude hypoxia to establish the normal values of the 10 targets.

Adolescent ; Asian Continental Ancestry Group ; Asthma ; blood ; diagnosis ; genetics ; Chemokine CCL11 ; genetics ; Chemokine CCL2 ; genetics ; Chemokine CCL5 ; genetics ; Child ; Child, Preschool ; China ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Predisposition to Disease ; Humans ; Immunoglobulin E ; blood ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

AIMTo develop a capillary gas chromatographic method for the determination and pharmacokinetic study of patchouli alcohol in rat plasma after iv administration.

METHODSThe drug was extracted with ethyl acetate. Eugenol was used as internal standard. The separation was carried out on a HP-5MS quartz capillary column, with high-purity nitrogen as carrier gas and flame ionization detector (FID) as detector. The column temperature was maintained at 80 degrees C for 1 min and then programmed to 200 degrees C at a rate of 15 degrees C x min(-1); it was held at 200 degrees C for 1 min, and then programmed to 290 degrees C at a rate of 60 degrees C x min(-1); the final temperature was held for 1 min. The temperature of both injector and detector was set at 290 degrees C.

RESULTSThe standard curve was linear from 25 to 5 000 microg x L(-1) in rat plasma. The recovery of this method was from 90.0% to 110.0% with satisfactory relative standard deviation (RSD) less than 10.0%. The pharmacokinetic parameters demonstrated patchouli alcohol were consistent with the two-compartment open model and showed linear pharmacokinetics. The T1/2beta, AUC and MRT of patchouli alcohol in patchouli oil were all higher than that of patchouli alcohol.

CONCLUSIONThis method is quick, precise and reliable. The pharmacokinetics of patchouli alcohol is different from that of patchouli alcohol in patchouli oil.

Animals ; Area Under Curve ; Injections, Intravenous ; Lamiaceae ; chemistry ; Male ; Oils, Volatile ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Sesquiterpenes ; blood ; isolation & purification ; pharmacokinetics

OBJECTIVETo investigate the involvement of transient receptor potential vanilloid receptor 1 (TRPV1) in the facial inflammatory pain in relation to thermal hyperalgesia and cold pain sensation.

METHODSFacial inflammatory pain model was developed by subcutaneous injection of turpentine oil (TO) into rat facial area. Head withdrawal thermal latency (HWTL) and head withdrawal cold latency (HWCL) were measured once a day for 21 d after TO treatment using thermal and cold measurement apparatus. The immunohistochemical staining, cell-size frequency analysis and the survey of average optical density (OD) value were used to observe the changes of TRPV1 expression in the neurons of the trigeminal ganglion (TG), peripheral nerve fibers in the vibrissal pad, and central projection processes in the trigeminal sensory nuclei caudalis (Vc) on day 3, 5, 7, 14, and 21 after TO injection.

RESULTSHWTL and HWCL decreased significantly from day 1 to day 14 after TO injection with the lowest value on day 5 and day 3, respectively, and both recovered on day 21. The number of TRPV1-labeled neurons increased remarkably from day 1 to day 14 with a peak on day 7, and returned back to the normal level on day 21. In control rats, only small and medium-sized TG neurons were immunoreactive (IR) to TRPV1, and the TRPV1-IR terminals were abundant in both the vibrissal pad and the Vc. Within 2 weeks of inflammation, the expression of TRPV1 in small and medium-sized TG neurons increased obviously. Also the TRPV1 stained terminals and fibers appeared more frequent and denser in both the vibrissal pad skin and throughout laminae I and the outer zone of laminae II (IIo) of Vc.

CONCLUSIONFacial inflammatory pain could induce hyperalgesia to noxious heat and cold stimuli, and result in increase of the numbers of TRPV1 positive TG neurons and the peripheral and central terminals of TG. These results suggest that the phenotypic changes of TRPV1 expression in small and medium-sized TG neurons and terminals might play an important role in the development and maintenance of TO-induced inflammatory thermal hyperalgesia and cold pain sensation.

Animals ; Cold Temperature ; Facial Pain ; chemically induced ; metabolism ; physiopathology ; Hot Temperature ; Immunohistochemistry ; Male ; Neurons ; cytology ; metabolism ; Pain Threshold ; physiology ; Rats ; Rats, Sprague-Dawley ; Statistics, Nonparametric ; TRPV Cation Channels ; metabolism ; Thermosensing ; physiology ; Trigeminal Ganglion ; cytology ; metabolism ; Turpentine ; administration & dosage

OBJECTIVETo develop a method for determining artemisinin in rat plasma in vivo.

METHODHPLC-MS was adopted. Estazolam was selected as an internal standard (I.S.). The sample and I.S. were extracted using methyl tertbutyl ether and measured at m/z of 305 and 296, respectively.

RESULTWithin the linear range of 5-500 microg x L(-1), the ratio of artemisinin's peak area and I.S. peak area and the concentration showed good linearity, thus the minimum concentration was set to be 5 mictrog x L(-1).

CONCLUSIONThe methodology proved that the method is so suitable for determining the drug concentration in rat blood that it can be used for studying pharmacokinetics in animals.

Animals ; Artemisinins ; pharmacokinetics ; Chromatography, High Pressure Liquid ; Mass Spectrometry ; Rats