Peroxidsome proliferator-activated rreceptor γ(PPARγ) has characteristics of regulation of adipocyte differentiation and lipid metabolism.In recent years,more and more evidences suggest that PPARγ plays an important role in the regulation of immune and inflammatory response.The PPARγ expression increased in airway of asthma patients, and PPARγ was involved in airway inflammation and airway hyperresponsiveneas. Recent studies have shown that PPARγ ligands may have a role in the treatment of asthma.

Phytantriol (PT), ethanol (ET) and water were used to prepare in situ cubic liquid crystal (ISV2). The pseudo-ternary phase diagram of PT-ET-water was constructed and isotropic solution formulations were chosen for further optimization. The physicochemical properties of isotropic solution formulations were evaluated to optimize the composition of ISV2. In situ hexagonal liquid crystals (ISH2) were prepared based on the composition of ISV2 with the addition of vitamin E acetate (VitEA) and the amount of VitEA was optimized by in vitro release behavior. The phase structures of liquid crystalline gels formed by ISV2 and ISH2 in excess water were confirmed by crossed polarized light microscopy and small angle X-ray scattering, respectively. Rheological properties of ISV2 and ISH2 were studied by a DHR-2 rheometer. In vitro drug release studies were conducted by using a dialysis membrane diffusion method. Pharmacokinetics was investigated by determination of sinomenine hydrochloride (SMH) concentration in synovial membrane after intra-articular injection of SMH-loaded ISH2 in adjuvant-induced arthritis rats. The optimal ISV2 (PT/ET/water, 64 : 16 : 20, w/w/w) loaded with 6 mg x g(-1) of SMH showed a suitable pH, injectable and formed a cubic liquid crystalline gel in situ with minimum water absorption in the shortest time. The optimal ISV2 was able to sustain the drug release for 144 h. The optimal ISH2 system was prepared by addition of 5% VitEA into PT in the optimal ISV2 system. This ISH2 (PT/VitEA/ET/water, 60.8 : 3.2 : 16 : 20, w/w/w/w) was an injectable isotropic solution with suitable pH. The new ISH2 was able to sustain the drug release for more than 240 h. Local pharmacokinetics study indicated that the retention time and AUC(0-∞) of ISH2 group were increased significantly compared with that of SMH solution group and the AUC(0-∞) of ISH2 group was 6.01 times higher than that of SMH solution group. The developed ISH2 was suitable for intra-articular injection that may apply to patients in the treatment of rheumatoid arthritis.

Aim To construct eukarytic vector for rat endostatin(endos) cDNA and observe its expression in C6 cell.Methods cDNA encoding rat endostatin was amplified from newborn brain tissue with RT-PCR and inserted into the eukarytic vector pBudCE 4.1.Recombinant was identified with KpnI,XhoI double digestion,PCR and nucleotide sequencing of the target gene.After successful reconstruction of the genes of endostatin,the recombinants was transfected into C6 cells with lipofectintechniques.The positive clones were screened out through zeocin resistance test.The endostatin in supernate of the positive clones was identified with Western-blot and MTT method.With immunocytochemistry,the endostatin in the positive clones was located.The quantities of VEGF in supernate of the positive clones were quantified with ELISA assay.Results The size of the amplified endostatin gene fragment was in accord with that we expected.And the gene sequence inserted into the eukarytic vector pBudCE 4.1 was consistent with the known sequence.Endostatin was secreted from the positive clone.Down-requlation of vascular endothelial growth factor(VEGF) was found in the positive clones.Conclusion The recombinant of rat endostatin gene clone had been established and inserted into the eukarytic vector pBudCE 4.1 successfully and endostatin was expressed in C6 cells.This provides a basis for further studies of endostatin effects in vivo,and creates the conditions for final clinical trial

Objective To evaluate the accuracy of an expiratory resistance device assisting pulse pressure variation (PPV) in predicting volume responsiveness in the spontaneously breathing patients.Methods Forty spontaneously breathing patients of both sexes,aged 22-61 yr,weighing 51-73 kg,of American Society of Anesthesiologists physical status Ⅰ or Ⅱ,undergoing elective operation,were enrolled.Before induction of anesthesia,mean arterial pressure (MAP),heart rate (HR),central venous pressure (CVP),cardiac index (CI) and pulse pressure variation (PPVB) were recorded after haemodynamics were stable.Then the expiratory resistance device was used,and MAP,HR,CVP,CI,and PPVA were recorded.The device was then removed.Volume expansion was carried out.6％ hydroxyethyl starch 130/0.4 6 ml/kg was infused over 10 min.MAP,HR,CVP,CI and PPVB were recorded within 3 min after volume expansion.The device was used again,and 1 min later MAP,HR,CVP,CI and PPVA were recorded.The device was then removed.The patients were divided into 2 groups according the percentage of increase in CI after volume expansion (△ CI):△ CI≥ 15％ considered to be a positive response group (group P),and ACI＜15％ considered to be a negative response group (group N).A receiver-operating characteristic (ROC) curve for PPV was plotted.According to the ROC curve,the diagnostic threshold,sensitivity,specificity,area under the curve,and 95％ confidence interval of the expiratory resistance device assisting PPV in predicting volume responsiveness were determined.Results The area under the curve (95％ confidence interval) of PPVA was 0.880 (0.70-0.98),the diagnostic threshold was 13.5％,and the sensitivity and specificity in determining volume responsiveness were 87％ and 88％,respectively.Compared with the value before administration of the loading dose,the CVP and CI were significantly increased,and PPVB and PPVA were decreased after volume expansion in group P,and the CVP and CI were significantly increased after volume expansion in group N (P＜0.05).Compared with group P,the PPVA was significantly decreased before volume expansion,and the CI was increased after volume expansion in group N (P＜0.05).Conclusion The expiratory resistance device can assist PPV in predicting volume responsiveness in the spontaneously breathing patients.

Serum vitamin E was determined using bathopllenanthroline by microspe-ctrophotometric method. The average recovery rate was 93.8 (92.2-98.0) per cent and the coefficients of variation were 2.5 and 7 per cent in high and low levels respectively. This method seems to be quite reliable and sensitive. Of the total 117 serum samples, 30 pairs matched blood for mother and cord, 27 cord blood, and 30 normal adults as control were studied. The mean level of vitamin E in the cord blood was 2.8 ug/ml (?0.9SD), which was about one third of that in the adult. The level of vitamin E in post-partum mother was 12 ?g/ml (?2.5 SD), which was significantly higher than that of nonpregnant women (p

Aim To investigate the influence of endostatin on pathological morphology of C_6 glioma and its molecular pharmacologic mechanism.Methods The C_6 cells,the C_6 clone stably transfected with endostatin cDNA(endo-C_6),which the endostatin with biological activities can be secreted from,and the C_6 clone stably transfected with empty vector pBudCE4.1 cDNA(pBud-C_6) were injected subcutaneous in nude mice to establish different tumoral model respectively.The morphologies of these tumoral tissues were observed and compared to each other under light and electron microscope.The expression of VEGF in tumor tissue was determined by ELISA.Results The expression of VEGF in endo-C_6 glioma(endo-C_6G) tissues was lower than that in C_6 glioma(C_6G) and pBud-C_6 glioma(pBud-C_6G).Moreover,endo-C_6G tissue was characteristic of a mimetic envelope,no intratumor bleeding and cystis degeneration,apoptosis of tumor cells,and edema in and around tumor.Rarefied vessels were found in tumor,and no vessel like structure formed by tumor cells was observed.The large irregular necrosis focus was showed in tumor,but mild vascular reaction around necrosis focus and peritumor,rare surrounding invasion.The basal lamina was discontinued.The basemembrane(BM) was loose.Few vesicular vacuolar organelle(VVO) structures were observed in plasma of endothelial cells.In C_6G and pBud-C_6G,tumor lesions demonstrated significant vascular reaction,intratumor bleeding,necrosis,edema in and around tumor,and surrounding infiltration.Vessel like structure formed by tumor cells was also observed.When examined with electron microscope,plenty of VVO structures were observed in plasma of endothelial cells,the worse the edema,the more the VVO were,which coincided with the expression of VEGF.Mostly,loose basal lamina surrounded by small amounts of collagen fibers was multilayer and integrated and continuous.No correlation between gene transfection and fenestra formation or cleft of capillary endothelial cell was observed,no apoptosis of endothelial cells were found.Conclusion In glioma,the apoptosis of endothelial cells tissue was not induced directly by endostatin,but the angiogenesis and vascular reaction can be inhibit by endostatin by down-regulation of the expression of VEGF in C_6 glioma cells.

ObjectiveTo determine the subcellular localization of exogenous human papillomavirus type 16 E6 protein(HPV16 E6) and hDaxx in HeLa cells and their effects on tumor necrosis factor (TNF)-α-induced apoptosis.MethodsHeLa cells were transfected with plasmids pDsRed-monomer-C1/HPV16 E6,pEGFP-CI/hDaxx,pEGFP-C1 and pDsRed-monomer-C1 respectively.Subsequently,Western blot was carried out to quantify the expression of fusion proteins DsRed-HPV16E6 and EGFP-hDaxx in transfected cells,and laser scanning confocal microscopy to observe the subcellular distribution of HPV16 E6 protein and hDaxx.Some HeLa cells were divided into 5 groups:untransfected (control group),untransfected and treated with TNF-α(TNF-ot group),transfected with pcDNA3.1 (-) and treated with TNF-α(empty vector group),transfected with pcDNA3.1 (-)/HPV16 E6 and treated with TNF-α (HPV16 E6 group),cotransfected with pcDNA3.1(-)/HPV16 E6 and pcDNA3.1 (-)/hDaxx and treated with TNF-α (cotransfected group).After additional culture,the cells were collected and subjected to flow cytometry(FCM) to evaluate the apoptosis of cells as well as spectrophotometry to determine the relative activity of Caspase-8 and Caspase-3.ResultsWestern blot showed that both DsRed-HPV16 E6 and EGFP-hDaxx were expressed in HeLa cells.In Hela cells transfected with pDsRedmonomer-C1/HPV16 E6 or pEGFP-C1/hDaxx alone,the red fluorescence of HPV16 E6 was observed in the nucleus and cytoplasm,while the green fluorescence of hDaxx only in the nucleus; in those cotransfected with pDsRed-monomer-C1/HPVl6 E6,HPV16 E6 and hDaxx proteins were regionally aggregated near the nuclear membrane in nuclei,and hDaxx was partly translocated from the nucleus to the cytoplasm.The apoptosis rate and relative activity of Caspase-8 and Caspase-3 were statistically lower in HPV16 E6 group than in the empty vector group and cotransfected group(21.4％ ± 1.1％ vs.27.0％ ± 0.9％ and 32.5％ ± 2.1％,0.057 ± 0.003 vs.0.092 ±0.012 and 0.109 ± 0.013,0.054 ± 0.006 vs.0.093 ± 0.005 and 0.110 ± 0.004,all p＜ 0.01).Conclusions HPV16 E6 protein induces the partial translocation of hDaxx from the nucleus into the cytoplasm and colocalizes with hDaxx in the cells.The apoptosis of HeLa cells induced by TNF-α can be suppressed by HPV16 E6 protein,while the overexpression of hDaxx can attenuate the suppressing effect of HPV16 E6 protein on apoptosis in Hela cells.

ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.

Objective To verify the relationship between transforming growth factor-β1(TGF-β1) and interleukin-10 (IL-10) in breast milk and allergic diseases development in infants. Methods Totally 191 mothers (99 allergics and 92 controls) and their full-term newborns participated in this prospective study on development of children atopy. Maternal blood, cord blood, colostrum and mature milk were assayed for TGF-β1 and IL-10 by enzyme-linked immunosorbent assay. Infants underwent pediatrician evaluation for allergic diseases at six months old. Concentrations of TGF-β1 and IL-10 from allergic and non-allergic mothers and prevalence of allergic diseases of infants were compared. Results The level of IgE in allergic mothers was 30 750 IU/L(6600-410000 IU/L),lower than that in non-allergic mothers[50000 IU/L(7100-610000 IU/L)](Z=-3. 444,P=0. 001).No difference in the concentration of TGF-β1, IL-10 and IgE in mature milk was observed between allergic and non-allergic mothers. TGF-β1, IL-10 and IgE levels in colostrum of allergic mothers were 2300 pg/ml(620-7000 pg/ml), 12. 8 pg/ml(7.5-560.0 pg/ml)and 7000 IU/L(5100-56000 IU/L),significantly higher than those in non-allergic mothers[1830 pg/ml(1240-9400 pg/ml), 11. 1 pg/ml (7. 2-630.0 pg/ml)and 6700 IU/L(5200-35000 IU/L)] (Z=-2. 215, -2. 730 and -2. 706,P＜0.05).In both allergic and non-allergic mothers, TGF-β1 and IL-10 levels in cord blood were higher than those in maternal blood, while IgE was lower. TGF-βl and IL-10 and IgE levels in colostrum were higher than mature milk(P＜0.05). At six months old, the prevalence of allergic diseases of infants from allergic mothers(59. 6%, 59/99) was significantly higher than those from non-allergic mothers (21. 7%, 20/92)(x2= 28. 177, P= 0. 000). The prevalence of allergic diseases of infants who completed two weeks' colostrum-fed after birth (44.5 %, 73/164) was significantly higher than those who did not (22.2%,6/27)(x2 =4. 749,P-=0. 029). Conclusions High concentration of TGF-βl and IL-10 in colostrum does not show any protective effect against allergic diseases in infants. The prevalence of allergic diseases of colostrum-fed infants is significantly higher than non colostrum-fed infants, showing that colostrum-fed might play a role in allergic diseases development.

Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.