Objective To construct the uItrasound microbubbIes co-carrying recombinant adenovirus containing stromal cell derived factor 1 (SDF-1α) and bone morphogenetic protein 2 (BMP2),and to study the maximum efficiency of carrying adenovirus and the optimum proportion of double gene combined with ultrasound contrast agents.Methods Microbubbles were combined separately with recombinant adenovirus co-expression of enhanced green fluorescent protein and SDF-1α(pAd-EGFP/SDF-1α) as well as red fluorescence protein and BMP2 (pAd-RFP/BMP2) via biotin-streptavidin method,and the maximum efficiency of carrying DNA in microbubbles was detected.Three microbubbles with binary vectors were prepared by blending the two above-mentioned pAd at different ratio (1 ∶1,1 ∶ 2,2 ∶ 1) into the microbubbles.The microbubbles with binary vectors were evaluated though physiochemical properties,fluorescence microscope and flow cytometry to test the carrying rate of DNA in microbubbles.Results There was no significant difference in PH,average diameter and concentrations between targeted microbubbles and control group (P ＞0.05).The carrying efficiency of DNA increased with virus loads in microbubbles,but lowered if further increasing virus amount after reaching saturation.When the proportion of binary vectors and microbubbles was 1 ∶ 1,its efficiency of carrying SDF-1α gene and BMP-2 was approximately equal,and flow cytometry demonstrated that the positive rate of microbubbles labeled by both fluorescein isothiocyanate(FITC) and rhodamine was (65.6 ± 0.5)％.However,it was (59.0 ± 2.3)％ when their proportion was the 2 ∶ 1,which was significantly lower than those when other two proportions (1 ∶ 1 and 1 ∶ 2).Under the fluorescence microscope,the targeted microbubbles were equally surrounded by bright green or red fluorescence.Conclusions Ultrasound microbubbles of double genes carrying EGFP/SDF-1a and RFP/BMP2 is made successfully via biotin-streptavidin method.The optimal proportion of combining microbubbles with double gene is 1 ∶1,which can reveal the optimum load rate and stable combination.

Objective To explore the relevance of high density lipoprotein cholesterol (HDL-C) level and resistance of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in advanced non-small cell lung cancer (NSCLC).Methods In a total of 87 NSCLC patients harboring EGFR mutation treated with EGFR-TKI, the correlations of pre-treatment levels of soluble HDL-C between efficacy and resistance of EGFR-TKI were analyzed by Kaplan-Meier method and Cox proportional hazard model.Results Patients with normal HDL-C (≥0.94 mmol/L) had a better disease control rate (DCR) than those with low HDL-C (＜0.94 mmol/L) [94.5 ％ (52/55) vs 65.7 ％ (21/32), P ＜ 0.001].The results of univariate analysis showed that patients with normal HDL-C had longer progress free survival (PFS) (HR =0.27, 95 ％ CI 0.164-0.444, P ＜ 0.001).The results of multivariate Cox proportional hazard model analysis with sex, pathologic histology, smoking history and mutation type showed that only HDL-C group had a significant difference (P ＜ 0.001).The same results were showed when stratified by sex and smoking history (female: P ＜ 0.001, male: P =0.002;Smoker: P ＜ 0.001, non-smoker: P =0.014).Conclusion Normal pre-treatment serum levels of HDL-C are associated with a better outcome and longer progression-free survival in NSCLC patients treated with EGFR-TKI, which may be a predictive marker for a better response.

Object To study the chemical constituents of Hibiscus mutabilis L.Methods Isolation and purification were carried out on silica gel, or polyamide column chromatography etc. Constituents were identified and structurally elucidated by physicochemical properties and spectral analysis.Results Ten compounds were obtained, nine of them were determined as tetracosanoic acid (Ⅰ), ?-sitosterol (Ⅱ), daucosterol (Ⅲ), salicylic acid (Ⅳ), emodin (Ⅴ), rutin (Ⅵ), kaempferol-3-O-?-rutinoside (Ⅶ), kaempferol-3-O-?-robinobinoside (Ⅷ) and kaempferol-3-O-?-D-(6-E-p-hydroxycinnamoyl)-glucopyranoside (Ⅸ).Conclusion All compounds are isolated from the plant for the frist time except Ⅱ and Ⅵ.

Aim To investigate the myocardial hypertrophy,the expression of transforming growth factor ?1(TGF-?1),Smad3 and Smad7 proteins in spontaneous hypertensive rats(SHR)and the effects of benazepril and candesartan.Methods SHRs of 12 weeks old were given benazepril and candesartan for 12 weeks.The tail arterial pressure was measured every two weeks.At 12 th weekend,cardiac configuration,heart mass index,area of cadiocytes,concertrations of AngⅡin plasma and myocardium,expressions of TGF-?1、Smad3 and Smad7 proteins were measured respectively.Results The arterial pressure,wall thickness,heart mass index,area of cardiocytes and the expressions of TGF-?1,Smad3 proteins increased in SHRs but were attenuated after the treatment of benazepril or candesartan.After the combined treatment,the synergistic effect could be observed.The levels of cardiac tissue and plasma AngⅡwere reduced.The expressions of Smad 7 were up-regulated after the treatment of benazepril or candesartan,while they were stable after the combined use.Conclusion There is a synergistic effect of attenuating myocardial hypertrophy in SHRs by combined use of benazepril and candesartan.It may be related to the regulation of Ang Ⅱ,decreasing the expressions of TGF-?1 and Smad3.

Objective By comparing the proliferation and the human telomerase reverse transcriptase expression of bone marrow mesenchymal stem cells (BMSCs) from rheumatoid arthritis (RA) patients and normal human to explore the existence of BMSCs defects or variation in RA patients,and whether these cells could be used in autologous transplantation.Methods BMSCs were obtained from bone marrow of eight RA patients and healthy volunteers.The cells were isolated andcultured by gradient centrifugation and adherence cultivation,and the surface markers of BMSCs were identitfied by flow cytometertry.Firstly,BMSCs reproductive activity of the two groups were compared:the auxodrome and cell cycle of BMSCs were measured respectively,then the generation doubling time of cell colony and proliferation index were calculated in order to see whether there was a difference of the proliferation capability between the two groups.Furthermore,the hTERT were measured.Results BMSCs were no significant differences between morphology of RA patients and healthy control group.The cells were obtained proof MSCs by flow cytometry.The difference was not statistically significant (P＞0.05),RA group and the healthy control group P3-generation cell population doubling time (4.7±0.3) d,(4.8±0.3) d.The difference was statistically significant (P＜0.01),P6 generation of healthy control group cell population has doubling time (5.7±0.4) d,RA group (6.3±0.3) d,buut the group RA BMSCs population doubling time is longer than the healthy control group.The difference was not statistically significant (P＞0.05),on one hand of the healthy control group P3 BMSCs proliferation index (17.9±2.1)％,on the other hand RA group is (16.9±2.1)％.The difference was statistically significant (P＜0.01).P6-generation BMSCs proliferation index in healthy control group (10.7±1.8)％,RA group (6.2±2.2)％,RA group proliferation index than the control group.The difference was also significant (P＜0.01).The healthy control group P6 generation of MSCs hTERT expression was (10.6±1.5),RA group is (3.1±1.0),hTERT RA patients expression is lower compared with normal control group.Conclusion There is no significant difference in the proliferative capacity of the two groups at P3,but the BMSCs proliferation at P6 lags behind in the normal group in RA patients,which inferred that the senescences of the BMSCs of RA patients are earlier than healthy donors.The determination results of hTERT have shown that the difference between the two groups is statistically significant,and it proves that the premature aging may be associated with hTERT expression.

Objective To prepare a redox-responsive doxorubicin-loaded nanoparticle,and to study its in vitro realease behavior and targeting effect on heptoma cells.Methods Cystamine was grafted on the side chains of hyaluronic acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide catalyst,and then β-cyclodextrin (β-CD) was conjugated on the amine groups of the cystamine by Schiff's base reaction to prepare β-CD modified hyaluronic acid (HACD).The HACD/DOX nanoparticles were prepared by encapsulating DOX into HACD using dialysis method.The drug loading,encapsulation efficiency,particle size and distribution,zeta potential and other physical and chemical properties,as well as in vitro drug release behavior of the HACD/DOX nanoparticles were characterized.The cytotoxicity of HACD/DOX nanoparticles to HepG2 cells was studied by cell counting kit-8 (CCK-8) method.The targeting effect of HACD/DOX nanoparticles on HepG2 cells was studied using flow cytometry and confocal laser scanning microscopy (CLSM).Results HACD were successfully synthesized,which could carry DOX to form uniform homogeneous nanoparticles.The drug loading of DOX in the nanoparticles was (16.1±0.2)％ and the encapsulation efficiency was (64.2±0.9)％.The transmission electron microscope images indicated that the shape of the HACD/DOX nanoparticles was homogeneous sphere.The results of granularity analysis showed that the average size of the HACD/DOX nanoparticles was (203.1 ±2.5) nm with a narrow size distribution (PDI =0.202).The zeta potential of the HACD/DOX nanoparticles was (-29.1±0.8) mV.The in vitro release behavior of the nanoparticles exhibited obvious redox-sensitivity.The results of in vitro cytotoxicity showed that the blank carrier material HACD had no obvious toxicity to hepatoma cells,and the HACD/DOX nanoparticles could effectively kill hepatoma cells with the 0.38 μg/ml half maximal inhibitory concentration (IC50) value at 48 h.Flow cytometry and CLSM results demonstrated that the HACD/DOX nanoparticles could target hepatoma cells through the mediating effect of hyaluronic acid.Conclusions The prepared HACD/DOX nanoparticles have suitable particle size,high drug loading and encapsulation efficiency,and can release DOX under the stimulation of reducing agent.These nanoparticles have obvious targeting effect on hepatoma cells,which is expected to be applied as the drug delivery system of hepatocellular carcinoma (HCC) therapy.

Objective To observe the clinical efficacy of glucocorticoidcombined with the eradication of helicobactepylori (HP) in the treatmenof idiopathithrombocytopenipurpura(ITP) and itinfluence on platelecounand cell subset.MethodNinety-foucaseof ITP were randomly divided into two group.The control group(47 cases) wagiven prednisone treatmen, while the observation group(47 cases) wagiven prednisone combined with HP eradication treatmen.The clinical effecand recur-rence rate within 1 yeawere observed ,and the changeof platelecounand cell subsetwere tested .ResultThe total effective rate of the observation group wa89 .36% ,which wasignificantly highethan 70 .21% in the control group ;the 1-yearecurrence rate in the observation group wa36 .17% ,which wasignificantly lowethan 72 .34% in the control group ,the differencebetween the two grouphad statistical significance (P＜0 .05) .The platelecounaftetreatmenin the two groupwere significantly in-creased ,platelet-associated antibody(PAIgG) level wasignificantly decreased ,buthe improvemenin the observation group wamore significant(P＜0 .05) .The percentage of CD3+CD4+ % increased significantly ,the percentage of CD3+CD8+ % wadecreased significantly ,buthe improvemenin the observation group wamore obviou(P＜0 .05) .Conclusion Glucocorticoidcombined with HP eradication treatmenin treating ITP hadefinitely clinical efficacy ,can increase the platelecoun,improve cell subsetimbalance ,and iworthy to be clinically promoted .

Objective To study the clinical application value of humidified high flow nasal cannula(HHFNC) on bronchitis in children.Methods Total 85 cases of bronchitis that needed oxygen therapy in our department from Oct 2015 to Feb 2016 were randomly divided into three groups,HHFNC group,NCPAP group and nasal cannula oxygen group(control group).According to the blood gas results,FiO2 was adjusted to maintain PaO2 in 50~70mmHg(1mmHg=0.133kPa),TcSO2 90% to 95%.The main symptoms and signs(wheezing,shortness of breath,three depression sign,wheezing rale) disappeared time,length of oxygen inhalation and stay,the change of PaO2,PaCO2,respiratory rate were compared among the three groups.Results Compared with control group,the clinical symptoms and signs disappeared time and length of oxygen inhalation and stay were significantly shorter in HHFNC group(P<0.05).The recovery of blood gas and respiratory rate in HHFNC group were better than those in control group(P<0.05).No statistically significant differences existed between HHFNC group and NCPAP group(P>0.05).Conclusion HHFNC can significantly improve the clinical symptoms,signs and blood gas results in children with bronchitis,reduce the length of oxygen inhalation and stay.HHFNC is an effective and well-tolerated treatment for bronchitis in children.

Objective To explore the mucosal healing quality of hydrotalcite combined with esomeprazole in gastric ulcer.Methods Forty-two patients visiting from June 2014 to December 2015 and diagnosed with gastric ulcer were selected and divided into combination therapy group and single therapy group with 21 patients in each group.The patients of combination therapy group received esomeprazole combined with hydrotalcite,and the patients of single therapy group received esomeprazole alone.The total therapeutic course was eight weeks.At the same period,21 health check-up participants were enrolled as normal control group.The healing of gastric ulcer was observed under white light endoscopy.The morphological changes of gastric pits and microvessel of mucosal at peripheral mucosa around ulcer and normal gastric mucosal were observed under narrow band imaging magnifying endoscopy.The gastric mucosa tissues of the two groups before and after treatment,and normal gastric mucosa of healthy control group were taken.The amount of deposition and composition of collagen fibers,the expression level of factor Ⅷ,the level of transforming growth factor (TGF)-beta1 and the content of hydroxyproline were analyzed by Masson,immunofluorescent and immunohistochemistry staining as well as enzyme linked immunosorbent assay.Chi square test,one-way analysis of variance (ANOVA),least significant difference (LSD) method,Dunnett's T3 and Kruskal-Wallis test and other method were used for comparison.Results After treatment,18 patients of combination therapy group (21 patients) had regular microvessel nets (85.7％),which was significantly more than those of single therapy group (12 cases,57.1％) and healthy control group (10 cases,47.6 ％),and the differences were statistically significant (x2 =4.200,P=0.040).In the comparison of maturity of regenerative mucosa between combination therapy group and single therapy group after treatment,the ration between collagen type Ⅰ and Ⅱ,deposition of collagen fibers,the number of factor Ⅷ positive cells,the level of TGF-beta1 and the content of hydroxyproline were 36.05 and 23.14;269 375.63.± 171 608.63 and 137 693.14±98 330.93;34.91±8.40 and 28.24±6.93;104 498.71±40 487.96 and 70 757.11±19 323.95;(1 897.80±879.35) and (1 230.57±536.05) μg/L,respectively;while in healthy control group,the above parameters were 36.81,245 696.90 ± 224 687.00,23.10 ± 8.40,94 048.04 ±41 306.55 and 1 681.20 ± 423.61 μg/L,and the differences were statitically significant among these three groups (H=7.375,F=3.465,11.680,5.190,5.160;all P＜0.05).Those parameters of combination therapy group were significantly higher than those of single therapy group (H=2.416,LSD method;all P＜0.05).Conclusion Hydrotalcite combined with esomeprazole in the treatment of gastric ulcer could significantly improve microvessel morphology,maturity degree of regenerative mucosal structure and function,and the mucosal healing quality was also superior to single esomeprazole group.

Aim To develop a sensitive,rapid and ac-curate LC-MS /MS method for the simultaneous deter-mination of cytochrome P450 probe substrates,inclu-ding caffeine and its metabolite paraxanthine for CYP1 A2,omeprazole and its metabolite 5-hydroxyome-prazole for CYP2C1 9,dextromethorphan and its metab-olite dextrorphan for CYP2D6,midazolam and its me-tabolite 1 ′-hydroxymidazolam for CYP3A4.Methods Probe drugs with the IS diazepam were extracted using ethyl acetate.Gradient elution was performed on an Agilent Eclipse Plus-C1 8 column (50 mm ×2.1 mm, 3.5 μm).The mobile phase consisted of 0.01 % for-mic acid(1 mmol·L -1 ammonium formate)and aceto-nitrile.The flow rate was 0.3 mL·min -1 ,and the in-jection volume was 1 0 μL.The analyte was detected u-sing electrospray ionization(ESI)in positive multiple reaction monitoring(MRM+)mode.The reaction se-lected ions were 1 95.0 /1 38.1 m /z for caffeine, 1 81 .1 /1 24.1 m /z for paraxanthine,346.1 /1 98.1 m /z for omeprazole,362.1 /21 4.1 m /z for 5-hydroxyome-prazole, 272.2 /1 47.1 m /z for dextromethorphan, 258.1 /1 57.1 m /z for dextrorphan,326.1 /291 .1 m /z for midazolam,342.1 /324.1 m /z for 1 ′-hydroxymid-azolam and 285.1 /1 54.0 m /z for diazepam as internal standard.Results The linear ranges of caffeine,pa-raxanthine,omeprazole,5-hydroxyomeprazole,dextro-methorphan, dextrophan, midazolam and 1 ′-hydroxymidazolam were 1 .95 ~2 000,0.98 ~250, 0.48 ~2 000,0.98 ~250,0.98 ~2 000,0.48 ~1 25,1 .95 ~2 000 and 1 .95 ~250 μg·L -1 respec-tively.The RSD of all probe drugs was less than 1 5%and matrix effects in plasma on the ionization of probe drugs were negligible.Conclusion This sensitive and rapid LC-MS /MS method is suitable for determination of the drug/metabolite concentrations in plasma,so as to study the metabolism of CYP1 A2, CYP2C1 9, CYP2D6 and CYP3A4 in depth.