Objective To construct a plasmid DNA encoding G glycoprotein of respiratory syncytial virus(RSV) and investigate the protective immune response against RSV infection. Methods Recombinant plasmid DNA of pcDNA3.1~G was constructed by standard RT-PCR based cloning procedure. The immunogenicity of recombinant G protein transiently expressed in HEK293 cells was detected by Western blot. BABL/c mice were intramuscularly immunized with pcDNA3.1~G. Samples of lung, sera, bronchoalveolar lavage fluid(BALF) were collected before and after RSV challenge; virus titer in lung was detected by viral titration; sections of paraffin embedding lung tissues were stained by haematoxylin and eosin(HE) for histological analyses; sera anti-RSV IgG levels were examined by ELISA; Th1/Th2 cytokine were detected by ELISA kit, the T lymphocyte subsets of BALF was determined by immunefluorescence staining followed by flow cytometry. Results Plasmid DNA of pcDNA3.1~G was successfully constructed. The expressed target protein possesses immunogenicity. After challenge, pcDNA3.1~G immunized mice presented relieved pathological changes in lung as well as reduced lung viral titers. The RSV specific IgG was detected in sera of immunized mice. There was significantly increased number of CD25~+CD4~+ T cells in mice BALF. Conclusion We constructed a pcDNA3.1~G plasmid DNA vaccination which can induce evident protective cellular immunity against RSV infection in mice with the increased number of CD25~+CD4~+ T cell subpopulation.

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 1:16.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.

Aged ; Castleman Disease ; complications ; Hepatitis B ; complications ; Humans ; Liver Neoplasms ; complications ; virology ; Lymphoma, Non-Hodgkin ; complications ; Male

The purpose of this study is to investigate the effects of multiple-trough sampling design and nonlinear mixed effect modeling (NONMEM) algorithm on the estimation of population and individual pharmacokinetic parameters. Oxcarbazepine and tacrolimus were used as one-compartment and two-compartment model drugs, respectively. Seven sampling designs were investigated using various number of trough concentrations per individual ranging from 1-4. Monte Carlo simulations were performed to produce state-steady trough concentrations. One-compartment model was used to fit simulated data from oxcarbazepine and tacrolimus. The accuracy and precision of the estimated parameters were evaluated using the median prediction error (PE), the median absolute PE and boxplot. The results indicated that trough concentrations could yield reliable estimates of apparent clearance (CL/F). For oxcarbazepine, as the number of trough concentrations per subject increased, the accuracy and precision of CL/F, between-subject variability (BSV) of CL/F and residual variability (RUV) tended to be improved. For tacrolimus, however, although no improvement were observed in the accuracy of CL/F and BSV of CL/F, the PE distribution ranges were significantly narrowed and the RUV estimates were less bias and imprecise. In terms of algorithm, Monte Carlo importance sampling (IMP) and IMP assisted by mode a posteriori estimation (IMPMAP) were consistently better than other methods. Additionally, the sampling design had no significant effects on the individual parameter estimates, which were only depended on the interaction between BSV and RUV in various algorithms. Decreased in BSV and RUV levels can improve the accuracy and precision of the estimation for both population and individual pharmacokinetic parameter estimates.
Algorithms ; Bayes Theorem ; Carbamazepine ; analogs & derivatives ; pharmacokinetics ; Humans ; Immunosuppressive Agents ; pharmacokinetics ; Models, Biological ; Monte Carlo Method ; Nonlinear Dynamics ; Regression Analysis ; Tacrolimus ; pharmacokinetics

Objective To investigate the effect and molecular mechanism of cyclooxygenase-2 (COX-2) on the benign prostatic hyperplasia (BPH) in rat model. Methods Thirty-six SD rats were randomly divided into 3 groups:normal control (group A,n=12),BPH model (group B,n=12) and BPH+selective COX-2 inhibitor celecoxib (group C,n=12). At the 5th week after treatment, the weight of the prostates was measured, and the morphological changes were examined under light microscope.Detection of ki-67 and TUNEL in prostatic epithelial and stromal cells was undertaken to assess the proliferation and apoptosis status.The protein and mRNA expression of COX-2,epidermal growth factor (EGF),basic fibroblast growth factor (bFGF) and transforming growth factor-?1 (TGF-?1) were analyzed by means of immunohistochemisty and RT-PCR. Results The prostate index [prostate wet weight (mg)/rat body weight (g)] of group B was significantly higher compared with those in groups A and C (1.88?0.17 vs 1.70?0.09 and 1.74?0.16,P0.05). Conclusions The increased expression of COX-2 may play an important role in the pathogenesis of BPH by modulating the expression of growth factors and affecting the proliferation and apoptosis of prostate cells.

0.05). Conclusions The expression of angiotensinⅡincreases in BPH tissues in rats. It is suggested that angiotensinⅡmay affect cell proliferation but not significantly affect cell apoptosis of the rat prostate.

To compare the difference of total phenol of magnolia solid dispersion prepared by different methods. Hot melt extrusion, solvent evaporation method, and fusion-cooling method were used to prepare total phenol of Magnolia accessory solid dispersion, Plastone S-630 and HPC. The drug dispersion state in the prepared solid dispersion was evaluated with DSC and X-ray diffraction; FT-IR method was used to analyze the possible connections between drug and accessories. Finally, accelerated stability-in vivo dissolution test was use to compare the stability differences between these three processes. The results of DSC and X-ray diffraction showed that all of the drug in solid dispersion processed by three processes can exist in amorphous form; FT-IR results also could not distinguish the difference between the three processes; accelerated stability-in vivo dissolution test showed the stability of solid dispersion prepared by HPC was better than Plastone S-630, and the same kinds of materials solid dispersion prepared by hot melt extrusion showed a better stability than the other two processes.
Chemistry, Pharmaceutical ; methods ; Drugs, Chinese Herbal ; chemistry ; Magnolia ; chemistry ; Phenol ; chemistry ; Spectroscopy, Fourier Transform Infrared ; X-Ray Diffraction

Objective To explore the distribution of RAS,AGT,ACE,eNOS,ET-2,ANP and NPRC of es-sential hypertension in Yunnan Han healthy population.Methods Gene chip technology was used to detect the pol-ymorphism of AGT M235T (MM, MT,TT), ACE I/D (II, ID, DD ), eNOS Glu298Asp (EE, ED, DD), ET-2 A985G (AA,AG,GG) ,ANP T2238C(TT,TC,CC) and NPRC A-55C(AA,AC,CC) in 97 health subjects.Results The MM,MT and TT genotype frequency of AGT M235T was 0.052,0.381 and 0.567 ,alle frequency of M and T was 0.242 and 0.758 in 97 healthy subjects of Yunnan population;The II, ID and DD frequency of ACE I/D was 0.340, 0.598 and 0.062, alle frequency of I and D was 0.680 and 0.320 in 97 healthy subjects of Yunnan population ;EE, ED and DD frequency of eNOS Glu 298 Asp was 0.845,0.144 and 0.011 ,alle frequency of E and D was 0.918 and 0.082 in 97 healthy subjects of Yannan population;AA,AG,GG frequency of ET-2 A985G was 0.020,0.258 and 0.722, alle frequency of A and G was 0.149 and 0.851 in 97 healthy subjects of Yunnan population;TT and TC fre-quency of ANP T2238C was 0.959 and 0.041 ,and CC was not detected;alle frequency of T and C was 0.979 and 0.021 in 97 healthy subjects of Yunnan population;AC and CC frequency of NPRC A-55C was 0.763 and 0.237, and no AA was detected,alle frequency of A and C was 0.381 and 0.619 in 97 healthy subjects of Yunnan popula-tion.Conclusion The polymorpbism of ACT M235T,ACE I/D,eNOS Glu298Asp,ET-2 A985G,ANP T2238C and NPRC A-55C is locally distributed in Yunnan Han healthy population.

OBJECTIVE To investigate the feasibility and application of enzyme-linked bridging assay(ELBA)method to the pharmacokinetic evaluation of antisense strand siRNA drug. METHODS Antisense strand RNAs were diluted in LNCap cell lysates from 5 to 50 000 pmol·L-1 to construct the quantification curves. We transfected the intact double-strand siRNA at a final concentration 100 nmol·L-1 targeting Polo-like kinase into the LNCap cells and investigated the specificity of ELBA quantitating the siRNA antisense strand in cell supernatant,cell lysates and RNA-induced silencing complex( RlSC). Quantification curves were constructed and validated in biological matrices such as plasma (5-25 000 pmol·L-1 )and multiple tissues(liver,heart,spleen,and kidneys)(3-6250 pmol·L-1 ). The prostate specific membrane antigen aptamer siRNA delivery system with the intact siRNA concentration of 15 nmol·kg-1 was prepared. The siRNAs were delivered into the LNCap xenogrant tumor model in C57 mice by tail vein injection. The concentration of siRNA antisense strand was determined in plasma and tissues 30 min post administration by ELBA. RESULTS The quantitative range of antisense strand siRNA in cell lysates was 5-50 000 pmol·L-1 ,and ELBA method could quantify the siRNA antisense strand concentration from cell lysates and RlSC in LNCap cells transfected with double-strand siRNA. ln addition,ELBA could specifically reflect the single antisense strand concentration instead of intact siRNA double strands in plasma. The quantification range of siRNA antisense strand using ELBA in plasma was 5-25 000 pmol·L-1 and 3-3125 pmol·L-1 in tissues. About 30 min post administration of PSMA aptamer-siRNA,the antisense strand of siRNA was distributed mainly to the tumor,liver,kidneys,blood and spleen in sequence. The distribution profile might be attributed to the target delivery and siRNA pharma-codynamics. CONCLUSION The ELBA method is successfully applied to the siRNA antisense strand pharmacokinetic evaluation,which provides an alternative for pharmacokinetic studies of siRNA-based drugs.

Objective To determine the risk factors of ruptured hepatocellular carcinoma (HCC) and to study the prognostic factors of long-term survival.Methods Of the 4209 patients with HCC diagnosed and treated at the Eastern Hepatobiliary Surgery Hospital from Apr 2002 to Nov 2006,200 patients (4.8％) presented with ruptured HCC.These patients were studied retrospectively and the results of treatment were evaluated and compared with a randomly selected group of 202 patients who had no history of rupture and were treated during the study period.Results On multivariate logistic regression analysis,co-existing hypertension and cirrhosis,tumor size ＞5 cm,vascular thrombus and extrahepatic invasion were predictors of spontaneous rupture of HCC.For the 200 patients with spontaneous rupture of HCC,105 patients underwent elective one stage hepatic resection,33 received transcatheter arterial chemoembolization (TACE),and 62 were treated conservatively.The median survival time (MST) for patients with spontaneous rupture of HCC was 6 months (range,1-72 months),and the overall survival rates at 1-,3-and 5-year were 32.5％,10％ and 4％,respectively.The MST was 12 months (range,1-72 months) in the surgical group,4 months (range,1-30 months) in the TACE group and 1 month (range,0-19 months) in the conservative group.The 1-,3-and 5-year overall survival rates in patients with ruptured HCC who received partial hepatectomy were 57.1 ％,19.0％ and 7.6％,respectively,compared with 77.1％,59.8％ and 41.2％ in 98 patients who underwent partial hepatectomy for HCC without rupture (P＜0.001).Conclusions For patients with HCC who had underlying of hypertension and cirrhosis,extrahepatic invasion and tumor size ＞5 cm,there was a high propensity to rupture.Prolonged survival could be achieved in selected patients who received one-stage partial hepatectomy,although the survival results were inferior to the patients had no rupture.