BACKGROUND:Tripterygium wilfordi and its certain monomers have exact clinical effects on rheumatoid arthritis. However, there are few studies about a systematic discussion on pharmacodynamic material basis and molecular mechanism of Tripterygium wilfordi. OBJECTIVE:To explore the pharmacodynamic material basis and molecular mechanism ofTripterygium wilfordi in treating rheumatoid arthritis. METHODS: Based on the platform of Discovery Studio 4.0, the molecular set of Tripterygium wilfordiwas built and compared with the rheumatoid arthritis drug set from Therapeutic Target Database in chemical space. After that, network pharmacology was used to explore the interactions ofTripterygium wilfordi and therapeutic targets related to rheumatoid arthritis. RESULTS AND CONCLUSION:The molecular sets ofTripterygium wilfordi and drugs for treating rheumatoid arthritis had similar chemical space. The pharmacodynamic material basis ofTripterygium wilfordi had 46 compounds, such as celacinnine, epigalocatechin, euonine, triptolide. They could mediate inflammation, regulate immune response, inhibit cartilage and bone destruction, improve blood stasis-type rheumatoid arthritis by acting on 10 targets, such as tumor necrosis factor-α, JAK-1, matrix metaloproteinase-1, matrix metaloproteinase-3, matrix metaloproteinase-9. Computer simulation could intuitively trace out the multi-ingredient, multi-target and multi-pathway effects of Tripterygium wilfordi.

Objective To evaluate the value of detection of interleukin 28B (IL28B) rs12979860 by allele-specific PCR (AS-PCR) for the prediction of antiviral treatment hepatitis C patients.Methods One hundred seventy-four blood samples were random collected from hospitalized patients with hepatitis C,who came from department of infectious diseases,Renmin Hospital of Wuhan University from May 2011 to May 2012.Two pairs of specific primers were designed for rs12979860 gene polymorphisms,and one mutated base was introduced to the second or third site of the end of 3' with the reverse primer.rs12979860 gene polymorphism of 30 cases with hepatitis C was detected by AS-PCR,and gene sequencing was further used to verify the consistency of the two methods in parallel.Then,the frequency distribution of different rs12979860 genotypes with 174 cases were analyzed by the AS-PCR method in the population.Results The genotype CC,CT or TT of rs12979860 with 30 cases could be well identified by both AS-PCR and gene sequencing,and the coincidence rate was 100％ (x2 =60.0,P ＜ 0.01).Compared to gene sequencing,both of the sensitivity and specificity of AS-PCR were 100％.Compared to the control (CC genotype),TT genotype detection sensitivity by AS-PCR was 10-5,while sequencing sensitivity was 2 × 10-1.rs12979860 polymorphism in the TT,CC and CT genotype distribution in the Chinese population frequencies were 3.45％ (6/174),13.2％ (23/174) and 83.3％ (145/174),respectively.Conclusion AS-PCR can quickly,accurate,reliable,economic and efficiently detect IL28B rs12979860 gene polymorphism of hepatitis C in patients,which could predict the effect of antiviral therapy on patients with hepatitis C.

Objective To construct the RNA interference(RNAi)recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor 1?(HIF-1?)and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1.Methods The recombinant adenovirus Ad was constructed.HIF-1? inserted with HIF-1? RNAi fragment via AdEasy system.The virus was purifed by CsCl gradient centrifuge.The functional titer of recombinant adenovirus was measured by transfection test in HEK 293 cells.SPCA-1 cells were transducted with 2 multiplicity of infection(MOI)Ad.HIF1? in vitro,the expression rate of green fluorescence protein(GFP)was recorded by flow cytometry,HIF-1? mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry.ResultsThe recombinant shuttle plasmid PAdTrack.HIF-1? and adenovirus plasmid Ad.HIF-1? were all correct shown by enzyme digestion confirmation.The plasmid pAd.HIF-1? was transducted into HEK293 cells,15%GFP expressionwere seen after 3 days.The final titers of recombinant adenovirus were 5.0?1010 TU/mL.SPCA-1 cells was transducted by Ad.HIF-1? in vitro for 48 h,GFP expression rate was 92%,HIF-1? mRNA and protein level decreased 89% and 87%,respectively.Conclusion RNAi adenovirus vector of human HIF-1? gene has been successfully constructed,which could facilitate the research onHIF-1? gene related gene therapy for lung cancer.

OBJECTIVE: To determine the chlorpyrifos in human blood by liquid chromatography-tandem mass spectrometry and to validate its application in poisoning cases. METHODS: The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 MGII column (250 mm x 2.0 mm, 5 μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammonium acetate) and solvent B (methanol with 2 mmol/L ammonium acetate) at 5:95 V:V). RESULTS: The linear ranged from 5 to 500 ng/mL (r = 0.998 7). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 2 ng/mL and 4 ng/mL, respectively. For this method, the precision and accuracy of intra-day and inter-day were < 10% and 97.44%-101.10%, respectively. The results in stability test of long-term frozen were satisfied. The matrix effect, recovery and process efficiency were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively. CONCLUSION This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.
Chlorpyrifos/blood* ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid/methods* ; Humans ; Limit of Detection ; Poisoning ; Reproducibility of Results ; Tandem Mass Spectrometry/methods*

Objective To quantify the expressions of tumor necrosis factor-α (TNF-α),caspase-8 and caspase-3 in lichen planus (LP) lesions,and to investigate their significance.Methods Skin samples were collected from the lesions of 20 patients with LP and normal skin of 20 healthy human controls.Immunohistochemistry was used to determine the expressions of TNF-αt,caspase-8 and caspase-3,and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique to evaluate the apoptosis in keratinocytes,in these samples.Results The expression levels (expressed in integrated optical density,IOD)of TNF-α,caspase-8 and caspase-3 were (12.58 ± 2.33) × 103,(11.69 ± 3.52) × 103 and (11.45 ± 2.82) × 103 respectively in LP lesions,significantly higher than those in the normal skin ((5.12 ± 1.78) × 103,(3.87 ± 3.36)× 103,(4.76 ± 1.93) × 103,t =11.38,7.19,8.76,respectively,all P ＜ 0.01).Elevated apoptosis index was noted in keratinocytes from LP lesions compared with those from normal skin (71.35 ± 7.93 vs.33.62 ± 8.75,t =14.29,P ＜ 0.01).In LP lesions,the expressions of both TNF-α and caspase-8 were positively correlated with the apoptosis index of keratinocytes (r =0.72,0.75,respectively,both P ＜ 0.01) and the expression of caspase-3 (r =0.68,0.73,respectively,both P ＜ 0.01).Conclusion The up-regulated expressions of TNF-α,caspase-8 and caspase-3 may participate in the apoptosis in keratinocytes in LP.

Objective To explore the clinical characteristics and treatment of nodular panniculitis disease in children. Methods Clinical data of 13 cases with nodular panniculitis disease were reviewed retrospectively. Their etiology,clinical manifestation,misdiagnosis cause,pathologic characteristics, treatment and outcome were analyzed. Results Its clinical manifestation was multiform and showed mainly as fever and hypodermic nodule. Concomitant damages to digestive, respiratory, circulatory and renal system might occur in those children with the system type of this disorder. Conclusion Pediatric nodular panniculitis disease can be easily misdiagnosed and lack of specificity in the early stage, and complicates multiple organs damage.

Objective To explore the prognostic value of arterial blood lactate (Lac) levels and lactate clearance rate (LCR) in the patients with septic shock.Methods A retrospective study was conducted.Clinical data of 94 septic patients admitted in the Department of Critical Care Medicine in Subei People's Hospital from January 2011 to June 2014 were analyzed.The arterial blood Lac levels at the moment of diagnosis of septic shock (incipient value,0 hour) and early-stage after treatment (3,6 and 24 hours) were reviewed,and individual LCR was calculated at 3,6,24 hours for each patient.According to the outcome in intensive care unit (ICU),patients were divided into survival group (n =48) and death group (n =46).The Lac and LCR at different time points in two groups were analyzed,and the relationships between them and outcome were analyzed.The receiver-operating characteristic (ROC) curve was plotted to assess the value of Lac and LCR at different time points for predicting the outcome.Results Lac level after treatment in survival group was significantly lower than incipient value,but there was no obvious change in death group.Compared with death group,early Lac levels (mmol/L) in survival group were significantly reduced (0 hour:3.80 ± 2.14 vs.5.75±3.21,3 hours:2.05± 1.04 vs.5.03±2.53,6 hours:1.80±0.77 vs.4.40±2.02,24 hours:1.35±0.43 vs.4.90 ± 2.72,P ＜ 0.05 or P ＜ 0.01),the LCR was significantly increased [3 hours:50.00 (72.35)％ vs.13.51 (20.67)％,6 hours:41.43 (58.42)％ vs.22.00 (22.31)％,24 hours:58.73 (29.94)％ vs.18.92 (47.28)％,P ＜ 0.05 or P ＜ 0.01].The Lac levels at all time points were positively correlated with the outcome,and 6-hour and 24-hour LCR were negatively correlated with the outcome.According to the incipient Lac level,patients were divided into low Lac group (Lac ＜ 2 mmol/L),mild Lac group (Lac 2-3 mmol/L) and high Lac group (Lac ≥ 4 mmol/L).The mortality in low Lac group,mild Lac group,high Lac group was gradually increased [23.07％ (6/26),50.00％ (8/16),61.54％ (32/52),x2=10.270,P =0.006].ROC curves demonstrated that the area under ROC curve (AUC) of 24-hour Lac was the largest,0.944,and it was more sensitive and specific in the prognosis evaluation (100％ and 78.3％,respectively).According to the cut-off value of 24-hour Lac as 2.35 mmol/L,patients were divided into high Lac and low Lac groups,and mortality rate in high Lac group was significantly higher than that in low Lac group [100.0％ (36/36) vs.17.24％ (10/58),x2=30.441,P =0.000].The AUC of 24-hour LCR was the largest,0.865,and it was more sensitive and specific for the prognosis evaluation (83.3％ and 91.3％,respectively).According to the cut-off value of 24-hour LCR as 36.8％,patients were divided into high LCR group and low LCR group,and mortality rate in low LCR group was significantly higher than that in high LCR group [84.00％ (42/50) vs.9.09％ (4/44),x2=26.278,P =0.000].Conclusion Early high Lac in patients with septic shock prompts a poor prognosis,and 24-hour Lac levels and LCR are indicators of assessment of clinical therapeutic effect and prognosis of patients with septic shock.

Objective To summarize clinical features of local and regional failure of salivary gland carcinoma treating by 125I seed,and evaluate the clinical and histologic risk factors for its development.Methods Patients with salivary gland carcinoma treated by 125I seeds between Oct 2001 and Aug 2012 were analyzed retrospectively.The risk factors were analyzed statistically,including age,gender,tumor site,TNM stage,histological differentiation,radiotherapy,treatment,matched peripheral dose and primary or recurrent tumor.Results Ninety-four of 449 patients with salivary gland carcinoma treated by 125I seeds developed local and/or regional area recurrence.Of these,six patients failed in both local and regional area,77 patients failed in local area and eleven patients failed in regional area.The local and regional failure rate was 20.9％.The result of multivariate analysis showed that surgery,radiotherapy and matched peripheral dose were the protective factors(OR =0.458,0.297,0.982,P ＜ 0.05),while age and TNM stage were the risk factors(OR =1.250,1.483,P ＜ O.05).Conclusions The local and regional failure rate was 20.9％.Surgery,radiotherapy and matched peripheral dose were the protective factors;age and TNM stage were the risk factors.

M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot.20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2 subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.

Objective To evaluate and analyze the clinical application value of detection of Warfarin-related gene polymorphisms,cytochrome P450 2C9 (CYP2C9) and Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms.Methods From July of 2011 to July of 2012,the blood samples were randomly collected from 140 lung cancer patients from Department of Oncology in Renmin Hospital of Wuhan University.These lung cancer patients were diagnosed through imaging examination and pathological examination.CYP2C9 and VKORC1 polymorphisms were detected in 70 patients (studied group) but not detected in the other 70 patients (control group) before they used warfarin.According to known gene sequences of CYP2C9 and VKORC1,specific primers were designed to genotype the CYP2C9 *2 and CYP2C9 * 3 alleles as well as the VKORC1-1639G ＞ A polymorphism through PCR amplification and DNA sequencing.Meanwhile,the distribution of these alleles in the studied group was analyzed.The clinical significance of detection of these polymorphisms was evaluated by comparing the proportion of patients within the therapeutic INR (International Normalized Ratio) range between control and genotype-guided dosing groups using Chi square test after 2 and 4 weeks of Warfarin therapy.Results Based on the results of agarose gel electrophoresis of PCR products and DNA sequencing,the primers for CYP2C9 and VKORC1 polymorphisms were indeed specific to these SNPs (CYP2C9 * 1,CYP2C9 * 2 and CYP2C9 * 3 ;VKORC1-1639GG,VKORC1-1639AG and VKORC1-1639AA) and both of the specificity and sensitivity of these primers are 100％,thus contributiug for genotyping these alleles.The distribution of CYP2C9 * 1/* 1 was 100％,CYP2C9 * 1/* 2,CYP2C * 1/* 3,CYP2C9 * 2/* 2,CYP2C9 * 3/* 3 and CYP2C9 * 2/* 3 were 0％.The distribution of VKORC1-1639AG,VKORC1-1639AA and VKORC1-1639 GG were 10％,90％ and 0％ respectively.2 weeks after the treatment of Warfarin,85.7％ patients in the genotype-guided dosing group reached the stable therapeutic INR range,which was significantly higher than that in the control group (48.6％,x2 =21.9,P ＜ 0.01); 4 weeks later,all patients (100％) were inside the stable therapeutic INR range whereas only 65 patients (92.9％) in the control group reached the therapeutic INR range.No haemorrhage or thromboembolic events occurred in both groups.Conclusions CYP2C9 and VKORC1 polymorphisms can be accurately detected by PCR reaction with the designed primers and the subsequent DNA sequencing in patients with lung cancer.This method is validated to be reliable.The genotyping of the Warfarin-related genes detective method can effectively guide Warfarin-dosing.