To screen potential hamster chymase 2 inhibitors, a high-throughput screening (HTS) model was established. Recombinant hamster chymase 2 with active form was cloned and expressed in E. coli. The HTS model with total volume of 50 microL in 384-well microplate was based on fluorescence analysis and was proved sensitive as well as specific (Z' = 0.84). A total of 40 080 samples (including 28 060 compounds and 12 020 natural products) were screened, and 613 samples with inhibition greater than 90% were selected for further rescreening. Finally, compounds J16647 and J16648 were identified with high inhibitory activity on chymase 2, and whose IC50 values were 0.823 and 0.690 micromol x L(-1), respectively.

To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.

Objective To establish a high throughput screening assay for identifying human small molecular antagonists targeted IL-6R.Methods The full length gene of the human IL-6R extracellular region was amplified by PCR and cloned into a eukaryotic expression vector to construct recombination expression plasmid pABHis -IL6R that was then transfected transiently into HEK293T cells to prepare recombination protein IL-6R.Western blotting assay and receptor-ligand binding experiment were used to analyze the bioactivity of IL-6R.A new screening method based on ELISA was established using the function of IL-6R binding to its ligand and the characteristics of Fc fragment binding to IgG-HRP.Then Z′-factor was calculated and a known antagonist ab 47215 was used to assess the stability and reliability of the new assay .Results Recombination plasmid pABHis-IL6R was constructed and soluble IL-6R was prepared.IL-6R reported herein could be recognized by an anti-IL-6R antibody and specifically bind to its ligand in a dose response manner .A Z′-factor of 0.53 was obtained that could serve high throughput screening assay .Ab47215 , as a known specific antagonist , was able to block rhIL-6 from binding to the receptor in a dose-dependent manner in the new screening assay , the IC50 of which was (0.55 ± 0.11)μg/ml.Conclusion An innovative and easy screening assay for identifying human IL-6R antagonists is established , which might help discover potent and specific antagonists .

Background and purpose:As the development of the completion of the human genome project (HGP), the research focus is turning to the gene function research. At present, the domestic experimental research on the apoptosis of K562 cells induced by antisense olignonucleotides is rare. This study was aimed to investigate the effect of human chronic myelogenou leukemia (CML) bcr-abl fusion gene antisense oligonucletides on autophagy and apoptosis of CMLK562 cells in vitro. Methods:By liposome as the carrier, K562 cells were transfected with the bcr-abl gene antisense olignonucleotides. Hoechst staining method was used to observe the apoptosis inducing effect of different concentrations of oligonucleotides, the expressions of LC3-Ⅱ, autophagy-related protein, were determined by the Western blot method, the cell cycles were determined by lfow cytometry (FCM), and JEM-4000EX electron microscope technology was used to detect the apoptosis morphological changes. The apoptosis was detected by DNA agarose gel electrophoresis. Results:Hoechst staining results showed that the bcr-abl gene antisense oligonucletides signiifcantly promoted the apoptosis of K562 cells in a certain concentration dependent manner. Western blot showed that the expression level of LC3-Ⅱwas obviously higher in bcr-abl gene antisense oligonucletides transfected group than the control group, showing a promoting effect on cell autophagy. FCM test results showed that bcr-abl gene antisense oligonucleotides transfected K562 cells showed obvious cell cycle arrest, visible obvious apoptosis morphology under the electron microscope, and DNA Ladder showed obvious apoptosis fragments. Conclusion:The bcr-abl gene antisense olignonucleotides can signiifcantly induce the cell apoptosis of K562. This study provides a new method for CML therapy.

Nano magnetic microspheres prepared by chitosan and poly acylic acid were applied to purifying superoxide dismutase from blood erythrocyte. Chitosan-polyacyilc acid graft copolymer was synthesized by free radical graft copolymerization with potassium persulfate as inititator. To prepare Fe3O4 magnetic fluids with chemical coprecipitation, chitosan-polyacylic nano magnetic microspheres were prepared with glutaraldehyde as crosslinking agent. Structure of nano magnetic microspheres was detected by FT-IR spectrometer. Particle size and morphology were characterized by JEM-4000EX technology. Chitosan-polyacylic nanometer microspheres have good paticle cize distribution, magnetic responsiveness and protein adsoption. Activity, product yield and activity recovery of SOD after purification reached 6 727 U/mg, 21.1%, and 85.7% respectively. Purification of blood superoxide dismutase by chistosan-polyacylic acid microspheres has its renewable and feasible nature.
Chitosan ; chemistry ; Erythrocytes ; enzymology ; Glutaral ; chemistry ; Magnetics ; Microspheres ; Polymers ; Spectroscopy, Fourier Transform Infrared ; Superoxide Dismutase ; isolation & purification

This study was conducted to investigate the paclitaxel loaded by hydrazone bonds in poly(ethylene glycol)-poly(caprolactone) micelles (mPEG-PCL-PTX) on proliferation and apoptosis of human lung cancer A549 cells and its possible mechanisms of anti-tumor activity. The cell proliferation was measured with MTT assay. Flow cytometry were used to analyze the cell cycle. The cell apoptosis was analyzed using Hoechst/P staining. The expression levels of apoptotic genes expression in the mitochondrial apoptosis pathway were detected by RT-PCR and Western blotting, respectively. The mPEG-PCL-PTX could inhibit the proliferation of A549 cells and promote the apoptosis. The Bax, caspase-3 protein expression were increased while Bcl-2 protein expression was decreased in A549 cells. Results showed that the polymer containing hydrazone bond is non-toxic in vitro, the mPEG-PCL-PTX micelles can inhibit the proliferation and induce the apoptosis of A549 cells. Key words: paclitaxel; micelle; A549 cell; proliferation; cell cycle; apoptosis

This study is to investigate the effect of the effective components group of Xiaoshuantongluo (XECG) on neuronal injury induced by oxygen-glucose deprivation (OGD) in primary cortical cultures isolated from SD rat cortex at day 3 and the possible mechanism. Cells were divided into control group, OGD model group and XECG group (1, 3 and 10 mg x L(-1)). The cell viability was assessed with MTT assay and the LDH release rate was measured by enzyme label kit. The cell apoptosis was analyzed using Hoechst staining. RT-PCR was applied to detect the mRNA levels of JAK2 and STAT3. Western blotting was used to detect the expressions of Bcl-2, Bax, p-JAK2 and p-STAT3 proteins. Results showed that XECG resulted in an obvious resistance to oxygen-glucose deprivation-induced cell apoptosis and decrement of cell viability, decrease the cell LDH release rate. XECG could adjust the expression of Bcl-2 and Bax proteins and increase Bcl-2/Bax ratio, up-regulate the expression of p-JAK2 and p-STAT3. In conclusion, XECG could protect against the neuronal injury cells exposed to OGD, which may be relevant to the promotion of JAK2/STAT3 signaling pathway, and impact the expression of Bax and Bcl-2.

Objective To investigate the effect of individualized and comprehensive therapy in treatment of severe acute pancrea-titis in early stage for preventing MODS .Methods 85 patients with severe acute pancreatitis treated in this Chengde medical college affiliated hospital from January 2008 to January 2012 were divided into two groups .The control group included 42 patients ,all used conventional basic treatment ;the test group included 43 patients ,and all used individualized and comprehensive therapy in early stage .The APACHEⅡ ,MODS and Ranson scores ,and serum inflammatory cytokines indexes of patients in each group before and after admission were all recorded .Results The APACHEⅡ and MODS scores after admission of test group were significantly low-er than the control group(P< 0 .05) .The TNF α,CRP and IL 10 after admission of two groups had significant difference (P<0 .05) .The MODS incidence of control group was 64 .3% (27/42) ,pancreatic encephalopathy incidence was21 .4% (9/43) ,the inci-dence of pancreatic infection was 33 .3% (14/42) ,hospital mortality incidence was 19 .1% (8/42) and length of hospital stay was (30 .4 ± 5 .7) d .The MODS incidence of test group was 27 .9% (12/43) ,pancreatic encephalopathy incidence was11 .6% (5/43) , pancreatic infection rate was 14 .0% (6/43) ,hospital mortality incidence was 4 .7% (2/43) and length of hospital stay was (23 .5 ± 4 .3) d .The incidence of MODS ,hospital mortality and pancreatic infection rates of test group were significantly lower than that of the control group (P<0 .05) .The average length of stay of test group were significantly lower than the control group (P<0 .05) . Conclusion To use individualized and comprehensive therapy in treatment of severe acute pancreatitis in early stage can reduce MODS ,protect organ function ,and reduce mortality .

Objective To analyze the prognostic value of age in patients with early stage breast cancer. Methods The clinical characteristics of 1030 patients with early stage breast cancer (the number of positive axillary lymph nodes was less than 3) were retrospectively reviewed. Of all the patients, 468(stage Ⅰ, n = 227; and stage Ⅱ , n = 241) received breast conserving surgery (BCS) and 562 (stage Ⅰ, n =184; and stage Ⅱ, n= 378) received modified mastectomy. Patients were divided into young-age group (≤35,136 patients), middle-age group (> 35-≤60,738 patients) and old-age group (> 60,156 patients).The number of patients without postoperative radiation therapy after BCS is 16, 60 and 39 in the three groups, respectively. Two-dimensional conventional fractionated radiotherapy was administered. The prognostic value of the tumor size, status of axillary lymph nodes or hormonal receptors, postoperative radiation therapy were analyzed. Results The follow-up rate was 97.86%. Of 795 patients followed up more than 5 years, 110,569 and 116 patients were devided into the three groups, respectively. There were 40, 202 and 87 patients without radiation therapy in the three groups. The 5-year recurrence rates of the three groups were 6. 2%, 8. 7% and 10. 4% (χ2 = 1.14, P= 0.567). The 5-year distant metastasis rates were4.3% , 9.5 % and2. 5% (χ2 = 5.31 , P = 0. 070) . The5 - year survival rates were9l. 2% , 92. 6%and 82. 1% (χ2 = 6. 83, P = 0.033). The young-age group had more tumors smaller than 2. 0 cm (65.4%), less positive axillary lymph nodes (13.2%), poorer differential tumor and less positive hormone acceptors (48.0%). Of patients with tumor larger than 2. 0 cm who had no radiotherapy after BCS, the 5-year survival rates were 94%, 87% and 71% (χ2= 20.69, P= 0.000) in the three groups. The corresponding recurrence rates were 23%, 18% ,7%, (χ2 = 9. 97, P = 0. 007), and distant metastasis rates were23%, 25% and 10% (χ2 =8.51, P=0. 014). Conclusions The age is an important prognostic factor in patients with early stage breast cancer undergoing BCS, but not in those undergoing modified mastectomy.

Objective To understand the prevalence and the factors associated with depression and anxiety among patients attending the community-based methadone maintenance treatment(MMT) program.Methods 9 MMT clinics in 3 provinces were randomly selected,in which 1 301 MMT patients who met the criteria of recruitment were all interviewed.A cross-sectional study by administrative questionnaire was employed to investigate the demographics,drug abuse and MMT treatment status of the respondents.The 1301 respondents were also evaluated for depression and anxiety by SDS(Self-Rating Depression Scale) and SAS(Self-Rating Anxiety Scale) respectively.ANOVA and multivariate linear regressions analysis was used to verify the factors associated with depression and anxiety respectively.Results The prevalence of depression and anxiety among the MMT patients were 38.3%(498/1 301) and 18.4%(239/1 301).The multivariates linear regression analysis indicated that at the level of ?=0.05,gender,employment status were associated with depression,while gender,employnment status,educational level,drug abuse and methadone daily dosage were associated with anxiety.Conclusions There are certain proportions of MMT clients who suffer from depression and anxiety.The MMT clinic staff should put more emphasis on the efforts to reduce psychological disorders and its potential risks by enhancing psychological councelling,intervention and social support towards the MMT patients.