Objective To construct ICAM-1 recombinant eukaryotic expression vector. Methods Human intercellular adhesion molecule-1 (ICAM-1) cDNA was obtained by RT-PCR of totol RNA extracted from human hepatocellular carcinoma tissue. Amplified ICAM-1 cDNA fragment was cloned into pGEM-T easy vector to construct pGEM-ICAM-1 vector. Then ICAM-1 cDNA from pGEM-ICAM-1 vector was cloned into eukaryotic expression pcDNA3.1hisB to construct recombinant pcDNA3.1hisB-ICAM-1 vector. Restriction endonuclease digestion and DNA sequencing were used to confirm the recombinant vector. Results 1622bp ICAM-1 cDNA was obtained by RT-PCR. The PCR product was successfully ligated with pGEM-I easy vector. Restriction endonuclease digestion analysis and DNA sequencing showed that recombinant pcDNA3.1HisB-ICAM-1 was successfully constructed. Conclusion Eukaryotic expression recombinant vector pCDNA3.1hisB-ICAM-1 was contructed.

Objective To carry on statistical analysis of tradtional mongolian medicine syndrome in angina pectoris of coronary heart disease, find out the significative indices for syndrome diagnosis, and establish the diagnostic criteria of tradtional mongolian medicine syndrome in angina pectoris of coronary heart disease. Methods Cases of angina pectoris of coronary heart disease were selected according to modern medical diagnostic criteria. Clinical observations were carried on and clinical information was filled in the forms of the clinical cases. Each case was differentiated and made corresponding diagnosis of syndrome according to tradtional mongolian medicine. Clinical cases were grouped according to syndrome of tradtional mongolian medicine and decided the significative indices by statistical analysis. Results Four hundred and ten cases were differentiated which contain tradtional mongolian medicine information about angina pectoris of coronary heart disease, including 358 cases of Chu Si syndrome, 23 cases of Nian Xie syndrome, and 29 cases of Wei Sha syndrome. Through statistical analysis of the various syndromes and the objective indices, diagnostic criteria of tradtional mongolian medicine syndrome in angina pectoris of coronary heart disease was established. Conclusion Diagnostic criteria of tradtional mongolian medicine syndrome in angina pectoris of coronary heart disease was summarized by combination of disease and syndrome, and provides a theoretical basis for the resonable application of mongolian herb.

Objective:To investigate the immunogenicity of pORF5 plasmid protein,and further to screen and identify its im-munodominant domian.Methods: 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamHⅠand NotⅠrestriction endonucleases.After identification by PCR and sequencing,the recombinant plasmids were transformed into XL1 Blue E.coli to express the GST fusion proteins.ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein.10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method.Results: pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human.The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de-stroyed.F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was.However,no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies.Conclusion:pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope,and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation.The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.

Objective To explore the enhancement effects and mechanisms of IL-32β on human cervical carcinoma cells C33A to hypoxic/hypoglycemic condition.Methods After cultured in hypoxia/hypoglycemic circumstance and normal circumstance for 20 hours respectively, the mRNA and protein expression of IL-32β in C33A cells were detected by real time-polymerase chain reaction (RT-PCR) and Western blotting respectively.Trypan blue stain was used to detect C33A cells viability in hypoxia/hypoglycemic circumstance and adding 10, 100,500 ng/ml IL-32β circumstance.The xenografted tumor of nude mice was established by intraperitoneal injection, and their volumes were tested for a given time after injecting 0, 1.0 mg/kg IL-32β.siRNA was used to construct IL-32β knockdown cells and detect the expression of VEGF.Results Under the hypoxia/hypoglycemic circumstance, the expressions of IL-32β mRNA were (6.12 ± 0.03) times of the normal circumstance (F =43.16, P ＜ 0.05), the expressions of IL-32β protein were (2.23 ± 0.04) times of the normal circumstance (F =22.32, P ＜ 0.05).The C33A cells viability in hypoxia/hypoglycemic circumstance was (51.92 ± 3.41) ％, whereas, viability in 10 ng/ml IL-32β group was (55.23 ± 3.92) ％ (F =14.25, P ＜ 0.05), viability in 100 ng/ml IL-32β group was (62.52 ± 4.14) ％ (F =35.53, P ＜ 0.01), viability in 500 ng/ml IL-32β group was (69.14 ± 2.45) ％ (F =56.28, P ＜ 0.01).After 28 days, the volume of xenografted tumor of 0 mg/kg IL-32β group was (578 ± 64)mm3, and 1.0 mg/kg IL-32β group up to (1 402 ± 142) mm3 (F =27.84, P ＜ 0.01).In addition, compared with control group, the expression of VEGF in IL-32β knockdown C33A cells was significantly decreased (F =36.85, P ＜ 0.05).Conclusion IL-32β can enhance the resistance to hypoxic/hypoglycemic condition of C33A cells, which is associated with the increase of VEGF.

Objective:To evaluate the expression of apoptosis related gene Fas Ligand(FasL) in human hepatocellular carcinoma(HCC) cells HepG2 and its significance in apoptosis.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1hisB- FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells were detected by immune histochemistry. After stained by annexin V and propidium iodine, cells were passed throw flow cytometer and examined by fluorescence microscope and sym-focus laser scaning microscope.Results:In comparison with untransfected cells,the soluble FasL could be detected in the supernatant of transfected cells, Fas L can be expressed in the membrane and cytoplasm of transfected cells. The apoptotic cell rate in transfected cells was 36.30%, as the control, untransfected cells was 11.53%.Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine stain.Conclusion:This supports a novel pathway of HCC cells were apoptotic itself via the CD95-CD95 ligand system without involvement of immune cells.

ObjectiveTo explore the relationship between the core self-evaluations,copying style and learning engagement of college students.Methods 250 college students were asked to complete the core self-evaluations scale,copying style scale and learning engagement scale.ResultsBivariate correlations results indicated that there was significantly positive correlation between the core self-evaluations,copying style and learning engagement in college students( r=0.179 ～ 0.586,P＜ 0.05 ) ; core self-evaluations affected motivation indirectly by the copying style; core self-evaluations had a direct influence on vigor and absorption; core self-evaluations had a indirect influence on vigor and absorption.ConclusionCopying style was a full mediator of the relationship of the core self-evaluations and motivation,but copying style partially mediated the relationship of the core self-evaluations and vigor and absorption.

To study the quantitative structure-activity relationship (QSAR) between the stuctures of 29 flavonoids and the inhibitory activity of their multidrug resistance-associated protein (MRP) 1 and 2 by using the comparative molecular similarity index analysis (CoMSIA). By studying the impact of the combination of different molecular force fields, researchers obtained the molecular force fields that played an important role in inhibiting the activity of MRP1 and MRP2, built the optimized QSAR model, and discussed the structural modification method for flavonoids' multidrug resistance-associated protein inhibitor. The results of the study could not only provide the guidance for new drug R&D, but also help partially discuss the synergy mechanism between MRP1 and MRP2 receptors and traditional Chinese medicines containing flavonoids.
Drugs, Chinese Herbal ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Humans ; Models, Molecular ; Multidrug Resistance-Associated Proteins ; antagonists & inhibitors ; chemistry ; Quantitative Structure-Activity Relationship

Purpose To prepare a novel fusion protein of CREKA and tTF as a universal carrier targeting to cancer,and to analyze its activities.Methods CREKA and tTF gene were acquired by PCR,and inserted into plasmid pET22b(+)to construct recombinant plasmid CREKA/tTF/pET22b(+),and the fusion gene was expressed in E.coli BL21.The fusion protein Wag purified through Nickel-affinity chromatography column.After purifying,the fusion protein was refold by subsequent dialysis.The activities of the fusion proteins were measured by coagulation timing and quantitative fluorescence test in vitro.Results The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was obtained.The fusion protein was highly expressed in E.coli BL21.The coagulation of the fusion protein Was determined by the coagulation test.And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.Conclusion The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was built.The fusion protein CREKA/tTF with both TF and CREKA activity was successfully obtained.

Nicotinic acid could increase high density lipoprotein and reduce serum total cholesterol, low density lipoprotein cholesterol and triglycerides in human bodies, thus is frequently applied in treating low high-density lipoprotein cholesterol and hypertriglyceridemia in clinic. However, according to the findings, nicotinic acid could also cause adverse effects, such as skin flush, beside its curative effects. In this study, bioisosterism, fragment-based search and Lipinski's Rule of Five were used to preliminarily screen out potential TCM ingredients that may have similar pharmacological effects with nicotinic acid from Traditional Chinese medicine database (TCMD). Afterwards, homology modeling and flexible docking were used to further screen out potential nicotinic acid receptor agonists. As a result, eleven candidate compounds were derived from eight commonly used traditional Chinese medicines. Specifically, all of the candidate compounds' interaction with nicotinic acid receptor was similar to nicotinic acid, and their docking scores were all higher than that of nicotinic acid, but their druggability remained to be further studied. Some of the eight source traditional Chinese medicines were used to lower lipid according to literature studies, implying that they may show effect through above means. In summary, this study provides basis and reference for extracting new nicotinic acid receptor agonists from traditional Chinese medicines and improving the medication status of hyperlipidemia.
Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; chemistry ; Humans ; Models, Molecular ; Molecular Structure ; Nicotinic Acids ; chemistry ; Nicotinic Agonists ; chemistry ; Protein Binding ; Receptors, G-Protein-Coupled ; agonists ; chemistry ; Receptors, Nicotinic ; chemistry

By using the pharmacophore model of mineralocorticoid receptor antagonists as a starting point, the experiment stud- ies the method of traditional Chinese medicine formula design for anti-hypertensive. Pharmacophore models were generated by 3D-QSAR pharmacophore (Hypogen) program of the DS3.5, based on the training set composed of 33 mineralocorticoid receptor antagonists. The best pharmacophore model consisted of two Hydrogen-bond acceptors, three Hydrophobic and four excluded volumes. Its correlation coefficient of training set and test set, N, and CAI value were 0.9534, 0.6748, 2.878, and 1.119. According to the database screening, 1700 active compounds from 86 source plant were obtained. Because of lacking of available anti-hypertensive medi cation strategy in traditional theory, this article takes advantage of patent retrieval in world traditional medicine patent database, in order to design drug formula. Finally, two formulae was obtained for antihypertensive.
Antihypertensive Agents ; chemistry ; pharmacology ; Databases, Factual ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Medicine, Chinese Traditional ; methods ; Mineralocorticoid Receptor Antagonists ; chemistry ; pharmacology ; Models, Molecular