Based on the gene sequence of AIV matrix protein 2(M2) and two cytotoxic T-lymphocyte epitopes derived from nucleoprotein,the prokaryotic expression vector pET-3M2e-NP1-NP2 was constructed.The target gene was expressed in the solvable form in BL21(DE3) when induced with 1.0 mmol/L IPTG and Western blot analysis showed that the expression product had good immunogencity.Purified fusion protein was mixed with various amount of adjuvant,including Freund's,Vash oil and chitosan,and then immunized 20-day-old chicken by intramuscular injection and boosted 3weeks later.Blood samples were collected weekly following the primary vaccination.The anti-M2e antibody was detected with ELISA method with the synthesized peptide as coated antigen;the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo,the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken was measured by flow cytometry.Results showed that the fusion protein can induce immunological reaction,and the antibody can bind with the viral M2 protein that expressed on the surface of MDCK cells.Flow cytometry result showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization(P

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 observe the effects of tissue factor pathway inhibitor(TFPI) on thrombosis formation in rabbit carotid arteries after ballon injury. Methods Fouty rabbits with the weight 2.5-3.0 kg were respectively divided into 4 groups, Ad-TFPI, Ad-LacZ, PBS and normal control groups. The normal control group was not given any treatment and other 3 groups were given 0.2 ml Ad-TFPI, Ad-LacZ or PBS reproduced by the Dispatch catheter respectively after the PTCA balloon iniury on the right carotid arteries. Ten days after gene transfer the repeated balloon injury was performed in the 3 groups, and the first balloon injury was performed in the normal control group by the same method. The carotid blood flow was recovered immediately after the injury. Thirty minutes later all the animals were sacrificed. The injured carotid arteries and one part of contralateral normal artery were cut down, scissored along the long axis, flattened and fixed in the 2% glutaral. The platelet aggregation and thrombosis formation on the luminal surfaces was observed under electron microscope. Results The electron microscope results showed that the vascular endothelial cell structure was integrated and lined up in order in the nomal artery which had no any injury. After the balloon injury in the normal control group, the structure of the endothelial cell was disintegrated, and there was some platelet aggregation but no fibrosis formation. A large amount of platelet aggregated but no fibrosis formed in Ad-TFPI group after the repeated balloon injury. A large amount of fibrosis formed and red cells piled up in the Ad-LacZ and PBS group. The positive rate of thrombosis formation among groups had siginificant differences(χ2=14.95, P<0.01). The positive rate in Ad-TFPI group(20%) was lower than that in Ad-LacZ group(80%, χ2=7.20, P<0.01) and PBS group(70%, χ2=5.05, P<0.05), but was higher than that in the normal control group(10%, χ2=0.39, P>0.05). The positive rate in Ad-LacZ group(80%) was higher than in the normal control group(10%, χ2=9.90, P<0.01) and in the PBS group(70%, χ2=0.27, P> 0.05). The positive rate in PBS group(70%) was higher than that in the normal control group(10%, χ2=7.50, P< 0.01). Conclusions The repeated balloon injury method can cause a large amount of fibrosis formation in the rabbit carotid. TFPI gene inhibits thrombosis formation in balloon-injured rabbit carotid arteries.

OBJECTIVE:To provide reference for the establishment and improvement of work system for clinical pharmacist in PIVAS of our province even the country. METHODS:Questionnaire was conducted to investigate the PIVAS directors in the 65 third-grade class A hospitals in China,and the data was statistically analyzed. RESULTS:Totally 65 questionnaires were sent out, 63 were effectively received,with effective rate of 96.9%. 98.4% respondents thought PIVAS should equip clinical pharmacist,but the actual situation in respondent's hospital was not ideal. 100% thought clinical pharmacist in PIVAS should have the ability of prescription checking and prescription evaluation;more than 95% thought clinical pharmacist in PIVAS should assume the medi-cine publicity and education and need to have good communication with clinic;only 42.9% thought clinical pharmacist in PIVAS need to take part in the routine drug dispensing. CONCLUSIONS:It is necessary to equip clinical pharmacist with professional training in PIVAS. Clinical pharmacist in PIVAS should focus their ability on prescription checking and evaluation,medicine public-ity and education,communication with clinic.

Objective To investigate the effects of ischemic pre- and postconditioning on cerebral glycogen synthase kinase-3 beta (GSK-3β) activity in a rat model of global cerebral ischemia-reperfusion (I/R).Methods Forty male Wistar rats weighing 200-230 g were randomly allocated into 4 groups (n =10 each) : Ⅰ group sham operation (group S); Ⅱ group I/R; Ⅲ group ischemic preconditioning (group IPR) and Ⅳ group ischemic postconditioning (group IPO). The animals were anesthetized with intraperitoneal 10% chloral hydrate 0.4 ml/100 g. Global cerebral ischemia was induced by four-vessel-occlusion in group Ⅱ , Ⅲ and Ⅳ. Bilateral vertebral arteries were cauterized and bilateral carotid arteries were occluded for 10 min. In group IPR cerebral ischemia was preceded by 3 cycles of 10 s ischemia followed by 30 s reperfusion. The group IPO received 3 cycles of 30 s reperfusion followed by 10 s ischemia at the end of 10 min cerebral ischemia. The animals were killed 2 days later. The brains were immediately removed for determination of neuronal apoptosis in the cortex (by TUNEL), the infarct size (by TTC), p-GSK-3β activity (by spectrum assay) and the expression of Bcl-2, Bax and Caspase-3 (by SP). Linear correlation of p-GSK-3β activity with the number of apoptotic neurons in the cortex and cerebral infarct size was analyzed. Results Cerebral I/R significantly increased the number of apoptotic neurons in the cortex and infarct size, decreased p-GSK-3β activity, down-regulated Bcl-2 expression and up-regulated Bax and Caspase-3 expression in group I/R as compared with group S. Ischemic pre- and postconditioning significantly attenuated these cerebral I/R-induced changes. The p-GSK-3β activity was negatively correlated with the number of apoptotic neurons in the cortex and cerebral infarct size. Conclusion Ischemic pre- and postconditioning reduces cerebral I/R injury through inhibiting the activity of GSK-3β.

The complex level of constructing biopharmaceutics classification system of Chinese materia medica CMMBCS) was the study of traditional Chinese compound, on the premise of insisting that the multicomponent simultaneous determination, when carrying out the study of intestinal permeability, the primary task was to define the source of the components that was absorbed through the intestinal wall, namely, which medicinal material the components belonged to in traditional Chinese compound. The technology of chemical fingerprint and in vitro everted gut sac model were used in this research to make multicomponent an intuitive source attribution which permeated the intestine in the classic formula Gegen Qinlian decoction, and to lay the foundation for the further qualitative and quantitative research of intestinal permeability.
Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Intestines ; metabolism ; Male ; Permeability ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar

Objective To investigate the relationship between coagulation function and the tumor size, clinical stage and metastasis status in renal cell carcinoma (RCC) patients. Methods A total of 290 RCC patients from 2004 to 2009 were included in present study. There were 181 male patients and 109 female patients. The average ages was (56.3± 13. 5) years. There were 252 cases of clear cell carcinoma, 19 cases of papillary carcinoma, 5 cases of chromophobe cell carcinoma, 3 cases of cystic RCC, and 11 cases of other types. TNM classification: stage Ⅰ 202 patients, stage Ⅱ32 patients , stage Ⅲ 32 cases, stage Ⅳ 24 cases. There were N0 264 patients, N1 11 patients and N2 15 cases. There were M0 273 cases, M1 17 cases. One hundred and eighty-six cases of benign renal tumors were set as the control group. Fibrinogen (Fib), prothrombin time (PT), activated partial thromboplastin time (APTT) and international normalized ratio (INR) were detected. Results The preoperative serum Fib of RCC patients was (39. 6±15.6) g/L, the control group was (32. 8±8. 2)g/L. There was significant difference between them (P<0. 05). The values of preoperative APTT,INR, and PT were (31.7±5.2)s, (1.0±0. 1), (11.2±1.3)s in RCC group and (32. 4±4.2)s,(1.0±0. 1), (11.1±1.3)s in the control group. There were no significant differences between them (P＜0.05). The values of Fib in stage Ⅰa, Ⅰb, Ⅱ, Ⅲ, Ⅳ groups were (32. 6±6. 6), (36. 1±8. 7),(48.8±21.6), (49.9± 17.8) and (59.7± 19.2)g/L, respectively. There was no significant difference between stage Ⅰ, and the control group. But the other stages showed significant difference with the control group (P<0.01). Hyperfibrinogenemia (Fib>44.0 g/L) in the RCC group accounted for 74 cases (25.5%). If the value of Fib ≤44. 0 g/L, 92.1% of patients can be excluded from the probability of metastasis. Conclusions Preoperative plasma Fib levels could be elevated in RCC patients with distant metastasis or lymph node metastasis. Increased preoperative plasma Fib levels, especially hyperfibrinogenemia may be a predictor of metastasis.

A strain was isolated from internal organ of died porcine about 8 weeks with purulent pneumonia,arthritis,pyogenic arthritis and endocarditis in April 2007.Objectives of the study are to confirm the genus of the strain,pathopoiesis,and drug sensitivity.The mainly study methods:the first,the strain was identified by the phenotype and the characteristics of the biochemistry,sequence 16S rDNA genes of the strain was analyzed by molecular biology technology,finally animal experiment and drug sensitivity testing were done.The results of the phenotype and the characteristics of the biochemistry showed that it is greatly similar to Actinomyces hyovaginalis,16S rRNA sequence analysis exhibited the homology achieved to 99.2% com-pared with group III strains of Actinomyces hyovaginalis,and the phyletic evolution analysis also indicated that it has mostly relationship with group III strains of Actinomyces hyovaginalis.Animal experiment dis-covered it has highly pathogenicity to Mus musculus albus;Drug sensitivity testing showed that it is hyper-sensitive to Erycin,Gentamicin and Amikacin.So,the result of the study confirmed that the strain is Actin-omyces hyovaginalis III with the pathogenicity.

Objective To evaluate the clinical value of color doppler flow image (CDFI) and three-dimension color power angiography(3D-CPA) in the diagnosis of thyroid nodules. Methods A total of 62 pathologically confirmed thyroid nodules of 30 patients were analyzed. All the patients underwent ultrasound examination and operation in the Second Affiliated Hospital of Harbin Medical University between January, 2008 and May, 2009. Both CDFI and 3D-CPA were performed before the operation. All the nodules were divided into three groups including nodular goiters, thyroid adenomas and thyroid cancers according to the pathological results. The hemodynamic features and the vascular morphology characteristics of nodules in different groups were compared. Results 3D-CPA showed that blood vessels of nodular goiters commonly distributed surrounding the mass and the inner vessels were thin and regular, thyroid adenomas were bulb-shaped and netted structure, and malignant thyroid nodules displayed distorted and irregular distributed vessels. Peak systolic velocity (PSV)of the three groups were (39.43±11.17a), (46.39±12.98) and (65.17±9.23)cm/s, respectively. Resistance index(RI) of the three groups were (0.32±0.08), (0.41±0.06) and (0.69±0.07)cm/s, respectively. Both PSV and RI in malignant thyroid nodules were higher than in nodular goiters and in thyroid adenomas and the difference were statistical significant (all P < 0.05). The blood flow grade of malignant nodules was also higher in malignant nodules than in other two groups(χ2 = 17.11, 12.79, 23.05, 15.41, P< 0.01). Conclusions CDFI and 3D-CPA could visually demonstrate the characteristic and distribution of the inner and outer blood vessels, display the vessels structures, and they are benefit the differential diagnosis of thyroid nodules.

AIM : To construct and analyze eukaryotic expression plasmid inserted by Cx50 with V64G mutation through bioinformatics software.METHODS: The full coding domain sequence of Cx50 with V64G mutation was acquired from the blood of patients with cataract and was cloned into pcDNA3.1 /Amp (+).The constructed plasmid was identified with PCR , enzyme digestion and sequencing. The analysis of Cx50 with V64G mutation was performed with bioinformatics software.RESULTS : Cx50 with V64G mutation was successfully amplified and its eukaryotic expression plasmid was constructed. Valine-64 is well conserved in the first extracellular loop of connexin 50 in different species and also in different human α -type gap junctional proteins.CONCLUSION : The successive reconstruction and verification of eukaryotic expression plasmid containing Cx50 with V64G mutation established the foundation for further studying the mechanism of cataract.