Objective:To construct high level prokaryotic cell expression vector carrying human upstream regulatory element binding protein1 (hureb1) gene for producing GST hureb1 fusion protein and generating anti hureb1 antibody Methods: After the Xho I/Not I fragment from hureb1 open reading frame was recombined into the prokaryotic expression vector pGEX 4T 2, the vector was transformed into E coli BL21(DE3) strain and induced with IPTG to express the fusion protein, GST hureb1 The crudely isolated fusion protein was applied to immunize rabbit and mouse for generating anti hureb1 polyclonal antibody and monoclonal antibody respectively Results:The prokaryotic expression vector expressing GST hureb1 fusion protein was constructed and induced to produce high level GST hureb1 that was detected up to 33 45% of the total bacterial protein expressed The GST hureb1 fusion protein immunizing animals generated high titer and specific anti hureb1 antibodies Conclusion:Recombinant hureb1 protein and anti hureb1 antibodies can be used to analyze the biological functions of hureb1

This paper is discussed about course system construction of Microbiology, teaching method, in- struction means and experimental teaching mode. Teaching practice indicated that reform the pattern of Mi- crobiology educational mode can stimulate students’ interest in studying the course, cultivate their inde- pendent ability to solve questions, develop their creative thinking. It is an important way to train high-caliber talents.

Aim To investigate the effect and mecha-nism of quercetin combined with cisplatin on prolifera-tion and apoptosis of human osteosarcoma cell line MG-63 . Methods MG-63 cells were treated with quercetin alone or combined with cisplatin. Cellular morphologic changes were observed under inverted phase contrast microscope. The effects of proliferation inhibition were assayed by CCK-8 method. The combination effect was judged through Chou-Talaly analysis. The apoptosis ra-tios of cells were analyzed by flow cytometry. The gene expression of Bcl-2 and caspase-3 was detected by RT-PCR assay. The protein expression of Bcl-2 and caspase-3 was measured by Western blot assay. Re-sults Quercetin alone or combined with cisplatin could inhibit the proliferation, but induce the apoptosis of MG-63 cells. Combination of quercetin and cisplatin revealed a synergistic effect on cell proliferation and apoptosis as it reduced the expression of Bcl-2 but en-hanced that of caspase-3 at both gene and protein lev-els. Conclusion Synergistic effect of quercetin com-bined with cisplatin on cell proliferation and apoptosis of MG-63 cells is possibly due to reduction of Bcl-2 and enhancement of caspase-3 expression.

Objective To compare the effects of Baimai ointment and baclofen in stroke patients with spas-ticity.Methods 84 cases accompanied by limb spasticity in stroke patients by digital table were randomly divided into Baimai ointment group and baclofen group,42 cases in each group.The Baimai ointment group were treated with Baimai ointment on the spastic limbs,the baclofen group received oral baclofen tablets 30 -75mg/days for 2 weeks, 4 weeks,8 weeks.The curative effects of the two groups were compared before and after treatment.Results Before and after treatment in the two groups,the levels of spasticity,pain and activities of daily living (ADL)differences were statistically significant and Baimai ointment in the treatment of spasm.After 4 weeks and 8 weeks,the Ashworth score of the Baimai ointment group were (1.59 ±0.46)points,(0.89 ±0.56)points,and those of baclofen group were (1.75 ±0.64)points,(1.45 ±0.48)points,the differences were statistically significant(t values were 2.916, 3.367,all P <0.05).After 2 weeks,4 weeks and 8 weeks,the VAS score of the Baimai ointment group were (2.72 ± 0.54)points,(2.02 ±0.24)points,(1.24 ±0.12)points,and baclofen group were (3.56 ±0.44)points,(3.15 ± 0.48)points,(2.58 ±0.26)points,the differences were statistically significant(t values were 2.975,3.359,5.416, all P <0.05),activities of daily living (ADL)was higher than that of the baclofen group.After 8 weeks,the MBI score of the Baimai ointment group was (64.46 ±10.78)points,and baclofen group was (50.74 ±9.18)points,the difference was statistically significant between the two groups (t values was 3.562,P <0.05).Conclusion Baimai ointment has the better antispasmodic effect than baclofen in patients with stroke.

PURPOSE: This study was to explore the cognitive function of Korean-Chinese stroke patient in China. METHOD: The study sample was 100 who were possible to communicate and agreed. The data were collected from one Brain's hospital at Yanji in China and by trained nurse from December 12, 2005 to April 28, 2006. The measurement tools were Digit span, Trail making, and MMSE-K. The data were analysed by SPSS Win 11.5 using frequency, t-test, ANOVA, and Pearson's correlation coefficients. RESULTS: The mean score of DSF was 5.07, 3.42 of DSB, 161.37 of TMA, 229.28 of TMB, 22.64 of MMSE-K. There was a significant difference in DSF (F=6.35, p=.001), DSB (F=6.10, p=.001), TMA (F=3.53, p=.018), TMB (F=3.26, p=.025), MMSE-K score (F=12.97, p=.000) according to age, and DSF (F=6.67, p=.000), DSB (F=6.01, p=.000), TMA (F=5.82, p=.001), TMB (F=6.23, p=.001), and MMSE-K score (F=13.02, p=.000) according to educational level, and DSF (F=5.35, p=.006), DSB (F=3.16, p=.047), TMA (F=3.30, p=.041), TMB (F=3.42, p=.037), and MMSE-K score (F=4.95, p=.009) according to duration of disease, and DSB (F=3.54, p=.018), and MMSE-K (F=6.05, p=.001) according to frequencies of hospitalization. There was high correlation between DSF and DSB (r=.581, p=.000), TMA and TMB (r=.936, p=.000), MMSE-K and DSF (r=.579, p=.000), MMSE-K and DSB (r=.591, p=.000), DSF and TMA (r=.727, p=.000), and DSF and TMB (r=.721, p=.000). CONCLUSION: The cognitive evaluation score of Korean-Chinese stroke patients in China was in normal limit. The age, educational level, duration of disease and income were significant demographic characteristic affecting cognitive function. Further study need to compare the cognitive function of Korean-Chinese stoke patients in China and stoke patients in Korea.
China* ; Hospitalization ; Humans ; Korea ; Stroke*

Objective The clinical manifestations of cerebral infarction caused by acute basilar arterial occlusion are complex.The purpose of this study is to explore the relationship between lesion location and onset symptoms of cerebral infarction caused by acute basilar arterial occlusion.Methods Fifty three patients diagnosed with cerebral infarction caused by acute artery occlusion were collected from Nanjing Stroke Registry.They were hospitalized in Jinling Hospital from January 2007 to July 2016 and were divided into sudden onset group and progressive onset group.Their clinical and digital subtraction angiography data were analyzed retrospectively.Results Middle and distal segment of the basilar artery occlusions were usually found in sudden onset group.Patients in progressive onset group were more likely to present with proximal segment of the basilar artery occlusions.Significant statistical difference was found between two groups (P<0.05).Logistic regression analysis showed that the symptoms of patients with proximal segment basilar artery occlusion tended to be progressive onset, compared with patients affected by distal segment occlusion (OR=14.77,95%CI:1.57-139.00, P=0.019).Conclusion There was significant relationship between lesion location and onset symptoms of cerebral infarction caused by acute basilar arterial occlusion.Early diagnosis and timely treatment may improve clinical prognosis in patients.

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.

AIM:To investigate protective effects of novel extract of Ginkgo biloba(nGBE) in different administration modes on glutamate-induced neuronal damage.METHODS: The values of Essential nGBE were obtained by supercritical CO_2 fluid extraction.Based on glutamate excitotoxicity of primary cultures from neonatal Wistar rats hippocampal,by use of Trypan blue dye staining,testing the lactate dehydrogenase leakage from cultured neurons and terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) method,we examined glutamate-induced necrosis and apoptosis.The protective effects of nGBE in different administration modes(pre-treatment and post-treatment) were adopted and compared with the NMDA receptor uncompetitive antagonist MK-801 in acute-treatment.RESULTS:Treatment with nGBE in two administration modes all could increase ratio of surviving neuron,decrease LDH efflux and reduce ratio of neuron apoptosis in different degree,and the benefit of pre-treatment was superior to post-treatment,but inferior to MK-801.CONCLUSION:The extracts of Ginkgo biloba used nowadays in cerebrovascular disease as post-treatment have no prominent effect as far as their mechanism of antiexcitotoxicity.However they may have more value if they were used for precautionary intervention in high-risk population.

OBJECTIVETo explore the inhibition and molecular mechanism of icaritin (ICT) combined doxorubicin (DOX) on human osteosarcoma MG-63 cells in vitro.

METHODSThe control group, ICT groups (10, 20, 40, 80, and 160 µmol/L), DOX groups (1, 2, 4, 8, and 16 µg/mL), and combination groups (20 µmol/ L ICT +1 µg/mL DOX, 20 µmol/L ICT +2 µg/mL DOX, 20 µmol/L ICT +4 µg/mL DOX, 40 µmol/L ICT +1 µg/mL DOX, 40 µmol/L ICT +2 µg/mL DOX, 40 µmol/L ICT +4 µg/mL DOX, 80 µmol/L ICT +1 µg/mL DOX, 80 µmol/L ICT +2 µg/mL DOX, 80 µmol/L ICT +4 µg/mL DOX) were set up. Human osteosarcoma MG-63 cells were respectively cultured and their effects on morphological changes were observed using inverted phase contrast microscope after 24-and 48-h intervention. The cell proliferation inhibition rate of each group was de- termined using CCK-8, and IC50 calculated. The MG-63 apoptosis rate was detected using Annexin V-FITC/ PI double dye flow cytometry. Expression levels of bcl-2, caspase-3, and p21 were detected using RT-PCR.

RESULTSICT and DOX could obviously inhibit the proliferation of MG-63 cell. Along with ICT concentration increasing from 10 µmol/L to 160 µmol/L, the cell proliferation inhibition rate also increased gradually from 9.67% ± 3.62% to 89.18% ± 9.66%. The IC50 was 46.93 µmol/L and 3.87 µg/mL respectively. ICT and DOX could cause either early or late stage apoptosis, down-regulate Bcl-2 gene expression, and up-regulate gene expressions of Caspase-3 and p21 respectively (P < 0.05). Aforesaid changes were more obviously seen in combination groups than in lCT groups and DOX groups (P < 0.05).

CONCLUSIONCT combined DOX had additive or synergistic inhibition effect for the proliferation of osteosarcoma MG-63 cells, which might be related with regulating gene expressions of bcl-2, caspase-3, and p21.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Down-Regulation ; Doxorubicin ; pharmacology ; Drug Synergism ; Flavonoids ; pharmacology ; Humans ; Osteosarcoma ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

Exosomes, small vesicles of endocytic origin, have attracted attention in bone regeneration field. The vesicles travel between cells and deliver functional cargoes like proteins and RNAs, thereby regulating targeted cells dif-ferentiation, commitment, function, and proliferation. Exosomes directly regulate MSCs into the osteoblastic lineage, stimulate bone regeneration by directly regulating osteoblast proliferation and activity, regulate osteoclast maturation and activity and stimulate bone growth and regeneration by increasing vessel formation. Meantime, exosomes resolves toxicity and immunogenicity problems caused by biomaterial treatment. Furthermore, compared with living cell trans-plantation, exosomes present a lower risk for severe complication(such as tumors, emboli formation).