Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA.Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers.A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments.Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively.HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor.Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups.Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression.ResultsRestriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully.The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+mmu-miR-30b, pmirGLO+mmu-miR-30b inhibitor, pmirGLO-mFoxO3+mmu-miR-30b, pmirGLO-mFoxO3+mmu-miR-30b+mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively.Compared with pmirGLO+mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+mmu-miR-30b group (P<0.05), but showed no significant change in pmirGLO-mFoxO3 mt+mmu-miR-30b group (P>0.05).In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05).Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05).Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b.There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA.

Objective: To construct a high effective eukaryotic expre ss ing vector containing mcpr1 gene. Methods: mcpr1 gene w as amplified by PCR from the plasmid T-easy/ mcpr1, then PCR product was in serted into eukaryotic expressing vector pcDNA3.1/V5-His B. The positive recomb inant was identified by PCR analysis, HindIII and BamHI restriction analysis and Sequence analysis. Results: A 400 bp DNA fragment was amplified from the recombinant. Sequence analysis and restriction digest demonstrated tha t the mcpr1 gene was successfully inserted into pcDNA3.1/V5-His B plasmid. Conclusion: The eukaryotic expressed vector pcDNA3.1/V5-His B/ mcpr1 has been successfully reconstructed.

Objective To establish a test method of dichloromethane,1,1-dichloroethylene,1,2-dichloroethylene,1,2-dichloroethane in drinking water with headspace gas chromatography.Methods Halogenated alkane hydrocarbon in the water was extracted by headspace technique,then analyzed with DB-624 capillary column.In the same time,determined with GC by controlling the temperature.Retention time of the peaks was used for qualitative analysis,while external standard method was used for quantitative analysis.Results The linear ranges for dichloromethane,1,1-dichloroethylene,1,2-dichloroethylene,trans-1,2-dichloroethylene,1,2-dichloroethane were 0.85-168,0.07-12.1,0.40-77.8,0.53-119 and 1.2-265 ?g/L respectively,the determination limits were 0.83,0.07,0.40,0.53 and 1.10?g/L respectively,r≥0.999 5,the rate of recovery were 102.5%-113.8%,and RSDs were 5.5%-11.8%.Conclusion This method is simple,rapid and sensitive,can efficiently separate and accurately determine 5 kinds of halogenated alkane hydrocarbon in the water and only takes 5.2 minutes.

Objective To explore the anticoagulation effect of low molecular weight heparin (LMWH) combination with warfarin at the early stage at the treatment of intracranial venous sinus thrombosis.Methods 80 cases of intracranial venous sinus thrombosis patients in people's hospital of Xinjiang Uygur autononous region from January 2010 to December 2014 were chosen to be analyzed retrospectively.37 cases in observation group were treated with low molecular heparin (LMWH) in combination with warfarin, and 43 cases in control group were treated with warfarin.The clinical curative effect between two groups was compared post-treatment.Results The effective rate in observation group was 91.89%, which was significantly higher than 67.44% in control group (P<0.05) .The recanalization rate of involved intracerebral venous in observation group was 89.19%, which was significantly higher than 67.29% in control group(P<0.05).After treatment, the prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were higher and fibrinogen (FBG) in both groups was lower than those pre-treatment(P<0.05).The PT and APTT was higher and FBG was lower in observation group than those in control group(P<0.05).Conclusion Low molecular weight heparin in combination with warfarin worked well than single warfarin in the treatment of intracranial venous sinus thrombosis.

In order to enhance the students’knowledge of clinical medicine and lay the foundation for using clinical medicine,we adjusted the content and compiled pharmacognosy teaching materials,aiming at its professional features of clinical pharmacy of Xuzhou Medical College.The pharmacognosy teaching material has been applied in several rounds.Feedback from students suggested that using this teaching material was well-targeted,clear and practical.

Objective To observe the effects ofLinggan Wuwei Jiangxin Decoction on contents of cAMP, cAMP dependent PKA and AQP5 of model rats with clod phlegm-fluid lying latent in the lung asthma;To discuss its mechanism.Methods Ovalbumin intraperitoneal injection and atomization motivation + cold stimulation were used to establish models. Ninety rats were randomly divided into blank control group, model group, hexadecadrol group, andLinggan Wuwei Jiangxin Decoction low-, medium-, high-dose groups. All administration groups were treated with relevant medicine. The levels of cAMP, PKA, and AQP5 were detected by ELISA.Results Compared with the blank control group, the levels of cAMP, PKA, and AQP5 of rats in model group decreased obviously (P<0.05). Compared with the model group, the levels of cAMP, PKA, and AQP5 of rats in all administration groups increased (P<0.05), which increased dramatically inLinggan Wuwei Jiangxin Decoction high-dose group.ConclusionLinggan Wuwei Jiangxin Decoction can regulate the normal secretion of cAMP, PKA, and AQP5 in serum of rats with clod phlegm-fluid lying latent in the lung asthma, increase secretion of air passage liquid and decrease mucoprotein concentration, through which plays the role of treating asthma.

Objective: To establish an anti-tumor drug sensitivity test by micocapsulated tumor cells in vivo and in vitro.Methods: Alginate-polylysine-alginate(APA) microcapsule technique was used to encapsulate tongue cancer Tca8113 cells. MTT method was used to detect the cytotoxic effect of methotrexate(MTX)and fluorouracil (FU) on encapsulated cells, then IC 50 was calculated. Encapsulated cells were also seeded into the abdominal cavities of adult Kunming mice and FU was injected through tail vein. After two weeks, microcapsules were recollected and histological examination was performed . Results: Encapsulated tumor cells derived from tongue could survive and proliferate in clusters. MTX and FU inhibited the cell growth in a dose-dependant way. IC 50 of MTX and FU was 200 ?mol/L and 0.85 mg/ml respectively. Necrosis of the cells in microcapsules and fibrotic encapsulation of microcapsules were observed in the in vivo tests. There was no significant difference between treatment group and control group. Conclusion: Microcapsulted tumor cells may be used in the anti-tumor drugs sensitive experiment in vitro.

Objective To investigate the expression of NF-κBp65 in hippocampus after the XST intervention therapy in the SD rats with global cerebral I/R injury and testify the protective effect of XST after global cerebral I/R injury.Methods 72 healthy SD rats were randomly divided into 3 groups,sham operation(SO) group ( n =24),I/R group( n =24) and XST group( n =24).The model of acute global cerebral ischemia/reperfusion (including:I/R and XST group) injury was produced by means of simple Pulsinelli- brierley's four arteries occlusion method.H.E.staining was performed to detect the number of surviving neurons and TUNEL was used to detect the rate of neurons apoptosis.The expression activation of NF-κB p65 in hippocampus comu-ammonis ( CA1 ) region were examined by immunohistochemical method (SABC).Results The survival pyramidal neurons in the XST group continued to increase,and it was significantly more than the I/R group at each time-point after reperfusion[ (99.23 ±4.22)/mm vs (75.83 ±7.17 )/mm,(80.93 ± 5.36)/mm vs (51.50 ± 8.26 )/mm,(103.24 ± 5.48 )/mm vs (35.67 ± 13.17 )/mm,( 126.22 ± 7.54 )/mm vs (9.83 ± 4.71 )/mm ],the differences were statistically significant ( P ＜0.01 ).The apoptosis rate of pyramidal cell in the XST group at each time-point were more significantly reduced than the I/R group [ ( 8.82 ± 2.71 ) ％ vs ( 22.58 ± 4.68 ) ％.( 19.15 ± 6.23 ) ％ vs (42.68 ± 3.04 ) ％,( 11.82 ± 2.87 ) ％ vs ( 55.51 ± 6.81 ) ％,( 8.44 ± 3.23 ) ％ vs ( 71.69 ± 7.71 ) ％ ],the differences were statistically significant ( P ＜0.01 ).The positive neurons of NF-κBp65 expression in the XST group at different time-points were significantly less than the L/R group[ ( 13.20 ±2.50) vs ( 18.00 ± 1.87),(8.20 ±5.31) vs (41.60±3.65),(6.70±3.36) vs (55.30±5.10),(7.10±3.57) vs (72.80 ±4.71)],the differences were statistically significant ( P ＜ 0.05,P ＜ 0.01 ).Conclusions After global cerebral ischemia/reperfusion,XST could protect the brain from global cerebral ischemia/reperfusion injury by holding up the expression of NF- kappaB p65,and inhibiting neuronal apoptosis,and increasing the number of surviving neurons.Thus,the results of this experiment could provide a powerful and weighty objective indication for XST being used during cerebral resuscitation.

Objective To analyse the etiology and clinical characteristics of 26 critically ill children with severe hand foot and mouth disease (HFMD) of Shanxi province in 2010.Methods We retrospectively analysed the clinical data of 26 children with severe HFMD from Mar to Sep 2010.Nucleic acid of enterovirus 71 ( EV71 ) and Coxsackie virus A 16 ( CoxA16) were detected in 20 out of 26 children with HFMD by reversed real time polymerase chain reaction (rRT-PCR),and the whole VP1 gene of EV71 deriving from 6 different areas of Shanxi province was amplified,sequenced,and compared with strains from other areas in china.Results EV71 nucleic acid were positive in 18 out of 20 children,while the other two were negative for EV71 and CoxAl6.Among all the critical cases,20 cases (76.9％) occurred in Weinan area,four in Xianyang area,and two in Xi'an urban area.Compared with those of Fuyang Anhui,Hong Kong China,Guangzhou,Shenzhen,Shandong,Beijing,the homology of the whole VP1 gene sequence from 6 strains of Shanxi area was 96％ ～ 100％.Most of the critical children were under 3-year-old,and the incidence rate of male children was higher than that of female children.All affected children had persisted fever,poor energy,hyperarousal,hypersomnia,and limb shaking.Meanwhile their peripheral blood leukocytes,C-reactive protein and blood glucose were markedly increased,but renal injuries were rare.Eighteen children clinically recovered on discharge,among which 2 cases had sequelae of limb activity obstacle,and 8 cases died.Conclusion Weinan is the area with the highest incidence rate of critical HFMD cases in Shanxi Province,and the major etiological organism is EV71,which is highly homologous to EV71 found in other regions of mainland China.As many cases are in dangerous condition,thus early identification and intervention could inhibit the disease progression,and play a key role in reducing the mortality.

Objective To investigate the effect of CDH1 3'-UTR + 54C/T single nucleotide polymorphism(SNP) on expression of luciferase reporter gene and its association with susceptibility to cervical cancer. Methods The luciferase gene expression vectors containing CDH1 3'-UTR +54C/T SNP C or T allelotype were constructed. The effect of CDH1 3'-UTR + 54C/T SNP on expression of luciferase reporter gene in 293 T cells were tested by daul lucfferase reporter assay system. The CDH1 3'-UTR + 54C/ T SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 280 cervical cancer patients and 330 healthy controls. Results The lucfferase activity analysis showed that the relative luciferase activity (RLA) of 293T cells with C allelotype was 1.46, which was significantly lower than that of the 293 T cells with T allelotype (3.01; t=2. 94, P =0. 042). There was no significant difference in age distribution between the cervical cancer patients and the healthy controls. The genotype frequency distribution of CDH1 3 '-UTR + 54C/T SNP in healthy controls did not significantly differ from that expected by Hardy-Weinberg equilibrium (P>0.05). The C allelotype frequency of CDH1 in cervical cancer patients was 80. 7%, which was significantly higher than that in healthy controls (74. 5%;χ2 =6.59, P=0.010). The T/T, T/C and C/C genotype frequencies of cervical cancer patients and healthy controls were 4. 3%, 30. 0%, 65. 7% and 5. 8%, 39. 4%, 54. 8%, respectively, which were significandy different (χ2=7.45, P =0.024). Compared with individuals with T/T or T/C genotypa, individuals with C/C genotype had significantly higher risks of developing cervical cancer (OR = 1. 578,95%CI=1.136 -2.191). Conclusion The C allelotypa of CDH1 3'-UTR + 54C/T SNP might decrease the expression of lucfferase reporter gene and the C/C genotypa might be a potential risk for cervical cancer development.