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.

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 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 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.

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 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 related brain areas and nucleus involved in the inhibition of vagus nerve stimulation (VNS) on epilepsy. Methods Using the kainic acid kindling epilepsy rats model,we observed the distribution of Fos positive neurons in the brain after VNS treatment combined with immunohistochemical method. Results VNS induced a significant increase in Fos immunoreactivity in the bilateral nucleus of solitary tract,the locus coeruleus,parabrachial nucleus,periaqueductal gray of midbrain,lateral habenular nucleus,paraventricular thalamic nucleus,rhomoid thalamic nucleus,paraventricular hypothalamic nucleus.Dense Fos immunoreactive staining was also seen in the central nucleus of amygdala,bed nucleus of stria terminalis,lateral septal nucleus and prepirifiorm cortex.Pretreatment with electric stimulation on cervical vagual nerve stem, c fos expressing of hippocampus formation,cingulate gyrus and frontal,parietal,temporal lobus significantly diminished after KA injection. Conclusion This finding may suggest that VNS activates various brain structure that could be involved in the regulation of seizures.

Objective To develop an albumin aptamer that may potentially serve as a selective ligand for albumin removal from experimental samples.Methods A single-stranded 59nt DNA library that contains 21 random oligo nucleotides was synthesized in vitro.An albumin aptamer A6 was developed by SELEX technique using bovine serum albumin (BSA) as target.The enrichment of aptamer and evaluation of its binding properties were monitored by flow cytometry.The secondary structure of A6 was predicted by MFord software.Results The aptamer A6 strongly bound to BSA with a Kd of 77.4 nmol/L,and had minimal cross reactivity with control proteins including ovalbu min,IgG,and trypsin.Conclusions Aptamer A6 may be a potential tool in albumin removal.

Objective To investigate the change and clinical significance of serum hepcidin,serum ferritin (SF),transferrin re-ceptor (sTfR)and serum iron (SI)in patients in type 2 diabetes(T2DM).Methods 130 patients with T2DM were divided into 2 groups according to the 24 hour urine microalbumin (mAlb)quantitative:group A for trace microalbumin group 45 ca-ses (mAlb30~300 mg/24 h),group B for normal albuminuria group of 85 cases,an alternate period of 45 cases of healthy physical examination for group C (control group).Results Serum hepcidin and SF of group A (42.27±32.12 ng/ml,211.6 ±107.2 ng/ml)were significantly higher than those in group B (26.12 ± 18.36 ng/ml,179.1 ± 109.7 ng/ml)and the healthy control group (P <0.05),hepcidin and SF of group B was significantly higher than that of the control group (9.47 ±1.65 ng/ml,84.41±47.10 ng/ml,P <0.01),SI and transferrin receptor(sTfR)has no statistical significance between the three groups (P >0.05).Correlation analysis showed that patients with type 2 diabetes hepcidin was positively related with SF (P <0.05),hepcidin and sTfR,SI had no significant correlation.Conclusion These results indicated that there existed serum hepcidin and SF increased iron overload and iron metabolism disorder in type 2 diabetes.Therefore,detection of serum iron and SF can be used as a predictor of diabetes early renal damage.