Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.

Objective To investigate the expression differences of mRNA and protein of early B-cell factor 3(EBF3) in the tissues between hepatocellular carcinoma(HCC) and distant noncancerous tissues and the clinical significance.Methods The expression levels of EBF3 mRNA in 18 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by real-time fluorescence quantitative polymerase chain reaction(FQ-RT-PCR).Western blotting was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues.Results The ratios of EBF3 mRNA tobeta2 microglobulin mRNA in eighteen liver cancerous tissues(0.52?0.17) were significantly higher than those in distant liver noncancerous tissues(0.28?0.23,t=3.56,P=0.0011).The average gray scale level of EBF3 in five liver cancerous tissues(26.35?14.06)were significantly higher than those in distant noncancerous tissues(7.86?8.47,t=2.52,P=0.036).Conclusion Theexpression levels of EBF3 mRNA and protein up-regulated in primary hepatocellular carcinoma,suggestting that EBF3 may contribute to occurrence of primary hepatocellular carcinoma.

Objective To study pharmacokinetics of curcumin in Curcuma phaeocaulis in rats in vivo.Methods HPLC method was used to determine the curcumin in rat plasma.The conditions were column: Lichrospher-5-C_(18)(250 mm?4.6 mm, 5 ?m); column temperature: 25 ℃;mobile phase: CH_3CN-5% HAc water solution(45∶55);flow: 1 mL/min;detection wavelength: 420 nm.Results The calibration curve was liner(r=0.999 5) at the range of 6.5—104 ?g/mL.The average recovery was 98.5%.RSD was 2.41%(n=5).The pharmacokinetic parameters of curcumin were as follows: k_a was 0.53/h,k_e was 0.10/h,t_(1/2ka) was 1.32 h,t_(1/2k) was 6.89 h,t_(peak) was 3.89 h,C_(max) was 93.15 ng/mL,AUC was(1 369.38) ng/mL.Conclusion This method is stable,simple,and reliable,which can be applied for the determination of curcumin in plasma and pharmacokinetic studies.

The objectives of this study are to prepare resveratrol loaded mixed micelles composed of poloxamer 403 and poloxamer 407, and optimize the formulation in order to achieve higher drug solubility and sustained drug release. Firstly, a thin-film hydration method was utilized to prepare the micelles. By using drug-loading, encapsulation yield and particle size of the micelles as criteria, influence of three variables, namely poloxamer 407 mass fraction, amount of water and feeding of resveratrol, on the quality of the micelles was optimized with a central composite design method. Steady fluorescence measurement was carried out to evaluate the critical micelle concentration of the carriers. Micelle stability upon dilution with simulated gastric fluid and simulated intestinal fluid was investigated. The in vitro release of resveratrol from the mixed micelles was monitored by dialysis method. It was observed that the particle size of the optimized micelle formulation was 24 nm, with drug-loading 11.78%, and encapsulation yield 82.51%. The mixed micelles increased the solubility of resveratrol for about 197 times. Moreover, the mixed micelles had a low critical micelle concentration of 0.05 mg · mL(-1) in water and no apparent changes in particle size and drug content were observed upon micelles dilution, indicating improved kinetic stability. Resveratrol was released from the micelles in a controlled manner for over 20 h, and the release process can be well described by Higuchi equation. Therefore, resveratrol-loaded poloxamer 403/407 mixed micelles could improve the solubility of resveratrol significantly and sustained drug release behavior can be achieved.

Vascular smooth muscle cells (VSMCs) proliferation is the critical pathological process of in-stent restenosis (ISR). Recent researches indicate that the traditional Chinese medicine curcuma zedoary (E’zhu) can inhibit VSMCs proliferation, with a potential value in preventing and treating ISR. The major ac-tive components of curcuma zedoary are curcuma and β-elemene. The underlying mechanisms involve the inhibition of hemeoxygen-ase-1 expression, blockade of extracellular signal-regulated ki-nase – MAPK and Akt pathways, and subsequent cell cycle ar-rest. This article reviews the recent progress on curcuma zedoary extracts regulating VSMCs proliferation in ISR.

Objective To discuss the relationship of drip velocity of nitrate on blood pressure while treating coronary disease,in order to provide appropriate drip velocity for clinical treatment.Methods 155 patients with coronary disease using nitrate to lower blood pressure were selected.They were divided into the nitro glycerin group(85 cases) and the isosorbide mononitrate group( 70 cases) according to difference of medication.The velocity of drugs was adjusted on basis of blood pressure changes.The blood pressure changes at different drip velocities were observed and compared.Results The systolic pressure and the diastolic pressure between two groups showed no difference at 20 drops/min,but the results were the opposite at 30 drops/min.The systolic pressure and the diastolic pressure in the nitro glycerin group showed evident changes at different drip velocities,but in the isosorbide mononitrate group,these changes were not so significant.9 patients in the nitro glycerin group had headache during treatment,no headache occurred in the isosorbide mononitrate group.Conclusions Intravenous use of nitrate at a velocity of 20 drops/min is relative secure.The risk of hypotension will increase if the medication speed increases.lsosorbide mononitrate has little influence on blood pressure.

Objective To evaluate the role of real-time three-dimensional transesophageal echocardiography(RT-3D TEE)in left atrial appendage (LAA)occlusion.Methods Consecutive 10 atrial fibrillation (AF)patients (CHADS2 ≥ 2 )with high risk bleeding underwent LAA occlusion under the guidance of TEE.The LAA orifice shape and characteristics of lobes were assessed,the size of LAA with RT-3D TEE wee measured before closer implanation,and the position of the LAA occlusion device were evaluated by RT-3D TEE.The correlational analysis between LAA diameter and occluder size was conducted.Results Among 10 patients,the test results revealed 8 cases with complete LAA occlusion and 1 case with incomplete occlusion,and 1 case with failed occlusion.Five cases showed approximate round LAA ostium,and the other 5 showed approximate oval ostium.The average number of LAA lobes were 2.2±0.7. LAA ostium long diameter were larger by 3D TEE compared with 2D TEE[(21 .8±5.1)mm vs (20.8±4.1) mm],and ostium short diameter were smaller by 3D TEE compared with 2D TEE [(16.1 ± 3.0 )mm vs (1 7.0±2.6)mm],however there were no significant differences between 2D and 3D TEE measurements,and the mean ostium diameter and LAA depth were comparable between two methods.LAA ostium long diameter,short diameter,average diameter and LAA depth assessed by 3D TEE and 2D TEE showed good correlation with occluder diameter (3D TEE:r =0.719,0.690,0.791 ,0.71 1 ,and P =0.029,0.040,0.01 1 , 0.032,respectively;2D TEE:r = 0.887,0.894,0.932,0.896,and P = 0.001 ,0.001 ,0.000,0.000, respectively).LAA occlusion device position assessed by RT-3D:6 cases with appropriate position, acceptable position with 2 cases,and 1 case with malposition.Conclusions RT-3D TEE can play important role in evaluating the morphology of LAA,accurately judging LAA ostium shape and size and position of the occlusion device.

Objective To compare the reliability and validity of 2 incontinence- associated dermatitis (IAD) risk assessment tools (IAD risk assessment scale and PAT) in patients with incontinence, and to search for the best risk assessment tool. Methods A total of 101 IAD cases were chosen from the Affiliated Hospital of Nantong University.Reliability was evaluated by the inter- rater reliability,internal consistency,item sensitivity analysis and retest reliability; validity was evaluated by the structure validity and predictive validity. Results Cronbach α was 0.313 (IAD risk assessment scale) and 0.421(PAT);Cronbach α coefficient was the highest after the items that Change sheets and mats and relevant factors were removed respectively in two tools,which were 0.431, 0.428; The correlation between the total scores were 0.711 (P<0.01)(IAD risk assessment scale)and 0.498 (PAT)(P<0.01);Area under curve were 0.661 (IAD risk assessment scale),0.864 (PAT); the best cut-off value to forecast a poor prognosis was 53.5 (IAD risk assessment scale),6.5 (PAT). Conclusions The reliability and validity of IAD risk assessment scale and PAT are low. The change of sheets and mats and Related influencing factors are needed to be adjusted and revised.

Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.

Objective To assess the value of transesophageal echocardiography (TEE) in left atrial appendage (LAA) closer with Amplatzer Cardiac Plug (ACP).Methods Consecutive 32 atrial fibrillation patients (CHADS2≥1) with high risk bleeding underwent LAA occlusion with ACP LAA occlusion device under the guidance of TEE.Measurements of LAA anchoring area diameter (AAD) and LAA anatomical orifice diameter (AOD) including maximum,minimum values,and LAA depth on 2 dimentional TEE (2D TEE) were conducted before closer device implantation.The outcomes of LAA occlusion were analyzed.Results Among 32 patients,27 cases achieved successful LAA occlusion,including 2 cases with peripheral leakage ≤5 mm,and 5 cases failed occlusion.Among 5 failed closed patients,3 cases with LAA AAD≥ 30 mm,1 case with LAA of short depth,and 1 case with anatomical variation of LAA of low position that access sheath could not be positioned in the LAA.LAA AAD maximum,minimum and LAA depth were (25.9±4.9)mm,(20.0±3.8)mm,(31.0±5.6)mm,respectively,and LAA AOD maximum,minimum diameters were (26.2±6.2)mm,(19.4±4.3)mm,respectively.Among 10 cases with LAA AAD≥30 mm,7 cases achieved successful LAA occlusion,but 3 cases failed.The implanted device diameter was (26.4±3.8)mm,and device compression rate was (7.6±5.5)%.The coefficient of correlation between device diameter and large LAA AAD and AOD is 0.770 and 0.717,respectively.There was no complication but 1 case with pericardial effusion.Conclusions Two dimentional TEE measurements of LAA have clinical guiding significance in the selection of proper size of ACP LAA occluder.The AAD of 2D TEE has the good correlation with ACP occluder′s size,the AADs are important factors which affect the success of LAA occlusion.