Objective To investigate the effect of deoxyribonucleic acid (DNA) methylation on the expression of hepatocyte nuclear factor-4α (HNF4c) and its role in the expression of HNF4α regulated by transforming growth factor-β1 (TGF-β1).Methods The expression of HNF4αP1 mRNA in six human hepatoma cell lines (HepG2,Huh-7,Hep3B,SMMC-7721,BEL-7405 and FOCUS),20 hepatic carcinoma specimens and corresponding adjacent tissues was detected by real-time reverse transcription polymerse chain reaction (real-time RT-PCR).The methylation status of the promoter region of HNF4αP1 in six human hepatoma cell lines was examined by bisulfite sequencing PCR (BSP).FOCUS cells were treated with 5-aza-2'-deoxycytidine (5-AZA-CdR) and then the methylation status of the promoter region of HNF4αP1 was examined by BSP.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR.The six human hepatoma cell lines were treated with TGFβ1 and the expression of HNF4αP1 mRNA was detected by real-time RT-PCR.FOCUS cells were cotreated with 5-AZA-CdR,TGF-β1 and 5-AZA-CdR.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR,and t test was performed for statistical analysis.Results Among 20 human hepatic carcinoma specimens and corresponding adjacent tissues,the expression of HNF4αP1 mRNA of 13 human hepatic carcinoma specimens was lower than that of corresponding adjacent tissues (t=2.350,P＜0.05).The relative quantity of the expression of HNF4αP1 mRNA was higher in Hep3B,HepG2 and Huh-7 cells,whereas that in SMMC-7721,BEL-7405 and FOCUS cells was lower.The methylation of the promoter region of HNF4αP1 in HepG2,Huh-7 and Hep3B was lower,but that in SMMC-7721,BEL-7405 and FOCUS was higher.Along with the increasing of the concentration of 5-AZA-CdR (0,0.1,1.0 and 2.5 μmol/L),the degree of the methylation of the promoter region of HNF4αP1 in FOCUS cells gradually decreased (61％,46％,32％ and 27％),and however the relative quantity of the expression of HNF4αP1 mRNA gradually increased ((9.661 ± 0.336)×10-7,(2.001±0.432)×10-6,(3.689±0.714)×10-6and (4.732±2.451)×10-6).After stimulated with TGF-β1,the relative quantity of the expression of HNF4αP1 mRNA was downregulated in HepG2,Huh-7 and Hep3B cells in which the methylation of the promoter region was low (t=12.994,8.441,and 9.032,all P＜0.01).There was no significant difference in the relative quantity of the expression of HNF4αP1 mRNA in SMMC-7721,BEL-7405 and FOCUS cells in which the methylation of the promoter region was high (all P ＞ 0.05).The relative quantity of the expression of HNF4αP1 mRNA in 5-AZA-CdR treated FOCUS cells ((4.972±0.035) × 10-6) was higher than that of control group ((1.411 ± 0.104) × 10-6) and the difference was statistically significant (t=13.212,P＜0.01).The relative quantity of the expression of HNF4αP1 mRNA in FOCUS cells co-treated with 5-AZA-CdR and TGF-β1 was lower than that in cells treated with 5-AZA-CdR alone and the difference was statistically significant ((1.181 ± 0.132) × 10-6 vs (4.972 ± 0.035) × 10-6,t=13.873,P＜0.01).Conclusions The expression of HNF4αP1 is down-regulated in hepatic carcinoma tissues.DNA methylation may regulate the expression of HNF4αP1 in hepatoma cells.The methylation of HNF4αP1 promoter region inhibits the regulating function of TGF-β1 in the expression of HNF4αP1.

OBJECTIVE:To study the stability of amoxicillin sodium and clavulanate potassium for injection in sodium chloride injection solution so as to offer a scientific basis for rational use of drug in clinical practice.METHODS:The external appearance and pH values of the mixed solution were observed,and the contents of Amoxicillin sodium and Clavulanate potas?sium were determined by HPLC method at5℃,25℃and37℃within3hours.RESULTS:The stability was related to the temperature and sunlight.CONCLUSION:The mixed solution is instable and should not be put in storage for long time.

Objective To define the nuclear agrresome formation is determinated by which part of the UL76 of
HCMV. Full-length, conserved N terminal and unconserved C terminal of pUL76 were constructed to eukaryotic ex-pression plasmid pEGFP-N1 . Methods Primers were designed to amplify full-length and different part of pUL76 according to standard sequence of HCMV AD169 which had been submitted to GenBank(FJ527563. 1). These frag-ments were constructed to eukaryotic expression plasmid pEGFP-N1 . The recombinant plasmids were designated pEGFP-UL76,pEGFP-UL76N, pEGFP-UL76C respectively. Double digestion and sequencing were performed to verify the accuracy of recombinant plasmids construction. Empty vector and three recombinant plasmids were transi-ent transfected to HELF and HepG-2 cells respectively. Reverse transcriptation PCR and Western blot were per-formed to detect the RNA and protein expression level respectively. Different nuclear aggresome formations were visualized with an Olympus fluorescence microscopy. Results pEGFP-N1 and pEGFP-UL76N were unable to in-duce nuclear aggresome formation, whereas pEGFP-UL76 and pEGFP-UL76C were able to elicit nuclear aggresome formation. Conclusion The unconserved C terminal of pUL76 is sufficient to induce nuclear aggresome formation.

Objective To evaluate the effect of sevoflurane postconditioning on the expression of CCAAT/enhancer-binding protein homologous protein (CHOP) in a rat model of hemorrhagic shock and resuscitation.Methods Thirty-six healthy adult male Sprague-Dawley rats,weighing 300-350 g,were divided into 3 groups (n=12 each) using a random number table:sham operation group (group S),hemorrhagic shock and resuscitation group (group HSR) and sevoflurane postconditioning group (group SP).Hemorrhagic shock was induced by withdrawing 40% of the total blood volume from the right carotid artery over an interval of 30 min,and 1 h later the removed blood was reinfused via the left jugular vein for resuscitation.Group SP inhaled 2.4% sevoflurane for 30 min starting from the onset of reinfusion.Mean arterial pressure was monitored and recorded at a 10 min interval.Before withdrawing blood (T0),immediately after the end of withdrawing blood(T1), at 1 h after the end of withdrawing blood(T2) and immediately after the end of reinfusion (T3),blood samples were collected from the common carotid artery for blood gas analysis.At 4 days after reinfusion,6 rats of each group were selected to detect spatial learning and memory ability by using Morris water maze test.The animals were then sacrificed,brains were removed for determination of neuronal apoptosis in hippocampal CA1 area using TUNEL.The rest 6 rats in each group were sacrificed at 72 h after reinfusion,and the hippocampus was isolated to detect the expression of CHOP by Western blot.Results Compared with group S,mean arterial pressure was significantly decreased,and lactic acid concentrations were increased at T1,2 in HSR and SP groups,and the escape latency was significantly prolonged,the percentage of time staying at the target quadrant was decreased,the number of apoptotic neurons in hippocampal CA1 area was increased,and the expression of CHOP was up-regulated in group HSR (P<0.05).Compared with group HSR,the escape latency was significantly shortened,the percentage of time staying at the target quadrant was increased,the number of apoptotic neurons in hippocampal CA1 area was decreased,and the expression of CHOP was down-regulated in group SP (P<0.05).Conclusion The mechanism by which sevoflurane postconditioning improves cognitive function is related to down-regulation of CHOP expression and inhibition of apoptosis in hippocampal neurons in a rat model of hemorrhagic shock and resuscitation.

Objective: To explore the mechanisms of acupuncture in improving osteoporosis. Methods: In this study, the expression of OPG and BMP-2 proteins of femur in SAMP6 control group, SAMP6 acupuncture group, SAMP6 sham-acupuncture group and SAMR1 control group were determined by Western Blot technique. Results: Compared with SAMR1 control group, the expression of OPG and BMP-2 proteins of the femur in SAMP6 control mice decreased obviously. Acupuncture could stimulate the expression of OPG and BMP-2 and no differences were detected between the SAMR1 control group and SAMP6 acupuncture group. Conclusion: Osteoporosis observed in SAMP6 was correlated with low expression of OPG and BMP-2. After acupuncture treatment, both of them showed increased expression, it suggested that the curative effects of acupuncture on osteoporosis were partly due to stimulating skeleton formation, inducing the production of some local factors of bone metabolism and decreasing the bone turnover rate.

Objective:To investigate the effects of Adp53 and F56 on the growth and lung metastasis of breast cancer.Methods:The BICR-H1 cells were inoculated into the mammary fatty pad of BALB/C nude mice and NOD/SCID mice to establish breast cancer model.Then the nude mice with xenograft tumor were randomized into group Adp53+F56,Adp53,F56 and control.The NOD/SCID mice with xenograft tumor were randomized into group Adp53+F56,Adp53,F56,Adlacz and control.They were theated for 3 weeks according to the plan,diversity of the volume and histopathology of xenograft tumor of nude mice was observed and the expressions of p53 and VEGF gene,and microvessel density(MVD)were detected by immunohistochemistry.Lung metastasis of breast cancer in NOD/SCID mice was observed.Results:(1)Intratumoral injections of Adp53,F56,and their combination resulted in an inhibition on the growth of xenograft tumor of BICR-H1 cells.The ultimate relative growth volumes of groups Adp53+F56,Adp53,F56 and control were 2.47,4.37,4.69 and 12.49 respectively.(2)After treatment,P53 positive rate of group Adp53+F56,Adp53 increased 9.4%,6.3% than before respectively,but compared with control group,the difference is not significant(P=0.693);VEGF protein of group Adp53+F56,Adp53 and F56 decreased 21.9%,9.4% and 3.1% than before respectively,but compared with control group,the difference was not significant(P=0.284).Necrosis and decrease of vessel in the tumor and morphological change of endothelium were observed under light microscope in the groups Adp53+F56,Adp53 and F56.MVD estimated by FⅧ-RA staining of group Adp53+F56,Adp53 and F56 were 14.50?2.54,16.28?3.44 and 18.06?7.66,compared with control group(24.93?6.53),the difference is significant(P=0.000).(3)The average number of lung metastasis of NOD/SCID mice in group Adp53+F56,Adp53 and F56 were 1.143?0.378,2.750?0.886 and 3.375?0.518 respectively,lower than Adlacz group(5.000?0.816)and control group(5.670?0.817)obviously(P=0.000).Conclusion:Adp53 combined with F56 can greatly inhibit growth and matastasis of breast cancer in vivo.The mechanism of anti-tumor effects of Adp53 and F56 may be related to the anti-angiogenesis effect on malignant tumor through inhibiting the expression and activity of VEGF.

Objective To preliminarily investigate the effect of ribosomal protein S7 on apoptosis of HeLa cervical cancer cells.Methods The previously constructed recombinant plasmid pIRES2-EGFP-RPS7 was transfected into HeLa cells,the empty vector pIRES2-EGFP transfected cells as control.Enhanced green fluorescent protein(EGFP)expressing cells were quantified by flow cytometry,and RPS7 protein level was also determined by Western blotting.Cell apoptosis of both RPS7 over-expression cells and knockdown cells were evaluated by flow cytometry after staining using allophycocyanin labeled Annexin-V.Results Apoptotic cell level in the obtained RPS7 transient over-expression HeLa cells was significantly higher than that of vector con-trol cells [(1 0.00 ±0.60)% vs (5.73 ±0.61 )%],with a statistic difference (t =8.63,P =0.001 ). Moreover,the apoptotic level in RPS7 knockdown cells was lower than that in control cells [(3.08 ± 0.49)% vs (5.97 ±0.63)%],with a statistic difference (t =6.40,P =0.003).Conclusion Up-regula-tion of RPS7 may promote apoptosis,while down-regulation of RPS7 may inhibit apoptosis of HeLa cells,indi-cating that RPS7 may play roles in regulating cell apoptosis.

Objective To verify the validity of the sucrose density gradient ultracentrifugation method for lipid rafts from cerebral cortex. Methods Extract lipid rafts from cerebral cortex in mouse were extracted by the sucrose density gradi-ent ultracentrifugation method. The properties of lipid rafts were detected by Western blotting method, double enzyme and light scattering methods. HPLC MS/MS proteomics and bioinformatics were used to locate proteins of lipid rafts in cells. Re-sults Lipid rafts from cerebral cortex were provided with the model properties of lipid rafts such as high light scattering and cholesterol and high expression of Flotillin-1. HPLC MS/MS proteomics identified total 647 proteins. Most of these pro-teins were from plasma membrane, endoplasmic reticulum, cytoskeleton and cytosol, however, there were 21% proteins among total 647 proteins were from nucleus, mitochondria and ribosomes. Conclusion The sucrose density gradient ultra-centrifugation method is a effective method to extract lipid rafts from cerebral cortex, however, the properties of mixture should be considered.

Objective To compare the effectiveness and safety of Foley catheter(FC)and vaginal prostaglandin E2 suppository(PGE2,Propess)for cervical ripening and labor induction in fullterm pregnant women with unfavorable cervix.Methods A prospective randomized controlled trial was conducted.Women with a term or post-term,live,singleton fetus in cephalic presentation,intact membranes,Bishop score＜6,not in labor,medically indicated for labor induction from June 2009 to December 2009 in Drum Tower Hospital of Nanjing University Medical School were randomly divided into two groups:FC group(n=64)and Propess group(n=62).In FC group,a 16-F Foley catheter was inserted into patient's cervical canal; once past the internal os,the balloon was inflated with 80 ml saline.Intravenous oxytocin was initiated after the balloon was spontaneously extruded from the cervix or after 24 hours.In Propess group,vaginal Propess was used.x2 or Fisher's exact test and t test were used to compare the outcomes,delivery mode and induction success rate between the two groups.Results There were no significant differences in gestational weeks,Bishop score,indication of induction,improvement of Bishop score,success rate of induction,rate of vaginal delivery,total duration of labor and volume of postpartum hemorrhage between the two groups(P ＞ 0.05,respectively).Propess group had a higher rate of vaginal birth within 24 hours[56.5％(35/62)vs 28.1％(18/64),t=10.37,P＜0.05],a higher risk for excessively frequent and hard uterine contraction[17.7％(11/62)vs 0.0％(0/64),P＜0.05]and lower incidence of oxytocin induction/augmentation during labor[21.0％(13/62)vs 87.5％(56/64),x2 =56.27,P＜0.05]than those of FC group.There were no differences in neonatal Apgar score,meconium staining and neonatal birth weight between the two groups.Puerperal infection occured in neither group.Conclusions Under strict control of indication and aseptic manipulation,Foley catheter was as effective and safe as Propess for cervical ripening with lower risk of excessive uterine activity.It is suggested that Foley catheter could be used for cervical ripening,especially in patients with economic difficulty.

OBJECTIVE:To study the chemical constitutes of Ligusticum chuanxiong and to establish the method for the content determination of ligustilide. METHODS:The compounds were extracted and percolated by ethanol. Then the samples were separated using silica gel and identified by 1H-NMR,13C-NMR data.HPLC was used to assay the contents of ligustilide. RESULTS:Three compounds were isolated and their structures were identified as Z-ligustilide,Z-6,8',7,3'-diligustilide and Z,Z'-6,6',7,3'a-diligustilide. The contents of ligustilide were no less than 0.70%.CONCLUSION:The separation method can be used to prepare high-purity ligustilide reference substance. And the determination method can be used for quality control of L. chuanxiong.