Objective To detection the methylation of p16INK4A in primary hepatocellular carcinoma, a nested bisulfite sequencing and methylation-specific polymerase chain reaction (BS-MSP) protocol was designed and used.Methods Bisulfite-modified DNA were amplified to evaluate the quality of templates with a pair of bisulfite sequencing primers in the first round of PCR, then subjected to methylation assay with corresponding methylation or unmethylation specific PCR primers.Representative PCR products were sequenced to confirm its correctness.Results 3 of 40 cases (7.5%) were failed to assay due to poor quality of templates, and 29 of 37 cases (78%) were detected p16INK4A methylation.Sequencing results confirmed that templates were correctly amplified.Conclusion BS-MSP technique might be valuable for methylation study on carcinogenesis and clinical assay.

Objective To analyze the significance of global end-diastolic volume index (GEDVI) in acute kidney injury (AKI) after septic shock.Methods A retrospective analysis of 61 patients was performed.The patients were diagnosed of septic shock in emergency ward of Shenyang Military Hospital from 2012 March to 2013 May and were monitored by pulse indicator continuous cardiac output (PiCCO).The patients were divided into two groups:low GEDVI group (GEDVI ＜ 700 ml/m2,29 cases) and high GEDVI group (GEDVI≥700 ml/m2,32 cases) by evaluating GEDVI of 24 hour after PiCCO.Several physiologic and biochemical indexes were recorded,including the hemodynamic parameters at the beginning and the 24 h of PiCCO monitoring,Scr,BUN,lactic acid,incidence and mortality of AKI,baseline glomerular filtration rate,baseline Scr,APACHE Ⅱ scores,mortality during the period of emergency ward or within 28 d after the diagnosis.Results A total of 26 cases in high GEDVI group (81.3％) were attacked with AKI,while 16 cases in low GEDVI group (55.2％) were attacked with AKI,the incidence of AKI in high GEDVI group was significantly higher than that in the low GEDVI group.A COX regression analysis of mortality was performed between the patients staying at emergency ward and during 28 d after diagnosis.The results indicated that AKI and GEDVI had no relation with patients' death.Therefore,AKI and GEDVI could not be considered as the risk factors for the prognosis.Conclusions High GEDVI can significantly increase the incidence of AKI after septic shock,therefore high GEDVI should be avoided as much as possible in the course of clinical treatment.

Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.

BACKGROUND:Many studies have demonstrated that restoration of femoral offset in revision total hip arthroplasty would contribute to the recovery of joint function.OBJECTIVE:To investigate the importance of restoration of femoral offset in revision total hip arthreplasty on the recovery of joint function.METHODS:An observational study was performed at the Department of Orthopedics,Third Affiliated Hospital of Guangzhou Medical University between February 2004 and May 2007.A total of 15 patients with the revision total hip arthroplasty,including 12 males and 3 females,aging 62 75 years,averaging 67 years old,were recruited into this study.Harris evaluation system was used to evaluate joint function.The femoral neck anteversion and the femoral offset were measured by the method of Sakai.The vertical distance from the teardrop line to the most prominent point of the lesser trochanter was measured from each radiograph.References were combined to investigate the effect of restoration of femoral offset in revision total hip arthroplasty on joint function.RESULTS AND CONCLUSION:All the 15 patients were recruited into this study.The duration of follow-up ranged from 24 months to 5 years.We measured the femoral offset on pre- and post-operative radiographs,and the results indicated that the femoral offset of 4 patients were above 4 mm.The femoral offset of 11 patients was restored.The femoral offset were 22-48 (32.21±0.64) mm pre- and 22-57 (36.13±0.82) mm post-operative radiographs,respectively.The mean difference in femoral offset post-operatively was significant (t=0.424,P=0.01 ).Harris scores were good in 4 cases,passable in 2 cases,and poor in 9 cases pre-operatively,and the scores were excellent in 8 cases,good in 4 cases,passable in 2 cases,and poor in 1 case post-operatively.The score of Harris evaluation system in the patient of restoration group and failed restoration group were 88.72±5.3 (80%) and 72.32±6.5 (27%) post-operative at 1 month respectively.The mean difference of the score was significant (χ~2=1.245,P<0.05).The 3 patients had complication,one was the dislocation of hip,and two had the pain of hip.All the patients with complication were in failed restoration of femoral offset,which was above 4 mm.The restoration of femoral offset contributes to the recovery of joint function and reduce complication occurrence after total hip arthroplasty revision.

Calcium hydroxide (CH) is applied to improve disinfection of root canals in most root canal retreatment. This study aimed to analyze the CH removal efficacy using 7 different root preparing files (K file, pre-curved K file, EndoActivator, Ultrasonic file, pre-curved ultrasonic file, F file and needle irrigation alone) with apical transportation. Standardized models of curved canal with such apical transportation or not were set up before applying CH to root canal for 7 days. Seven techniques described above were used for its removal. Then the roots were disassembled and digital photos were taken. The ratio of residual CH in the overall canal surface was calculated using the image analyzer image pro plus 6.0. The data were analyzed using one-way ANOVA with post hoc Tukey test. Results revealed that CH was effectively removed (P<0.05) by using all 6 mechanical methods except irrigation alone. In curved root canals with apical transportation, EndoActivator, pre-curved ultrasonic file and F file were found to be more effective in removing CH than the other four file (P<0.001), while there was no significant difference among EndoActivator, pre-curved ultrasonic file and F file groups (P>0.05). The percentage of residual CH in the canal with apical transportation was higher than that in the canal without apical transportation (P<0.05). In conclusion, CH can be hardly removed completely. Canal with apical transportation will result in insufficient CH removal. EndoActivator, pre-curved ultrasonic file and F file are more effective in the curved root canal with apical transportation.

In this study, we use pot experiment to evaluate the effect of plant growth regulator on plant morphology and biomass allocation of Salvia miltiorrhiza. Different concentrations of uniconazole were supplied to S. miltioohiza by means of foliar spray. Height, breadth and stem diameter were measured dynamically, the biomass of leaf, stem, flower and fruit, root biomass and biomass ratio were also examined at the harvest time. Owing to the treatment, plant morphology showed significant changes, the height had been greatly reduced and the breadth decreased largely. Meanwhile, the biomass allocation changed too. The biomass ratio of leaf and stem had been notably reduced while the biomass ratio of root had been increased remarkably. It appears that foliar application of uniconazole during vigorous growth period in S. miltioohiza has dramatic effect on dwarfing plant and improving resistant to lodging. This measure could also be applied to condensed cultivation of S. miltioohiza to increase production.
Biomass ; Plant Growth Regulators ; pharmacology ; Plant Leaves ; drug effects ; growth & development ; Plant Roots ; drug effects ; growth & development ; Plant Stems ; drug effects ; growth & development ; Salvia miltiorrhiza ; drug effects ; growth & development ; Triazoles ; pharmacology

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

Objective To investigate the spiral CT features of gastric carcinoma in the invasion and metastasis and its correlation with the expression of phosphatase and tensin horology deleted on chormosome ten(PTEN)and basic fibroblast grouth factor(bFGF).Methods Spiral CT plain scan and triphasic enhanced scans were performed in 83 patients.The postoperative specimens were embedded with paraffin to obtain 5?m thickness tissues and stained with HE and immunohistochemistry.Spiral CT findings were compared with the expression of phosphatase and tensin horology deleted on chormosome ten(PTEN)and basic fibroblast growth factor(bFGF).Results(1)The accuracy of spiral CT in T and N staging of gastric carcinoma was 94.0%(78/83)and 89.2%(74/83),respectively.(2)The expression of PTEN was 47.0%(39/83)in gastric carcinoma.The expression of PTEN in T_(3.4)(40.8%,29/71)and N_(1+2) (38.3%,23/60)gastric carcinoma was significantly lower than that of T_2(10/12)and N_0(16/23)gastric carcinoma,respectively(X~2=7.439,P=0.006;X~2=6.511,P=0.011).(3)The expression of bFGF was 63.9%(53/83)in gastric carcinoma.The expression of bFGF in T(3.4)(70.4%,50/71)and N_(1+2) (71.7%,43/60)gastric carcinoma was significantly higher than that of T_2(3/12)and N_0(10/23)gastric carcinoma,respectively(X~2=7.314,P=0.007;X~2=5.724,P=0.017).(4)Both PTEN-positive expression and bFGF-positive expression were detected in 16 specimens.The expression of PTEN(41.0%, 16/39)was negatively correlated with that of bFGF(30.2%,16/53)(r=-0.447,P=0.000). Conclusion Spiral CT triphasic enhanced scans combined with biologic characteristics can improve diagnostic accuracy of gastric carcinoma in the invasion,metastasis and prognosis.

Thermodynamic parameters of complexation of naphto-15-crown-5 with four alkaline earth ions in aqueous media was determined using titration microcalorimetry at 298.15 K. The stability of the complexes, thermal effect and entropy effect of the complexation is discussed on the basis of the guest ions structure and the solvent effect. The stability constants tendency to vary with ion radius was interpreted. Complex of naphtha-15-crown-5 with calcium ion is very stable due to the synergism of static electric interaction and size selectivity between the host and the guest.
Calorimetry ; methods ; Crown Ethers ; chemistry ; Ions ; chemistry ; Macromolecular Substances ; analysis ; chemistry ; Metals, Alkaline Earth ; chemistry ; Microchemistry ; methods

Objective To explore the risk factors of cerebral infarction in youth,and to provide evidence for prevention and control. Methods A total of 105 patients aged 35 -45 years old with cerebral infarction in the affiliated hospital of Shaoxing University from January 2010 to October 2014 were recruited as the cerebral infarction group . The healthy people without neurological symptoms were recruited as the control group. The two groups were compared for the risk factors of cerebral infarction in the youth. Results Logistic regression analysis showed that high blood pressure,diabetes,lipid disorders, smoking,obesity,unstable plaque,high homocysteine and anti - cardiolipin antibodies were the risk factors for cerebral infarction in the youth. The value of ORs and 95% CIs interval value for high blood pressure,diabetes,lipid disorders, smoking,obesity,unstable plaque,high homocysteine and anti - cardiolipin antibodies was 14. 614(0. 469 -47. 273),5. 129 (1. 541 -28. 466),44. 970(2. 789 -101. 549),26. 180(1. 085 - 51. 912),45. 196(2. 572 - 205. 674),258. 786(4. 892 -367. 678),14. 585(1. 770 - 49. 662)and 5. 145(1. 005 - 20. 293),respectively. Conclusion High blood pressure, diabetes,lipid disorders,smoking,obesity,unstable plaque,high homocysteine and anti - cardiolipin antibodies were closely related with cerebral infarction in the youth,and it is necessary to prevent and control the influencing factors and diseases.