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 investigate the levels of serum Golgi protein(GP73) (sGP73) in patients with HBV-related liver disease and investigate the role of sGP73 as an indicator for diagnosis of hepatocelluar carcinoma (HCC).Methods The concentration of sGP73 in patients with chronic hepatitis B (CHB,n =31),liver cirrhosis (LC,n =60),HCC (n =71),self-limited HBV infectors (n=21 ) and healthy controls (n =42) were tested by enzyme-linked immunosorbent assay (ELISA) assay and statistically analyzed in combination with relevant clinical indicators.Results The median of sGP73 in HBV-related liver disease group was significantly higher than that in the groups of self-limited HBV infectors and healthy controls respectively (P＜0.01).Among the groups with HBV-related liver disease,the median of sGP73 in LC group (231.13 ng/ml) was significantly higher than that in HCC without treatment group ( 117.63 ng/ml) (P ＜ 0.01 ) and CHB group (93.09 ng/ml) (P＜0.01).No significant difference was shown between HCC (without treatment) group and CHB group (P＞ 0.05),neither between self limited HBV infectors and healthy controls (respectively,36.79 ng/ml and 45.40 ng/ml) (P ＞ 0.05). The median of sGP73 in post-operation group (175.12 ng/ml,n=52) was significantly higher than in pre-operation HCC group (107.28 ng/ml,n=52) (P＜0.01).Along with the decreasing of liver function,sGP73 level was elevated in groups with HCC or LC.The receiver operating curve (ROC) constructed with the ratio of AFP and GP73 (AFP/GP73) showed a sensitivity of 78.87 ％ and specificity of 86.21 ％ with an area under the receiver operating curve (AUROC) of 0.878 (95％ CI:0.817-0.938) for diagnosis of HCC; comparably,a sensitivity of 67.61％ and specificity of 85.12％ were shown with a AUROC of 0.826 (95％ CI:0.755-0.897) when performed with AFP.Conclusion The level of sGP73 in HBV-related liver disease group is higher than that in the groups of self-limited HBV infector and healthy control,and it is positively correlated with the degree of hepatic impairment.For the diagnosis of HCC,joint detection of AFP and GP73 could achieve a better combination of sensitivity and specificity than the independent AFP test.

ObjectiveTo investigate hepatocyte growth factor (HGF) levels in the tissue and serum of patients with chronic hepatitis,cirrhosis or hepatocellular carcinoma (HCC),and analyze the clinical significances of HGF for HCC.MethodSurgical specimens from 97 patients were collected during Dec.2003 to Aug.2008 in the Third Central Hospital.The patients were prospectively enrolled and categorized into four groups:normal subjects ( n =11 ),chronic hepatitis B or C ( n =6＝,cirrhosis ( n =20)and HCC ( n =60 ) including well-differentiated ( n =21 ),moderately differentiated ( n =23 ),poorly differentiated (n =16) specimens.N0 (n =24),N1 (n =21 ),N2 (n =54) and N3 (n =43) were tissues respectively removed from liver at 0,1,2 or 3 cm beyond the margin of tumor.HGF mRNA expression in liver tissues was determined by real-time quantitative reverse transcription- (RT)-PCR.Serum HGF levels in the other cases of normal subjects ( n =20),chronic hepatitis B or C ( n =20),cirrhosis ( n =20) and HCC (n =57) were measured by ELISA.The Kaplan-Meier method with log-rank test was employed for survival analysis.Univariate and multivariate analyses were performed to identify prognostic factors in each group.ResultsThe HGF mRNA in normal subjects,chronic hepatitis,cirrhosis,N3,N2,N1,N0 and HCC were0.99(0.78-1.66),2.15(1.06-3.40),1.78(1.18-2.73),4.59(2.67 -8.63),3.86 ( 2.25 - 6.45 ),3.12 ( 1.59 - 5.74 ),2.92 ( 0.88 - 5.99 ) and 0.48 ( 0.19 - 1.06 ) respectively.The serum concentration of HGF in the normal subjects,chronic hepatitis,cirrhosis and HCC patients were (0.31 ± 0.05 ),(0.65 ± 0.07 ),( 1.27 ± 0.30 ) and ( 2.06 ± 0.66) μg/L respectively.The highest level of HGF mRNA was found in N3,while the HGF mRNA expression in HCC was [ (2.14 ± 0.52 ) μg/L] lower than that not only in the non-tumor tissues,but also in the normal control ( U =196.50,P =0.03 ).The serum concentration of HGF was significantly higher in patients with chronic hepatitis,cirrhosis or HCC than in normal subjects.The serum HGF level of HCC was bounced after hepatectomy (t =2.70,P ＜0.01 ).On the logistic regression analysis,the tumor numbers and Child-pugh were related with the levels of the tissue HGF mRNA and serum HGF of HCC,OR were0.15 (95％CI:0.03-0.72,P＜0.05) and0.13 (95％CI:0.27 -0.89,P ＜0.05 ),respectively.Univariate analysis using the Cox proportional hazards model in the complication groups revealed that the levels of the tissue HGF mRNA and serum HGF were significant risk factors of death for HCC,OR were 0.02 (95％ CI:0.00 - 0.52,P ＜ 0.05 ) and 10.01 (95％ CI:1.16 -86.23,P ＜ 0.05 ),respectively.On the Log-rank analysis,no statistically difference in the cumulative survival was found between the two groups categorized by median (0.49) of tissue HGF mRNA 2 - AACT (X2 =0.13,P =0.72).While the HCC patients were dichotomized by their the median(0.69 μg/L) of serum HGF concentration,the death risk for the patients with higher levels of HGF was increased 2.84 fold than those with lower levels (95％ CI:1.03 - 7.92,P ＜ 0.05 ).ConclusionHGF mRNA expression is decreased in tumor tissues,while its level in tumor adjacent live and serum is significantly elevated and is in association with shortened postoperative survival of HCC patients.

Objective To investigate the influence of marrow-derived mesenchymal stem cells(MSCs) on intercelluar adhension molecule-1(ICAM-1) in lung of neonatal rats suffered hyperoxia.Methods Marrow-derived MSCs were separated,cultured,amplificated and labeled with 5bromo 2′-deoxy-uridinel(BrdU);after suffered 95% oxygen for 7 days,24 three-day-old SD rats were randomly divided into group A,B and C,and they were injected intraperitoneally with MSCs of 1?10~4,5?10~4 PBS,respectively.Seven days later,immunocytochemisty was used to determine the expression of BrdU and ICAM-1,and value of radical alveolar counts(RAC) of lungs were counted for histopathological study under light microscope.Results Both group A and B,the labeled MSCs had been(detec)-ted in lungs,and there existed significant variance between two groups(P

The type of basic media and the contents of plant growth substances were investigated by orthogonal design experiment,and also the effects of different culture conditions on the growth of suspension cells and the accumulation of total flavonoids in Eucommia ulmoides were studied.The results showed that B5 medium supplemented with 0.5mg/L NAA,0.6mg/L 6-BA and 30g/L sucrose,at initial pH 5.0～5.5,20g(FW)/L inoculation quantity and 110 r/min of rotation speed was a preferable culture conditions for E.ulmoides suspension cells growth and flavonoids synthesis.The results of metabolic kinetics analysis for E.ulmoides cell suspension culture showed that the logistic and Luedeking-Piret equations can be used for describing the kinetics of cell growth,sucrose consumption and flavonoids production during the process.The maximum specific growth rate(?m),the actual growth yield based on sucrose(YG) and maintenance coefficient(m) were 0.417/d,0.619g/g and 0.0206g/(g?d-1) respectively.All these outcomes could give a basis for establishing the suspension cell culture of E.ulmoides and production of the natural active components in large-scale.

Objective:This study aimed to detect the special methylation profile in peripheral blood for hepatocellular carcinoma (HCC). Methods:The methylation status of 12 tumor suppressor genes (TSGs) in the plasma of 55 HCCs and 54 chronic liver diseases (CLDs) was tested by methylation-specific PCR (MSP). Results:In HCC, the methylation frequencies were 78.18%in APC, 63.64%in cyclin D2, 58.18% in TFPI2, 49.09% in DKK3, 49.09% in GSTP1, 47.27% in p16, 40.00% in Sigma 14-3-3, 18.18% in SFRP2, 16.36% in ppENK, 9.09% in DKK2, 7.27% in NPTX2, and 5.45% in LHX1. In CLD, the methylation frequencies were 27.78% in APC, 22.22%in cyclin D2, 7.41%in TFPI2, 3.70%in DKK3, 16.67%in GSTP1, 37.04%in p16, 37.04%in Sigma 14-3-3, 11.11%in SFRP2, 20.37%in ppENK, 7.41%in DKK2, 7.41%in NPTX2, and 9.26%in LHX1. The methylation frequencies of APC, cyclin D2, TFPI2, DKK3, and GSTP1 were higher in HCC than in CLD (P<0.01). The methylation index (MI) of the five-gene methylation profile was statistically higher in HCC (median, 0.6;IQR, 0.4-0.8) than CLD (median, 0.2;IQR, 0-0.2) (P<0.01). In HCC, MI was statistically related to the patient's age. Older patients with HCC had a higher MI. No significant correlation was observed between MI and other clinicopathological data. Moreover, MI was not related to the disease free survival and the overall survival in HCC. Conclusion:This five-gene methylation profile may be a promising biomarker for the assistant diagnosis of HCC.

The chemical structures of P1 A was identified by complete acid hydrolysis, partial acid hydrolysis, periodate oxidation-Smith degradation, methylation analysis, IR and NMR. The results showed that P1 A had a backbone consisting rhamnose, mannose, glucose and galactose. The side chain possessed arabinose and xylose. 1-->, 1-->6 and non-reducing terminal linkages existed in polysaccharide P1A, but there are doubling amount of 1-->2 and 1-->4 linkages. Oxidable linkage of P1 A accounted for 45%, and inoxidable linkage of P1A accounted for 55%. Mannose, glucose and galactose were mainly linked by 1-->2 linkage. Rhamnose, arabinose and xylose were mainly linked by 1-->2 and 1-->4 linkages. PlA contained beta-Glc(1,6)-,beta-Gal(1,3)-,beta-Man(1,4)-beta-Rha,-Glc(1,4)-, Glc(1)-,-Gal(1,4)- and Man(1)-.
Acanthaceae ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Molecular Weight ; Polysaccharides ; chemistry

Objective To evaluate the relationship between myocardial energy expenditure(MEE) level and cardiac function in chronic heart failure (CHF) patients. Methods A total of 99 CHF patients were divided into 3 groups according to the LVEF ( HFNEF≥50% , n = 37; HFREF1 35. 1% -49.9% , n=30; HFREF2 ≤35% , n=32) or the New York Heart Association (NYHA Ⅱ, n=26;Ⅲ ,n=42; Ⅳ, n =31) criteria. Thirty patients with cardiovascular disease and without CHF served as controls. Routine examinations including serum CRP (ELISA) and plasma NT-proBNP (chemiluminescence sandwich ELJSA) were made on the next morning after admission; echocardiography was performed on the third day after admission. LVMW, LVMWI, RWT, LVIDd, LA, LV, LVEF, LVFS, E/A, EDT, IVRT, Tei index and MEE were measured or calculated. Results MEE was significantly higher in HFREF patients than in controls (P < 0.01) and similar between HFNEF patients and controls (P > 0.05). MEE increased in proportion to decrease of LVEF and increase of NYHA grades in CHF patients (all P < 0.05 ) . Bivariate analysis confirmed that MEE was significant correlated with LVMW, LVMWI, RWT, LVIDd, LA, LV, LVEF (r=- 0.540, P<0.01), LVFS (r= -0.454, P<0.01), E/A, EDT, IVRT, Tei index, NYHA grades, CRP and NT-proBNP. Conclusion MEE derived from standard echocardiographic measurements is an effective indicator for myocardial bioenergetics and significantly correlated with cardiac function in CHF patients, especially in CHF patients with reduced LVEF.

To identify acupuncture resources in six databases of Cochrane Library (CL) with computer retrieve. Seventy-two literatures were identified in Cochrane Database of Systematic Reviews (CDSR). Among them, 12 Cochrane systematic review (CSR) verified the effectiveness of acupuncture, 29 concerning the indeterminacy of the efficacy of acupuncture with 1 didn't support acupuncture for epilepsy and 31 remained as protocols; 121 literatures were found in Database of Abstracts of Reviews of Effects (DARE) with more types of diseases or symptoms and rich modality comparing to CSR; 4218 randomized controlled trials and clinical controlled trials were identified in Cochrane Central Register of Controlled Trials (CCRCT); 43 literatures in Cochrane Methodology Register Database (CMRD) which focused on blindness study, quality assessment of methodology of research and publication bias and so on; 25 literatures in Health Technology Assessment Database (HTAD) and 18 in NHS Economic Evaluation Database (NHS EED) which were centered on acupuncture analgesia. Consequently, acupuncture literatures in 6 databases of CL do provide good resources for acupuncture researchers due to its abundant content, concrete classification and high quality evidence.
Acupuncture Therapy ; Databases, Factual ; Health Resources ; Humans ; Libraries

OBJECTIVETo investigate the effect of intravenous infusion of rat bone marrow-derived mesenchymal stem cells (MSCs) against lung injuries in neonatal exposed to hyperoxia.

METHODSRat bone marrow-derived MSCs were separated, cultured, amplified, and labeled with BrdU. Thirty-two 3-day-old SD rats were randomized into 4 equal groups (groups A, B, C and D), and the rats in groups A and B were exposed to 7-day 95% oxygen, while those in groups C and D were not. In groups A and C, the rats received injection with 5x10(4) MSCs via the tail vein, and those in groups B and D were given PBS only. Seventy-two hours after housing in normal air, all the rats were killed to determine the radial alveolar count (RAC) under light microscope. Immunohistochemistry was used to detect BrdU expression in the lung tissue, where the levels of tumor necrosis factoralpha(TNFalpha) and transforming growth factor beta1 (TGFbeta1) were detected using enzyme-linked immunosorbent assay.

RESULTSCompared to air exposure groups, the levels of TNFalpha and TGFbeta1 in the homogenate of the lungs increased while RACs decreased significantly in the two hyperoxia exposure groups. Groups A and B showed significant differences in the fields of RACs and the levels of TNFalpha and TGFbeta1 in the lung tissue homogenate, and BrdU-positive cells were detected only in the lungs of groups A and C, between which a significant quantitative difference was seen.

CONCLUSIONIntravenously injected MSCs may reside in the lungs of neonatal rats, which is subject to influences by the exposure conditions, and the transplanted MSCs may offer effective protection against lung injuries induced by hyperoxia.

Animals ; Animals, Newborn ; Bone Marrow Cells ; cytology ; Hyperoxia ; pathology ; Infusions, Intravenous ; Lung Injury ; prevention & control ; Mesenchymal Stem Cell Transplantation ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism