According to bias in codon choice of Pichia pastoris, The phytase phyA gene from Aspergillus niger N25 was mutated without changing its amino acid sequence. The expression plasmid pPIC9k-phyAm was constructed and transformed into GS115 strain. Positive clones,of which the chromosomes were integrated with phyA gene,were identified by the phenotype and PCR. SDS-PAGE analysis suggust that the size of enzyme protein of the expression product was about 70.15kDa.Southern blotting analysis to the yeast transformants showed that phyA gene was intergrated into the chromosome genome. The phytase activity of PP-NP m-4-4 with codons optimized reached 136 000U/ml in malt wort culture medium after being induced with 36h, which was the 2.8 times of the original strain PP-NPm-8.

Objective To establish and verify the prediction model for ischemic cardiovascular disease(ICVD)among the elderly population who were under the current health care programs. Methods Statistical analysis on data from physical examination, hospitalization of the past years, from questionnaire and telephone interview was carried out in May, 2003. Data was from was randomly selected to generate both module group and verification group. Baseline data was induced to make the verification group into regression model of module group and to generate the predictive value. Distinguished ability with area under ROC curve and the predictive veracity were verified through comparing the predictive incidence rate and actual incidence rate of every deciles group by Hosmer- Lemeshow test. Predictive veracity of the prediction model at population level was verified through comparing the predictive 6-year incidence rates of ICVD with actual 6-year accumulative incidence rates of ICVD with error rate calculated. Results The samples included 2271 males over the age of 65 with 1817 people for modeling population and 454 for verified population.All of the samples were stratified into two layers to establish hierarchical Cox proportional hazard regression model, including one advanced age group(greater than or equal to 75 years old), and another elderly group(less than 75 years old). Data from the statically analysis showed that the risk factors in aged group were age, systolic blood pressure, serum creatinine level, fasting blood glucose level, while protective factor was high density lipoprotein; in advanced age group, the risk factors were body weight index, systolic blood pressure, serum total cholesterol level, serum creatinine level, fasting blood glucose level, while protective factor was HDL-C. The area under the ROC curve (AUC)and 95%CI were 0.723 and 0.687-0.759 respectively. Discriminating power was good. All individual predictive ICVD cumulative incidence and actual incidence were analyzed using Hosmer-Lemshow test, x 2=1.43, P=0.786, showing that the predictive veracity was good.Conclusion The stratified Cox Hazards Regression model was used to establish prediction model of the aged male population under a certain health care program. The common prediction factor of the two age groups were: systolic blood pressure, serum creatinine level, asting blood glucose level and HDL-C. Thc area under the ROWC curve of the verification group was 0.723, showing that the distinguished ability was good and the predict ability at the individual level and at the group level were also satisfactory. It was feasible to using Cox Proportional Hazards Regression Model for predicting the population groups.

Objective To investigate the 6-year accumulative incidence rate of ischemic cardiovascular disease (ICVD) and its related risk factors. Methods Baseline population was all from an elderly population with members all born before 1-1,1938 and under health care programm.Data was recorded in a database of a hospital and baseline of subjects under research was aged ＞65years. People that had had ICVD at baseline were excluded. Risk factors under research would include: age at the baseline study, gender, body mass index, systolic blood pressure, serum cholesterol level, serum triglyceride level, serum high-density lipoprotein level, serum triglyceride level, serum apolipoprotein A1 level, history of diabetes mellitus and cigarette smoking etc. Single factor analysis was carried out using person-years as time of study, then calculating the person-year incidence and the accumulative incidence rate at different levels related to baseline risk factors.Multivariate analysis was under Cox Proportional Hazards Regression Model. Results Baseline population included 2271 elderly men, with the 6-year cumulative prevalence rate of ICVD as 23.56%, and the person-year prevalence rate was 45.41‰. Single factor analysis showed that the ICVD events positively related to variables which including systolic blood pressure, body mass index,serum cholesterol level and serum triglyceride level, serum apolipoprotein A1 level, status of diabetes mellitus and cigarette smoking. The ICVD events negatively related to variables as serum high-density lipoprotein levcl and serum creatinine level. Results of multivariate analysis showed that high systolic pressure, diabetes mellitus, serum creatinine level, Body Mass Index were the main risk factors. The serum high-density lipoprotein level served as the main protective factor. Conclusion The aged male population under health care programme was high in the ICVD prevalence rate, suggesting that programmes as controlling blood pressure, blood glucose, and increasing serum high-density lipoprotein levels were effective measures to decrease the ICVD events.

OBJECTIVETo investigate polymorphism of HLA-DRB1 in Chinese Lahu population in Yunnan.

METHODSPolymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-DRB1 genotypes of 110 unrelated healthy Lahu minority people of Yunnan Province.

RESULTSSixteen alleles of DRB1 were detected in this study. The results of test showed that the genotype distributions observed were corresponded with the Hardy-Weinberg equilibrium.

CONCLUSIONThis study has obtained a more comprehensive and accurate data set of the normal allele frequencies of HLA-DRB1 in Chinese Lahu population in Yunnan Province, which may be of significance in the studies on population genetics and disease association.

Asian Continental Ancestry Group ; DNA Primers ; Ethnic Groups ; Female ; Gene Frequency ; Genotype ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Male ; Polymerase Chain Reaction ; methods

The relationship of HLA-A, -Cw alleles on HIV infection and AIDS disease progression in the Chinese Yi ethnic group of Sichuan province were investigated. The genetic polymorphisms of HLA-A, -Cw alleles of 102 unrelated healthy Chinese Yi ethnic individuals, 68 HIV-1 infected and 21 HIV positive long-time survivors were typed by PCR-SSP assay. Statistic signifiance was determined by the χ2 test with the SPSS software. No significant differences were observed between the HLA-A, -Cw alleles of the 68 HIV-1 infected and 102 non-infected Chinese Yi control individuals. Whereas the prevalence of A*3601,Cw*14(01-03)and Cw*0304 was significantly higher in 21 long time survivors compared with 102 healthy controls with P values of 0.016, 0.016 and 0.000 by χ2 or the Fisher exact test respectively. The result implies that A*3601,Cw*14(01-03) and Cw*0304 may be associated with slow AIDS disease progression in the Chinese Yi ethnic group, further studies on this association may yield insight on the pathogenesis of HIV-1 infection.

OBJECTIVETo investigate the clinicopathological and molecular genetic characteristics of gastrointestinal stromal tumor (GISTs) with significant cystic changes, and to assess their biological behavior.

METHODSClinicopathological features of 7 patients with cystic GISTs treated at the Zhongshan Hospital of Fudan University from February 2005 to January 2010 were summarized retrospectively. The mutations status of c-kit and PDGFR-α were analyzed.

RESULTSThere were 2 males and 5 females aged from 46 to 76 years old. Primary site of GISTs included stomach(n=4), duodenum(n=1), and small intestinal(n=2). Tumor size ranged from 6 to 16 cm with obviously cystic changes. Tumor cells were found in the solid components under microscope, of which epithelioid cell type were found in 4 case and spindle cell type in 3 cases. The mitotic figures were no more than 3/50 HPF in all the patients. According to the NIH criteria, 4 were high-risk and 3 were low-risk. Based on morphological characteristics, 3 cases were as borderline tumor, 3 moderate-risk, and 1 moderate-risk. Gene mutation of exon 11 of c-kit were identified in 3 cases. During the follow up ranging from 9 to 80 months, all the 7 patients had cancer-free survival.

CONCLUSIONThe biological behavior of cystic GIST is indolent with a low risk of malignancy and favorable prognosis.

Aged ; Female ; Gastrointestinal Stromal Tumors ; diagnosis ; pathology ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies

OBJECTIVETo investigate the interrelationship of genetic polymorphisms in folate metabolic enzymes (MTHFRC677T, MTHFRA1298C, MTRA2756G and MTRRA66G) and their combinative effects with colorectal cancer (CRC).

METHODSA nested case-control study was designed and carried out. 140 CRC patients and 343 control subjects were included in this study. Polymorphisms of folate metabolic enzyme genes were genotyped by PCR-restriction fragment length polymorphism method. Risk of CRC was estimated by unconditional logistic model, and P value for interaction was calculated by likelihood test.

RESULTSThe allele of MTR2756G showed a positive association with CRC (OR = 2.04, 95% CI = 1.22 - 3.40). Those with MTHFR1298AA and MTR 2756AG/GG genotypes had an elevated risk with CRC (OR = 2.57, 95% CI, 1.42 -4.65), and their combinative effect showed a significant association with CRC (P = 0.04).

CONCLUSIONMTR2756G allele may be a risk factor of CRC, and interaction may exsit between polymorphisms of MTHFRA1298C and MTRA2756G. Further studies with larger sample and in different ethnic groups are needed.

5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase ; genetics ; Alleles ; Case-Control Studies ; Colorectal Neoplasms ; enzymology ; genetics ; Female ; Ferredoxin-NADP Reductase ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Middle Aged ; Odds Ratio ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length

OBJECTIVETo explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer.

METHODSRecombinant adenovirus rAd-hTERT and rAd-TRF2 expressing siRNA-hTERT and siRNA-TRF2 was constructed, and the vectors were transfected into MCF-7 cells. Than the expressions of hTERT and TRF2 proteins were detected by Western blot, the inhibition of MCF-7 cell proliferation by MTT colorimetry, and the changes of MCF-7 cell cycle by flow cytometry and the colony forming ability of MCF-7 cells by clone form test.

RESULTSAt 48 h after transfection, the relative expression amounts of hTERT protein of the PBS control group, rAd-blank group, rAd-HK control group, rAd-hTERT group, rAd-TRF2 group and rAd-hTERT and rAd-TRF2 group were 1.00, 0.94 +/- 0.02, 0.95 +/- 0.04, 0.18 +/- 0.04, 0.95 +/- 0.01 and 0.18 +/- 0.04, respectively. The relative expression amounts of TRF2 protein were 1.00, 1.01 +/- 0.08, 0.96 +/- 0.02, 0.95 +/- 0.08, 0.22 +/- 0.01 and 0.26 +/- 0.02, respectively. After transfection of rAd-hTERT or rAd-TRF2 into MCF-7 cells separately, the inhibition rate of cell proliferation was only 54.6% and 48.4%, there was 8.9% +/- 1.2% or 9.2% +/- 2.3% of MCF-7 cells into M phase, 66.4% +/- 1.5% or 64.6% +/- 1.9% of MCF-7 cells was arrested at G(0)/G(1) phase, and the cell colony forming ability was decreased significantly (cell colony number from 100 in PBS control group down to 41.3 +/- 5.1 and 43.7 +/- 6.4). But after transfection by rAd-hTERT and rAd-TRF2 simultaneously, the inhibition rate of cell proliferation was about 82.1%, and M phase cells was significantly reduced to 4.4% +/- 1.2%. Large numbers of cells were arrested at G(0)/G(1) phase (81.4% +/- 1.3%), and the cell colony forming ability was more significantly decreased (cell colony number there were only 29.2 +/- 3.9).

CONCLUSIONMore effective effect of tumor gene therapy can be achieved by combination of interference hTERT and TRF2 genes as compared with interference by either of the single gene alone.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Telomerase ; genetics ; metabolism ; Telomeric Repeat Binding Protein 2 ; genetics ; metabolism ; Transfection ; Tumor Stem Cell Assay

Objective To explore the correlation of liver depression grading with estrogen receptor alpha and beta(ERα,ERβ)and G protein-couple receptor 30(GPR30)of the perimenopausal women suffering from non-organic insomnia. Methods A total of 127 perimenopausal women suffering from non-organic insomnia were enrolled into the study. The data collected by four diagnostic methods and the scores of Pittsburgh Sleep Quality Index(PSQI)were used for the scoring and grading of liver depression according to Syndrome Element Differentiation. The mRNA and protein expression levels of ERα,ERβand GPR30 in the mononuclear cells of peripheral blood from the patients were detected with real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB)method respectively. Results (1)The differences of mRNA levels of ERα,ERβ and GPR30 were insignificant among the patients with different degrees of liver depression (P > 0.05). ERα mRNA/ERβ mRNA ratio was decreased with the increase of liver depression grading,the differences being significant (F = 6.710, P < 0.001). (2) The differences of protein levels of ERα, ERβ and GPR30 were insignificant among the patients with different degrees of liver depression (P > 0.05);ERα/ERβ protein gray value ratio was decreased with the increase of liver depression grading, and the differences were significant (P < 0.05) . Conclusion The decline of ERα/ERβ is probably contributed to the pathological basis of liver depression in perimenopausal non-organic insomnia women.

Objective To compare the vision and visual quality after implantation of refractive rotationally asymmetric mutifocal IOL (SBL-3) and diffractive mutifocal IOL (Zeiss809) in cataract surgery.Methods Totally 80 patients (100 eyes) who underwent cataract surgery in our hospital from May 2016 to June 2017 were chosen in the research,followed by the implantation of SBL-3 in 39 patients (50 eyes,SBL group) and Zeiss809 in other 41 patients (50 eyes,Zeiss group).The gender,age,length of optic axis and corneal curvature between the both groups were not significantly different (all P ＞ 0.05) before surgery.Variables including far,middle and near uncorrected vision acuity,defocus curves,vision quality were observed 3 months after surgery.Objective scattering index (OSI),modulation transfer function cutoff frequency(MTFcutoff) and Strehl ratio (SR) were detected,and objective optical quality analysis system was conducted.Results Both groups has no ocular hypertension and complications after surgery.The far uncorrected vision acuity of both groups in 3 months after surgery showed significant statistic difference from preoperation (both P ＜ 0.01).There has no statistic difference between both groups in far and near uncorrected vision acuity 3 months after surgery (both P ＞0.05).SBL group showed better outcomes than that in Zeiss group in the middle uncorrected vision acuity 3 months after surgery (P =0.04).Defocus curves showed the better middle uncorrected vision acuity in SBL group than that in Zeiss group when the degree was-1.50 D (70 cm).The OSI,MTFcutoff and SR in both groups significantly improved after surgery when compared with before surgery (all P ＜0.05).And the OSI,MTFcutoff and SR 3 months after surgery had no statistic difference between the two groups (all P ＞ 0.05).Questionnaire results showed there were 3 patients in SBL group and 4 in Zeiss group complaining glare at night,and there was no statistic difference between the two groups (P ＞ 0.05).Conclusion Both refractive rotationally asymmetric mutifocal IOL and diffractive multifocal IOL shows satisfactory vision acuity after cataract surgery.Significant improvement of vision quality in both groups can be presented after IOL implantation.