To reverse drug resistance mediated by the MDR1 gene product P-glycoprotein(P-gp) in tumor cells in a specific manner, a hammerhead ribozymes which can cleave the GUC sequence in codon 196 of MDR1 mRNA was designed and cloned into a recombinant retroviral vector pDOR-neo at BamH I restriction sites and packaged with packaging cell line PA317 cells.The viral supernatant was used to infect the multidrug-resistant human hepatocarcinoma cell line BEL-7402/DOX. After selection with G418,resistant colonies were obtained.Stable expression of retroviruses in both PA317 and infected BEL-7402/DOX cells was confirmed by Northern Blot hybridization. Down-regulation of P-gp and even of MDR1 mRNA was found in BEL-7402/DOX infected with ribozyme construct. The rate of BEL - 7402/DOX infected cell defected by flow cytometric analysis was 8.2 ~ 14.6% while in uninfected cell it was 93 .4 ~ 97.5% . The BEL-7402/ DOX cell infected with ribozyme construct was found back to drug-sensitivity to a series of drugs by MTT colorimetric assay . The results demonstrated that the recombinant retroviral vector expressing ribozyme transfecting human hepatocarcinoma BEL-7402/DOX could inhibit MDR1 gene expression and reverse tumor MDR phenotype back to drug-sensitive condition.

Objective To investigate the relationship between fasting plasma glucose (FPG) and islet α-cell and β-cell function in patients with type 2 diabetes mellitus (T2DM).Methods Four hundred and thirty-seven patients with T2DM were divided into 3 groups according to the level of FPG:F1 group:FPG ≤ 6 mmol/L (73 cases),F2 group:6 mmol/L ＜ FPG ≤ 7 mmol/L (103 cases),and F3 group:FPG ＞ 7mmol/L (261 cases),and 30 cases of healthy people were selected as control group.Oral glucose tolerance test,insulin releasing test and glucagon releasing test were performed to observe the differences of glucagon,glucagon/ insulin,the ratio of 30 min insulin and blood glucose value after glucose load (△ I30/△ G30),and the area under curve of insulin (AUC1) among the 4 groups and the correlation analysis was performed between glucagon and other indicators.Results Glycosylated hemoglobin (HbA1c),plasma glucose 120 at min after glucose load in F1,F2 and F3 group were significantly higher than those in control group,and there were statistical differences (P ＜0.05).In F1,F2,F3 group,with the increase of the HbA1c,the course of disease and plasma glucose at 120 min after glucose load showed increasing trend.The triglyceride in F2 group and F3 group was significantly higher than that in F1 group and control group,and low density lipoprotein cholesterol in F3 group was significantly higher than that in F1 group,F2 group and control group,and there were statistical differences (P ＜ 0.05).The glucagon at 60,120 min after glucose load in F1 group,30,60,120 min after glucose load in F2 group,and 30,60,120,180 min after glucose load in F3 group was significantly higher than that in control group,and there were statistical differences (P ＜ 0.05).The glucagon at 60,120,180 min after glucose load in F2 group,at fasting and 30,60,120,180 rain after glucose load in F3 group was significantly higher than that in F1 group,and there were statistical differences (P ＜ 0.05).The glucagon at fasting and 30,60,120,180 min after glucose load in F3 group was significantly higher than that in F2 group,and there were statistical differences (P ＜ 0.05).The area under curve of glucagon in control group was 9.5 ±0.3,in F1 group was 9.7 ± 0.2,in F2 group was 9.9 ± 0.2,in F3 group was 10.2 ± 0.3,and there were statistical differences among the 4 groups (P ＜ 0.05).The glucagon/insulin at fasting and 30,60 min after glucose load in F1 groups,fasting and 30,60,120 min after glucose load in F2 group,fasting and 30,60,120 min after glucose load in F3 group was significantly higher than that in control group,and there were statistical differences (P＜ 0.05).The glucagon/insulin at fasting and 60,120 min after glucose load in F2 group,fasting and 30,60,120,180 min after glucose load in F3 group was significantly higher than that in F1 group,and there were statistical differences (P ＜ 0.05).The glucagon/insulin 30,60,120,180 min after glucose load in F3 group was significantly higher than that in F2 group,and there were statistical differences (P＜ 0.05).The homeostasis model of assessment for insulin resistance index (HOMA-IR) in F2 group and F3 group was significantly higher than that in control group and F1 group,in F3 group was significantly higher than that in F2 group,and there were statistical differences (P＜ 0.05).The insulin sensitivity index (ISI) in F2 group and F3 group was significantly lower than that in control group and F1 group,in F3 group was significantly lower than that in F2 group,and there were statistical differences (P ＜ 0.05).The homeostasis model of assessment for islet β-cell function index (HOMA-β) and △I30/△G30 in F1,F2,F3 group were significantly lower than those in control group,and there were statistical differences (P ＜ 0.05).The AUC1 in F2 group was significantly lower than that in control group,and AUC1 in F3 group was significantly lower than that in control group,F1 group and F2 group,there were statistical differences (P ＜0.05).The results of Pearson correlation analysis showed there was negative correlation between glucagon and △I30/△G30,HOMA-β,body mass index,ISI,AUC1 (r =-0.229,-0.153,-0.151,-0.146,-0.136,P＜0.01 or ＜0.05),and there was positive correlation between glucagon and FPG,area under curve of glucose (AUCG),HbA1c,course of disease and HOMA-IR (r =0.545,0.476,0.273,0.193,0.189,P ＜ 0.01).The results of multiplestepwise regression analysis showed there was positive correlation between glucagon and FPG,AUCG,HbA1c,course of disease (P ＜0.01 or ＜0.05),and there was negative correlation between glucagon and △I30/△ G30 (P ＜ 0.05).Conclusions Islet β-cell function is decreased with the increasing of FPG,while islet α-cell function is increased,especially in those with higher levels of FPG.Regulation of glucagon should be concerned to make the blood glucose target easier to reach,at the same time of protecting β-cell function.

Background and purpose: Reports showed that berbefine not only had an effect ofdetoxification and inflammation but also could induce the cell to apoptosis. Berberine may also inhibit the migration and metastasis of tumor. To investigate the effects and mechanism of berberine on migration and metastasis of A549 cells, the proliferation, mobility and adhesion of A549 cells were observed after incubation with berberine. Methods: The proliferation was determined by MTT. Wound healing assay and adhesion test were used to observe the mobility and adhesion rate. The mRNA and protein expressions of MMP2 and MMP9 were assessed by semi-quantitive RT-PCR and gelatin zymography, respectively. Results: Berberine could significantly inhibit the proliferation ofA549 within a certain range of treating time and dose (P<0.05). The mRNA and protein expression of MMP2 and MMP9 gene were decreased slightly by the treatment ofberberine (P<0.05), especially at dose of 100 μg/ml. Conclusion: Berberine can inhibit the migration and adhesion abilities of A549 cells, probably by down-regulating the expression and activation of MMP2 and MMP9.

Objective To investigate the association of single nucleotide polymorphism (SNP) rs13333226 in uromodulin (UMOD) gene with diabetic kidney diseases (DKD) in Han population in Tianjin,China.Methods A total of 210 type 2 diabetes (T2DM),90 normal controls (NC) and 280 DKD patients were recruited.According to the level of estimated glomerular filtration rate (eGFR),the DKD subjects were further subdivided into three groups:GFR≥90 ml/min group (n=105),60 ml/mim≤GFR ＜ 90 ml/min group (n=84) and GFR ＜ 60 ml/min group (n=91).Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for UMOD rs13333226C genotyping.Results The frequencies of AA,GA,GG genotype were 27.8％,58.9％,13.3％ in NC group and 41.0％,48.6％,10.5％ in T2DM group and 54.3％,36.1％,9.6％ in DKD group.The frequency of G allele was 42.8％ in NC group,34.8％ in T2DM group and 27.7％ in DKD group.The genotype distribution of UMOD was statistically significant between NC group and DKD group,and between T2DM group and DKD group (P ＜ 0.05).G allele of UMOD was an independent protective gene polymorphism of DKD in Logistic regression (B=-0.248,Wald=8.012,P=0.021,OR=0.780,95％ CI 0.612-0.968).Conclusion The G allele of UMOD gene may be an independent protective factor of DKD in Han population in Tianjin,China.

Objective To explore the application effect of sandwich teaching method in clini-cal teaching in the department of orthopedics. Methods Totally 150 clinical medicine students were divided into 2 groups: sandwich teaching group (n=75) and lecture-based learning group (n=75). Sandwich teaching method combined with bedside teaching method was used in sandwich teaching group while lecturing combined with bedside teaching method was used in control group. Theoretical examination, clinical operation skill test, clinical case analysis and questionnaire investigation were conducted after the course. Comparison between the two groups was made using independent sample t test and measurement data were expressed as x±s. P<0.05 signifies statistically significant differences. Results Theoretical examination score, clinical skill test score and clinical case analysis score were higher in sandwich teaching group than in control group. [(53.70±4.27) vs. (48.00±4.83);(15.70± 2.49) vs. (11.40±2.87);(17.10±1.52) vs. (13.80±1.32)]. Questionnaire showed that commutation and expression ability, cooperation ability and self thinking ability were better in sandwich teaching group than in control group , with statistical differences . Conclusions Sandwich teaching method achieves good teaching effect and it is worth recommending in clinical teaching.

Objective To evaluate the functions of pancreatic islet α-cells and β-cells in different disease courses of type 2 diabetes mellitus.Methods Two hundred and eighty three patients with type 2 diabetes mellitus were divided into 4 groups according to their disease courses:group A (course of disease ≤1 years),group B (1 years ＜ course ≤ 5 years),group C (5 years ＜ course ≤ 10 years) and group D (course ＞ 10 years).Oral glucose tolerance test (OGTT),insulin releasing test and glucagon releasing test were performed to observe the differences of glucagon,glucagon/insulin,ratio of insulin increment/glucose increment 30 min after glucose-load (△I30/△G30),area under curve (AUC) of insulin in receiver operational characteristic (ROC) curve of insulin (AUCI) and glucagon among 4 groups and the correlation analysis was performed between glucagon and other indicators.Results (1) Glucagon,glucagon/insulin and AUC of glucagon increased significantly with the prolonged course of disease (P ＜0.05),0、30、60、120、180 min of group A were (71 ± 20)、(106 ± 36)、(143 ± 54)、(133 ± 68) 和 (87 ± 55) ng/L respectively,glucagon increased significantly with the prolonged course of disease,0、30、60、120、180 min of group D (80 ±19)、(125 ± 36)、(167 ± 47)、(178 ± 64)、(129 ± 65) ng/L respectively.(2) There were no significant differences in homeostasis nodel assessment for insulin resistance index (HOMA-IR) and insulin sensitive index (ISI) among 4 groups (P ＞0.05); compared to group A,HOMA of β-cell function (HOMA-β),△I30/△G30,AUCI in groups B,C and D were significantly lower (F =3.75,3.77 and 3.07 respectively,all P ＜ 0.05).(3) Multiple stepwise regression analysis showed that glucagon was positively correlated with FPG and AUC of glucose (AUCG) (t =6.23 and 3.41,all P ＜ 0.05),and negatively correlated with AUCI/AUCG (t =-2.13,P ＜ 0.05).Conclusions In order to reach the blood glucose control target,in the early stage of diabetes attentions should be given to regulation of glucagon while protect the β-cell function.

Objective To observe the effect of Rhy-SLN on the proliferation of rat vascular smooth muscle cells (VSMC) induced by TGF-β1, and explore the mechanism. Methods The primary culture of rat thoracic aortic vascular smooth muscle cells was studied by tissue block culture method. The cells were divided into the control group, TGF-β1 group, TGF-β1+ the high, medium and low dosage groups of Rhy-SLN. In addition to the control group, the cells of the other groups were involved in the intervention of TGF-β1 of 20 g/L, and the high, medium and low dosage groups of Rhy-SLN cells were involved in the intervention of 25, 50, 100 mg/L of the hook teng solid lipid nanoparticles. After 24 hours of culture, MTT assay was used to detect cell proliferation inhibition rate in each group, and the cell cycle was detected by flow cytometry. The expression of c-myc and c-Fos protein in each group was detected by Western blot method. Results Compared with the TGF-β1 group, the absorbance value (0.457 ± 0.046 vs. 0.975 ± 0.049) of TGF-β1+ rhy-sln high dose group significantly decreased (P<0.01); the number of S phase cells (15.87% ± 2.47%, 15.23% ± 1.69%, 17.02% ± 2.87% vs.38.58% ± 2.68%)of TGF-β1+rhy-sln in each dose group significantly decreased(P<0.01);The c-myc(48.65 ±3.65,50.69 ± 4.16,55.29 ± 3.67 vs.68.21 ± 3.25)and c-Fos(38.78 ± 4.25,43.56 ± 3.69,46.58 ± 3.57 vs.66.54 ± 4.09) of the TGF-β1+ rhy-sln each dose group significantly decreased (P<0.01). Conclusions The Rhy-SLN can inhibit the proliferation of VSMC in rats induced by TGF-β1.Its mechanism is related to the conversion of G0/G1 phase to the S phase and the expression of the reduction of c-myc and c-fos protein.

[Summary] A total of 128 individuals with type 2 diabetes underwent continuous glucose monitoring for 3 consecutive days.The dawn phenomenon was defined by three different parameters according to the previous research:(1)the absolute increase of glucose level from nocturnal nadir to prebreakfast value(?G) above 20 mg/dl;(2)?G above 10 mg/dl;( 3 ) insulin requirement increased at least 20%.The participants were secondarily separated by presence/absence of a dawn phenomenon based on the definitions above.The impact on blood glucose fluctuation of different groups was assessed according to the standard deviation of blood glucose( SDBG) , the area under curve above 10 mmol/L ( AUC ) , and the mean amplitude of glycemic excursions ( MAGE ) , etc.The frequencies of dawn phenomenon were 64.8%(?G≥20mg/dl), 85.2%(?G≥10 mg/dl), and 59.4%(rise in insulin requirement≥20%)respectively.The impacts on SDBG, AUC, MAGE, and MODD were without statistical difference(P>0.05) between the presence and absence of the dawn phenomenon patients when?G≥10 mg/dl.However, the differences reached statistical significance(P<0.05) when ?G≥20 mg/dl and the increase in insulin requirement≥20%. Besides, the incidence of dawn phenomenon was positively correlated with HOMA-IR, HbA1C , and free C-peptide.Dawn phenomenon is a very frequent event in type 2 diabetes and not only impacts the overall glycemic control but also exaggerates glucose fluctuation.To be clinically relevant, ?G≥20mg/dl should be taken as the quantitative criterion of the dawn phenomenon.

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.
Crystallography, X-Ray ; Ebolavirus ; physiology ; Humans ; Nucleoproteins ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship ; Virus Assembly ; physiology

Objective To understand the status and correlative factors on menarche and first spermatorrhea among children and adolescents,in Chongqing,in order to provide theoretical basis for carrying out sex and health education in this population.Methods By random stratified and cluster sampling,10 498 students (5 372 boys and 5 126 girls),5 to 18 years old and living in Chongqing urban districts,were enrolled.General situation and physical features of the population were studied.Statistics analysis system included logistic regression methods,t-test and chi-square test.Results For urban kids,first experience of spermatorrhea was 0.218 years later than those living in the rural areas (Z=-73.287,P＜0.001),but median age for girls in urban areas was 0.073 years earlier than in rural areas (Z=-71.589,P＜0.001).Except for factor as mother' s education level in the family (x2=21.564,P＜0.001),other family or environment related factors did not show significant difference between the two groups of boys (P＞0.05).However,significant difference appeared in average family income (x2=6.175,P=0.046) between two groups of girls.Data from the logistic analysis showed that BMI,hip circumference,height,weight,number of children in the family,time of sleep and the diet structure were associated with menarche.Correlative factors of boys' first spermatorrhea would include:high-energy snacks,hip circumference,weight,height,school type and mother's education level (P＜ 0.05).Conclusion First spermatorrhea and menstruation of boys and girls were closely related to environment of the family,diet and the time of sleep.