Objective:To construct NBS1 microRNA expressing eukaryotic recombinants,and identify biological activity of recombinants in Hela cell after transfection.Methods:According to sequence of NBS1mRNA,the NBS1 pre-microRNA was designed and synthesized,then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cell line.To detect integrity of inset fragment through colony PCR and sequencing analysis.The biological activity of recombinants through identify interference efficiency of NBS1 microRNA recombinants by way of Real-Time PCR and Western blot were determined.Results:Sequences of inset fragment in four microRNA expressing recombinants were correct.NBS1 mRNA and protein expression of four microRNA recombinants were decrease,which is the lowest in the NBS1mi-2 group.Conclusion:Four NBS1-targeting microRNA expressing recombinants all have biological activity in Hela cell line,and NBS1mi-2 recombinant has the most interference efficiency.The microRNA expressing plasmid which were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.

Objective To investigate expression of glutathione S-transferase(GST) mRNA in peripheral blood of the population in coal-burning fluorosis area and to evaluate the effect of comprehensive control intervention. Methods Fifty samples of peripheral blood from patients in the coal-buring fluorosis area in Bijie county of Guizhou province were selected as fluorasis group and 50 samples of peripheral blood from patients in area with comprehensive management were selected as intervention group, respectively. Fifty samples from non-endemic fluorosis area were selected as the control group. Total RNA from blood was extracted and purified by the Trizol- Phenol-Chloroform one-step method. Expression of GST mRNA was detected by using SYBR Green I real-time fluorescence quantitative PCR. Results The data of GST mRNA in fluorosis group, intervention group and control group was 38.28±27.22,70.56±37.23 and 103.46 ± 46.62, respectively. There was a significant difference between the groups(F = 3.75, P < 0.05). Decreased expression of GST mRNA in fluorosis group and intervention group as compare to control was detected(all P < 0.05), and the expression of GST mRNA in intervention group was higher than that in fluorosis group(P < 0.05). Conclusion Coal-burning fluorosis possibly led to the decreased expression of GST mRNA in peripheral blood, and comprehensive control maybe prevent the decreased expression of GST in mRNA level.

OBJECTIVETo study the effects and mechanism of apigenin on the expression of vascular endothelial growth factor (VEGF) in human breast cancer MDA-MB-231 cells.

METHODSMDA-MB-231 cells were used as the study object. MTT assay was used to detect the effect of apigenin on the cell viability. ELISA was used to determine the protein level of VEGF. RT-PCR was used to detect VEGF at mRNA level. A double luciferase system was used to measure the transcription activity of VEGF. pCEP4-HIF-1alpha was transferred to explore the reversing effect of HIF-1alpha on the inhibitory effect of apigenin on the transcription activity of VEGF. Western blotting was used to detect the time-dependent and dose-dependent effect of apigenin on HIF-lalpha, p-AKT, p-ERK, and p53 expression at protein level.

RESULTSApigenin had no visible inhibitory effect on cell viability. Apigenin reduced the secretion of VEGF, mRNA levels of VEGF and transcription activity of VEGF. Furthermore, the inhibitory effect of apigenin on the transcription activity of VEGF could be reversed by transferring pCEP4-HIF-1alpha into the cells. Additionally, apigenin inhibited the expression of HIF-1alpha and p-AKT, induced the expression of p53, but had no effect on the expression of p-ERK1/2.

CONCLUSIONApigenin can inhibit VEGF expression in breast cancer cells, and this effect may be achieved through decreasing the expression of HIF-1alpha.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apigenin ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

The association of coagulation function with progressive proteinuria in type 2 diabetic patients was retrospectively analyzed.With increasing microalbuminuria,fibrinogen level was increased significantly.Fibrinogen was an independent risk factor of microalbuminuria. In patients as the early-stage diabetic nephropathy (DN)progressed to clinical-stage DN,the baseline level of fibrinogen was also increased [ ( 3.5 ± 0.9 vs 3.0 ± 0.6 ) g/L,P＜0.05 ].Fibrinogen may serve as a useful predictor of progressive proteinuria in type 2 diabetes.

Blood lipid level and its associations with insulin resistance were studied in patients with impaired glucose tolerance (IGT).Two hundred and twenty first degree relatives of type 2 diabetes mellitus were grouped into normal glucose tolerance (NGT) and IGT groups according to results of oral glucose tolerance test.Compared with the NGT group,the IGT patients had higher serum levels of total triglyceride (TG),total cholesterol (TC),low density lipoprotein-C (LDL-C) but a lower serum level of high density lipoprotein-C (HDL-C).Homeostasis model of assessment for insulin resistance index (HOMA-IR) and area under curve of insulin (AUCI) also increased.A positive relationship was found between TG and HOMA-IR (or AUCI),but a negative relationship existed between HDL-C and HOMA-IR.In conclusion,abnormal blood lipid metabolism is present in IGT patients and it has a close correlation with insulin resistance.

[Summary] To investigate the association between sleep disorder and ambulatory blood pressure rhythm in patients with type 2 diabetes. 418 patients with type 2 diabetes were divided into two groups according to Pittsburgh sleep quality index ( PSQI):patients without sleep disorder and patients with sleep disorder. Oral glucose tolerance test, insulin releasing test, and C-peptide releasing test were performed to investigate the differences in the β-cell function, the circadian rhythm of blood pressure, and blood pressure variation between the two groups after fasting and glucose-load. The correlation and regression analysis were performed between PSQI and other indicators. (1)The level of HbA1C , fasting plasma insulin, area under curve of insulin, fasting plasma C-peptide, area under curve of C-peptide, and homeostasis model assessment for insulin resistance ( HOMA-IR) were significantly higher in patients withsleepdisordercomparedtothoseinpatientswithoutsleepdisorder[(8.2±2.1)% vs(7.4±1.8)%,(13.42± 4.55vs11.86±4.52)mU/L,(8.51±0.54vs8.38±0.51)mU·L-1·min,(2.42±1.25vs1.79±0.73)ng/ml, (6.59±0.39vs6.49±0.43)μg·L-1·min,4.63±1.12vs3.86±0.97,allP<0.05]. Insulinsensitivityindex (ISI) was lower in patients with sleep disorder than that in patients without sleep disorder(-4. 26 ± 0. 78 vs-4. 05 ± 0.62,P<0.05). (2)Thelevelof24hmeansystolicanddiastolicbloodpressure,nocturalsystolicanddiastolicblood pressure, and systolic blood pressure during daytime and nighttime were significantly higher in patients with type 2 diabetes who were suffering from sleep disorder. The blood pressure variation was more marked in patients with sleep disorder. (3)Multiple stepwise regression analysis showed that PSQI score was positively related to area under curve of C-peptide, HOMA-IR, 24 h mean systolic blood pressure, and noctural systolic blood pressure (β=0. 242, 0. 293, 0. 352, 0. 413, all P<0. 05), and negatively related to ISI and decreasing ratio of noctural systolic blood pressure (β=-0. 124 and -0. 226, both P<0. 05). Sleep disorder may cause abnormal circadian rhythm of blood pressure through various mechanisms. Improving sleep disorder may help to ameliorate insulin resistance and restore normal circadian rhythm of blood pressure.

Through retrospective analysis of the clinical and laboratory data of 1 466 inpatients with type 2 diabetes mellitus(T2DM),we investigated the prevalence of chronic kidney disease (CKD) and analyzed the risk factors.The prevalence of CKD in hospitalized patients with T2DM was 52.25％.In the patients with CKD,protein urine was present in 93.47％ of the cases,27.93％ of them had glomerular filtration rate(eGFR) ≤60 ml · min-1 · 1.73 m-2,damage of renal tubular function was present in 24.28％,and abnormal renal imaging in 14.88％.Logistic regression showed that age,body mass index(BMI),duration of diabetes,systolic blood pressure,serum uric acid,low density lipoprotein-cholesterol (LDL-C),and smoking were independently associated with patients of T2 DM and CKD.The prevalence of CKD was increased with aging,diabetic course,BMI,and LDL-C.CKD is a common chronic complication in patients with T2DM,especially in patients with prolonged course,advanced age,and obesity.Much attention should be paid to early detection of CKD in patients with diabetes.In addition to detecting urinary protein and eGFR,renal tubular function and morphological examination should also be included.

Objective To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. Methods According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group( incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group( incubated with 100 μ mol/L atorvastatin), the fluvastatin group (incubated with 100 μ mol/L fluvastatin)and the pravastatin group (incubated with 100 μ mol/L pravastatin). Stimulated by 2. 8,5. 5,11.1,16. 7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 μ mol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37℃ bath incubation method and ATP content was measured by chemiluminescence method. Results Incubated with 100 μ mol/L atorvastatin for 30 minutes, in the presence of 16. 7 mmol/L glucose, the ATP content [(9. 54 ± 1. 64) pmol/islet vs ( 12. 33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0. 21 vs 2. 39 ± 0. 30) were significantly reduced in comparison with the control group (P＜0. 05). Cultured with 100 μmol/L fluvastatin for 24 hours, the ATP content [( 10. 24 ±2.01 )pmol/islet vs (12. 31 ±2. 16) pmol/islet] and GSIS (3. 12 ± 0. 32 vs 4. 17 ±0. 37 ) were all significantly decreased at the higher glucose concentration of 16. 7 mmol/L ( P ＜ 0. 05). Conclusion Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.

Objective To investigate plasma glutathione S-transferase(GSTs) activity and GSTP1 gene Ile105Val polymorphism in Bijie City, Guizhou Province, a coal-burning fluorosis endemic area. Methods One hundred and sixty villagers from Yachi Twon using non-improved cooking stoves were selected as the non-intervened group in Bijie City, Guizhou Province where coal-burning fluorosis was prevailing; 153 villagers as the intervented group were chosen from Changchun Twon, where cooking stoves were improved; 151 villagers were served as the control group from Baiyunshan Twon, Changshun County without endemic fluorosis. The activity of GSTs was tested by colorimetric analysis with spectrophotometer. The genotype of the GSTP1 gene Ile105Val polymorphism, presenting as either homozygous wild-type (AA), or heterozygous mutation type (AG), or homozygous mutation type (GG), was detected through the PCR-RFLP procedure. Results The activity of GSTs in plasma of non-intervened group [(12.44±4.97) kU/L]was significantly lower than that of intervened group (P < 0.05), and that of intervened group[(20.78±6.20)kU/L]was significantly lower than that of control group[(24.30±6.27)kU/L, P< 0.05]. The difference of the enzyme activity of three groups were statistically significant (F = 51.71, P < 0.05), but this enzyme activity did not vary significantly in each sex of each grnup(P > 0.05). Compared intervened group [AA:67.3%(103/153), AG:29.4%(45/153),GG:3.3%(5/153)]and non-intervened group[AA:66.9%(107/160), AG:30%(48/160), GG:3.1%(5/160)]with control group[AA:74.8%(113/151), AG:25.2%(38/151), GG:0 (0/151)], the Ile105Val polymorphism site of GSTP1 gene had significant difference(χ2= 6.04,6.07, both P< 0.05), but not significant between intervened and non-intervened groups(χ2 = 0.02, P>0.05). Conclusions Fluorosis can decrease the activity of GSTs and introduce the GSTP1 gene Ile105Val polymorphism, intervention with the fluorine intake will improve the effect of fluoride on the body.

Objective To investigate the association between interleukin-10 (IL-10) gene promoter microsatellite polymorphisms and the susceptibility to hepatitis B virus infection in Han,Yi and Yao ethnicities in GuiZhou province.Methods 500 volunteers were selected from Guizhou province.Ailelic frequency of IL-10.G and IL-10.R loci was identified by short tandom repeat polymerase chain reaction.The relativity between allelic frequency and HBV infection was analyzed.Results Genotype data from H-W analysis on all the IL-10 polymorphisms indicated that it was a random distribution.Very high HBV infection rates were found in the native ethnic minorities of Guizhou province.The overall HBV infection rate among the total population was 67.00％,with the HBV infection rates of Yi nationality in Weining,Yi nationality in Qianxi,Yao nationality in Libo and Han nationality in Libo as 51.85％,42.86％,79.52％ and 84.30％,respe~vely.The polymorphisms distribution of IL- 10.G and IL- 10.R were statistically different among the ethnic groups (P＜ 0.05 ).The polymorphisms distribution of IL-10.R had no significant difference between HBV infection group and non-infection group,as well as among HBV natural removal group and non-infected group in all the ethnic groups.The frequency of IL-10.G 459 bp (19CA) was significantly higher in non-infection group than in the infected group (P＜ 0.05 ).The frequency of IL-10.G 471 bp (25CA) was significantly higher in the non-infection group than in the HBV natural removal group(P＜0.05).The polymorphisms distribution of IL-10.G did not show significant difference between the HBV infection group and the HBV natural removal group in all the ethnic groups.We did not find any differences in allelic and genotypic frequencies of IL-10.G between infection group and non-infection group in Yi nationality in Weining,and Yao nationality in Libo (P＞0.05),as well as HBV natural removal group and non-infected group (P＞0.05).Conclusion The polymorphisms distribution of IL-10.R and IL-10.G did not show significant difference in Yi,Yao and Han ethnics population living in Guizhou province.IL-10.G seemed to influence the susceptibility of HBV infection in Han,Yao and Yi ethnics population of Guizhou province.