Objective To investigate the predictive values of hypoxia-induced factor(HIF)- 1α and vascular endothelial growth factor (VEGF) in patients with type 2 diabetic kidney disease, and its relationship between HIF-1α, VEGF and glomerular filtration rate (eGFR). Methods Seventy-six patients with type 2 diabetic kidney disease were divided into two groups:DKD1 group [eGFR>90 mL/(min·1.73 m2),n=48] and DKD2 group [60 mL/(min·1.73 m2)0.05). Serum levels of HIF-1α and VEGF were significantly higher in DKD2 group than those of DKD1 group (P<0.05). There were negative correlation between VEGF and HIF-1αlevels with eGFR level (r=-0.59, P<0.01;r=-0.55, P<0.01). ROC curve showed that serum VEGF and HIF-1αlevels were above the opportunity diagonal. The diagnostic sensitivities of HIF-1 to DKD1 and DKD2 were 92.1%and 87.4%. The diagnostic specificities of HIF-1 to DKD1 and DKD2 were 91.7%and 85.4%. The diagnostic sensitivities of VEGF to DKD1 and DKD2 were 96.1%and 86.2%, and specificities were 93.3%and 89.1%. Conclusion Serum levels of VEGF and HIF-1αchange with the severity of diabetic nephropathy are likely to be an early predictor for the progression of diabetic kidney disease.

Objective_ To investigate the effect of resting heart rate on the progression to diabetes in impaired glucose regulation patients. Methods A total of 638 patients with impaired glucose regulation, from January 2011 to December 2012 in our endocrinology clinic, were selected for the study. According to the resting heart rate, patients were divided into four groups:<66 beat/min group, 66 to 69 beat/min group, 70 to 75 beat/min group, and>75 beat/min groups. All patients'baseline data were collected. The incidences of diabetes in different resting heart rate groups were compared, and the relationship between resting heart rate and the progression to diabetes was estimated using multiple regression analysis. Results In 704 patients with impaired glucose regulation, 636 patients have been completed 2 years follow-up, or reached the end of follow-up ( diagnosed as diabetes ) , the follow-up rate was 90. 34%. During two years follow-up, the incidence of diabetes of<66 beat/min group, 66 to 69 beat/min group, 70 to 75 beat/min group, and>75 beat/min group were 16. 2%, 19. 4%, 25. 0%, and 33. 0%, respectivlely. And the Cochran Armitage trend test showed that χ2 =11. 109, P=0. 001, the difference was statistically significant ( P<0. 05). According to blood glucose monitoring, the 636 patients with impaired glucose regulation were divided into impaired fasting glucose group, impaired glucose tolerance group and impaired fasting glucose combined with impaired glucose tolerance group:the Cochran Armitage trend test showed that, with the resting heart rate accelerating, the incidence of diabetes increased. The incidence of diabetes in impaired fasting glucose combined with impaired glucose tolerance group was higher than that of impaired fasting glucose group and impaired glucose tolerance group ( P=0. 062, 0. 113). The average resting heart rate in 68 impaired glucose regulation patients progressed to diabetes was (79.8±8.3)beat/min,andin568non-diabetescases,itwas(74.5±7.2)beat/min(t=-5.043,P<0.01). With the use of patients progressing to diabetes as the dependent variable, different resting heart rate group as independent variables, and resting heart rate<66 beat/min group as a reference, the logistic regression analysis showed that the risk of the progression to diabetes increased with the resting heart rate levels. Conclusion Higher resting heart rate is linked to higher risk of diabetes in patients with impaired glucose regulation.

Objective To explore the influence of different dose of Helicobacter pylori on the expression of transforming growth factorβ(TGF-β1) and B7-H1 in the infected gastric mucosal epithelial cell and the bacterial factors which influence the expression of TGF-β1.To confirm that H.pylorican induce the expression of TGF-β1 and B7-H1 to inhibit the host immune function in gastric mucosal epithelial cell.Methods (1) We investigated the expression of TGF-β1 of human gastric mucosal epithelial cells infected with different concentration(1.0 × 109 CFU/ml,4.0 × 109 CFU/ml,8.0 × 109 C FU/ml) of H.pylori(NCTC 11637) in different time-point(0 h,0.5 h,1 h,1.5 h,2 h,4 h,8 h,12 h),and compared with the expression of TGF-β1 between the deactivated H.pylori group and activated H.pylori group.The blank group is the gastric mucosa epithelial cells which does not infect H.pylori.To detect expression of TGF-β1 in infected cell culture supernatant by enzyme-linked immunosorbent assay(ELISA) and the expression of B7-H1 mRNA by in situ hybridization.(2)At the same time,the middle concentration of deactivated H.pyloriand in vitro gastric mucosal cells were incubated for 2 h and 12 h,to detect expression of TGF-β1 in the cells and cell culture supernatant.(3)In vitro gastric mucosal cells were incubated with H.pylori bacterial supernatant and sedimentation by ultrasonic crushing and centrifugation and with H.pyloribacterial supernatant and sedimentation after boiling respectively,to detect expression of TGF-β1 in the cells and cell culture supernatant after 2 h,12 h.Results (1)Compared to the control group,the expression of TGF-β1 was significantly increased after stimulation with different concentration of activated H.pylori in different time-point(P ＜0.05).The expression of TGF-β1 secretion group has a similar dynamic trend,and the highest expression is the middle concentration group(P ＜0.05).(2)There was no difference between the middle concentration of deactivated H.pylori group and the same concentration of activated H.pylorigroup(P ＞ 0.05).(3) The expression of TGF-β1 in the H.pylori bacterial supernatant group was significantly increased higher than the blank group and the H.pylori bacterial sedimentation group(P ＜0.05),and the H.pylori bacterial supernatant group after boiling was significantly lower than the H.pylori bacterial supernatant group(P ＜ 0.05),but there was no difference between H.pylori sedimentation group after boiling and not boiling(P ＞ 0.05).The B7-H1 expression of different concentration groups which the H.pylori and gastric mucosal epithelial cells cocultured 12 h are higher than the blank group(P ＜ 0.05) by in situ hybridization,and the middle concentration group is the highest expression.TGF-β1 and B7-H1 mRNA are positively correlated(r,=0.628,P ＜0.01).Conclusion H.pylori can induce the gastric mucosal epithelial cells to express the TGF-β1,the factor was the soluble protein in the H.pylori thalline.At the same time,H.pylorican induce the B7-H1 expression increased.In gastric mucosal epithelial cells,TGF-β1 and B7-H1 mRNA are positively correlated.So H.pylori can inhibit the host immune response and participate the process of immune escape by increased the expressions of TGF-β1 and B7-H1.

BACKGROUND: It is easy to established human solid tumor nude mouse model, but for leukemia which is difficult. We inhibited immune system further by radioactive ray or CTX, to decrease cost and increase the stability.OBJECTIVE: To establish a human acute myeloblastic leukemia M2 Kasumi-1 models containing AML/ETO positive genes in BALB/c nude mouse. METHODS: Nude mice were randomly divided into three groups: CTX group was injected CTX 2 mg/day in abdominal cavity for two days, and injected 8×10~5/mouse Kasumi-1 cells in caudal vein next day; irradiation group was exposed to total body irradiation, and injected 8×10~5/mouse Kasumi-1 cells in caudal vein that day; untreated group was inoculated with 8×10~5/mouse Kasumi-1 cells by caudal vein injection. Three additional mice were considered as the normal control group. The blood smearing and bone morrow slides were detected, immunity type of BMC was detected using flow cytometry, loading of leukemic cellular tumor was detected using RT-PCR, and positive ratio of AML/ETO fusion gene was detected using FISH method. RESULTS AND CONCLUSION: After inoculated into untreated nude mice by caudal vein injection for 14 days, the ratio of leukemia cell in blood smearing was 3.5%, and over 40% in bone marrow slides, which was equal to the results of FISH and FCM. The increasing of tumor loading was time-dependent. For irradiation group and CTX treated group, the tumor loading was higher that untreated group, and the cells also survived more than 60 days. AML/ETO band was observed by RT-PCR in all experimental groups, for normal mice it was negative. The results indicated that the systemic disseminated leukemia model was established successfully by caudal vein injection 8×10~5/mouse Kasumi-1 cells in the three experimental groups.

On the basis of introducing FDA's regulatory measure and relevant requirement for life-cycle management of combination product, this paper aims to discuss corresponding countermeasure for supervision system construction in consideration of domestic drug-device combination product's current situation, in order to promote innovative development of relevant industries.
Device Approval ; Drug Approval ; United States ; United States Food and Drug Administration

Objective To observe RNA-Seq analysis ofgene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.Methods Retinal vascular endothelial cells were cultured in vitro,and the logarithmic growth phase cells were used for experiments.The cells were divided into the control group and high glucose group.The cells of two groups were cultured for 5 hours with 5,25 mmol/L glucose,respectively.And then,whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq.Now with biological big data obtained as a basis,to analyze the differentially expressed genes (DEGs).And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.Results The gene expression profiles of the two groups of cells were obtained.Through analysis,449 DEGs were found,including 297 upregulated and 152 downregulated ones.The functions of DEGs were influenced by regulations over molecular biological process,cellular energy metabolism and protein synthesis,etc.Among these genes,ITGB 1BP2,NCF 1 and UNC5C were related to production of inflammation;AKR1C4,ATP 1A3,CHST5,LCTL were related to energy metabolism of cells;DAB 1 and PRSS55 were related to protein synthesis;SMAD9 and BMP4 were related to the metabolism of extracellular matrix.GO enrichment analysis showed that DEGs mainly act in three ways:regulating biological behavior,organizing cellular component and performing molecular function,which were mainly concentrated in the system generation of biological process part and regulation of multicellular organisms.Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in transforming growth factor-β (TGF-β) signaling pathway,complement pathway and amino acid metabolism-related pathways have also been affected,such as tryptophan,serine and cyanide.Among them,leukocyte inhibitory factor 9 and bone morphogenetic protein 4 play a role through the TGF-β signaling pathway.Conclusions High glucose affects the function of retinal vascular endothelial cells by destroying transmembrane conduction of retinal vascular endothelial cells,metabolism of extracellular matrix,and transcription and translation of proteins.

Objective To observe the synergistic effect of metformin and anti-vascular endothelial growth factor (VEGF) in the treatment of diabetic retinopathy.Methods This study was composed of clinical data review and in vitro cell experiment.Ten patients (12 eyes) with diabetic macular edema treated with antiVEGF drugs were included in the study.Patients were randomly divided into the VEGF group (anti-VEGF drug therapy) and the combined treatment group (anti-VEGF drug combined with metformin).The changes of visual acuity and central retinal thickness (CRT) were compared between the two groups.As far as the in vitro experiment was concerned,vascular endothelial cells were divided into the control group (normal cells),the VEGF group (50 ng/ml VEGF),the anti-VEGF group (50 ng/ml VEGF+2.5 μg/ml of conbercept),and the combined group (50 ng/ml VEGF +2.5 μg/ml of conbercept +2.0 mmol/L of metforrnin).And then MTT cell viability assay,scratch assay and real-time quantitative polymerase chain reaction assay were performed to analyze the cell viability,cell migration and mRNA level of VEGFR2,protein kinase C (PKC)-α and PKC-β successively.Results Review of clinical trial shows that the CRT recovery rates in the combined treatment group were much higher than that in the VEGF group at 3 month after the operation,while the difference was statistically significant (t=-2.462,P＜0.05).In vitro cell experiment results showed that VEGF induction upregulated the viability and mobility of vascular endothelial cells obviously compared with control group,at the same time,the use of anti VEGF drugs can effectively reverse the trend,in contrast,combination of metformin and anti-VEGF showed a more superior effect to some extent (P＜0.05).In the VEGF group,the mRNA expression of VEGFR2,PKC-αand PKC-[β were significantly increased compared with the control group (P＜ 0.01);while the mRNA expression of VEGFR2,PKC-αand PKC-β in the combination group decreased significantly compared with the VEGF group and the control group (P＜0.05).However,in the anti-VEGF group,the mRNA expression of VEGFR2,PKC-αand PKC-β were decreased,but has failed to reach the level of statistical learn the difference.Conclusions The combination ofmetformin and anti-VEGF drugs can reduce the CRT of diabetic retinopathy patients and inhibit the proliferation and migration of retinal vascular endothelial cells which induced by VEGF.The synergistic mechanism may be related to the inhibitory effect of metformin on the expression of VEGFR and PKC.

Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).Methods Human retinal Müller cells cultured in vitro were divided into normal control group,model group (H2O2 group) and experimental group (H2O2+NBP group).The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μ mol/L H2O2 for 2 h.Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 tmol/L NBP.The normal control group was a conventional cultured cells.Müller cells were identified by immunofluorescence staining.Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes.MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention.Hoechst33258 staining was used to observe the apoptosis.LIVE/DEAD (R)cell activity/cytotoxicity kit was used to detect cell viability.Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells.One-way ANOVA combined with Dunnett statistical method were used for data analysis.Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group.MTT assay showed that after 24 h and 48 h of NBP intervention,the differences in cell viability between the normal control group and the H2O2 group,the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96,3.658,47.58,20.33;P＜0.001,0.022).The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced,and the blue fluorescence of the remaining cells was uniform.The LIVE/DEAD ~ cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly,and the number of viable cells with green fluorescence decreased significantly.In the H2O2+NBP group,the number of viable cells with green fluorescence increased,and the number of dead cells with red fluorescence decreased.The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced;the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.

Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.

Objective To observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).Methods Eighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue.Results In the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20,P<0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71,P<0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60;P<0.05).Conclusion Intravitreal injection of LV-191 inhibits RNV in mice model of OIR possibly through up-regulating p21.