Objective To determine the involvement of DNA demethylation in tumor necrosis factor-α(TNF-α)-induced matrix metalloproteinase 9 ( MMP9) expression in human epidermal keratinocytes. Methods Real-time RT-PCR, Western blot, and enzyme-linked immuno sorbent assay (ELISA) were performed to determine the mRNA and protein levels of MMP9 after human keratinocyte cell line (HaCaT) cells were treated with 10 ng/ ml TNF-α or 2. 5 μmol/ L DAC/ 300 nmol/ L TSA. Bisulfite sequencing PCR ( BSP) and Methylation-sensitive high-resolution melt analysis ( Ms-HRM) were used to detect significantly differentially demethylated CpG sites in the human MMP9 promoter region in cells exposed to TNF-α. Different sites methylation constructs of promoter-luciferase reporter gene were made to detect the influences of site-specific DNA demethylation on transcription activity of MMP9 promoter. Results Compared with PBS-treated control, TNF-α significantly increased the expression of MMP9 in HaCaT cells for indicated culture duration ( P < 0. 05 ). Real time PCR, Western blot, and ELISA analysis demonstrated that the mRNA and protein levels of MMP9 were increased initially, followed by a decline with prolonged incubation time. After TNF-α treatment, varied degrees of DNA demethylation occurred at 10 CpG sites in the promoter of MMP9, and the changes at the -36 bp site were statistically significant (P<0. 05). The demethylation at the -36 bp site greatly increased the transcription activity of MMP9. Conclusion TNF-α promotes MMP9 expression in HaCaT cells through inducing -36 bp site DNA demethylation on the promoter of MMP9.

[Objective]To explore the relationship between peripheral arterial disease(PAD)and diabetic retinopathy(DR)in type 2 diabetes patients.[Methods]A total of 99 patients diagnosed with PAD were classified into grade 1-3 by their total scores of peripheral arterial stenosis assessed by color doppler ultrasound examinations,where the degree of stenosis 30% ~ 49% scored 0, 50%~99%scored 1,lumen occlusion(i.e. degree of stenosis 100%)scored 2,and therefore the total score 0-2 was categorized into Grade 1 ,3~4 into Grade 2 ,5~12 into Grade 3. The bilateral anterior tibial artery ,posterior tibial artery and dorsalis pedis artery of these patients were analyzed. The presence of diabetic retinopathy(DR)was graded from retinal photographs using a standard protocol.[Results]Among 99 cases of type 2 diabetic patients with peripheral arterial disease ,58.6%of them were male with average age of 67.3 ± 7.9 years old. Patients of Grade 1,Grade2,Grade 3 lesion accounted for 45.4%,30.3%,24.2%,respectively. Age, gender,smoking history,SBP,DBP,BMI,FBG,TC,TG,LDL-C,HDL-C,HbA1C among 3 groups were not statistically signifi-cant. The associations of DM duration and HbA1C value were significantly larger in DR than in PAD. The proportion of DR patients increased with the severity degree of PAD(p for trend=0.004). Degree of stenosis Grade 2 and Grade 3 could be predictive for DR.[Conclusions]DR is associated with the severity degree of PAD in type 2 diabetes patients as evaluated by duplex ultrasonography.Degree of stenosis Grade 2 and 3 could be used for screening or finding DR. Strategies for optimum treatment and early prevention are needed.

Both sides of the picornavirus genome have 5'-untranslated region (5'UTR) and 3'- untranslated region (3'UTR). This study demontrated that both the 5'-and 3'-UTR can form complex structures, such as stem-loop, clover and pseudoknot structure, These structures play an important role in the regulaton of the replication and translation of the viruses. This article reviewed the progress of research on the structure and function of picornavirus' 3'-UTR over recent years.
3' Untranslated Regions ; Animals ; Humans ; Nucleic Acid Conformation ; Picornaviridae ; chemistry ; genetics ; metabolism ; Picornaviridae Infections ; virology ; RNA, Viral ; chemistry ; genetics ; metabolism

[ABSTRACT]AIM:Toinvestigatecellapoptosisindiabeticfootulcersandtheeffectofadvancedglycosylation end products (AGEs) on apoptosis in human fibroblast cells.METHODS: Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited .The clinical and biochemical features were compared by statistics methods . Skin biopsies were obtained from foot .Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex ( SABC ) staining.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL) technique was used to detect apoptosis of the skin tissues .Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose ( changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose).After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3.Other cells were trypsinized , washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis .RE-SULTS:Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration.The protein level of cleaved caspase-3 was signifi-cantly higher in diabetic group , suggesting that apoptosis was increased in diabetic skin tissues .TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%± 0.8%) , which further confirmed that cell apoptosis was increased in diabetic foot tissues .In human fibroblasts , the levels of cleaved caspase-3 in normal group , sustained high glucose group , fluctuant high glucose group and AGEs group were 0.80 ±0.13, 1.22 ±0.18, 1.46 ±0.32 and 1.83 ±0.25, respectively.The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99% ±0.24% and 6.83% ±0.36%, respectively.Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group .CONCLUSION:Increased apoptosis in diabetic foot ulcers is one of the most important reasons for im-paired wound healing .As compared to sustained high glucose and glucose fluctuations , AGEs induce greater apoptosis in human fibroblast cells .

Surgical robots, as a new means for surgeons, have been gradually applied in orthopedics. Initially, the development of orthopedic robots was stagnant for a long time because of limited techniques available, clumsy equipment, high costs, and low market demands. The recent decade has witnessed rapid growth of artificial intelligence in all walks of life, increasing investment in research and development, reducing manufacture costs and expanding demands for precise and individualized medical treatment so that a wide variety of novel and ingenious robotic systems have been proposed, prototyped, and commercialized in most of the major procedures in orthopedic surgery, including knee and hip replacements, cruciate ligaments surgery, spine surgery, corrective osteotomy, bone tumor surgery, and trauma surgery. This review depicts the history of development and future prospects in application of surgical robots in the field of orthopedics.

OBJECTIVETo study the diagnosis and proper treatment of focal nodular hyperplasia (FNH) of the liver.

METHODSForty-eight consecutive patients with FNH treated from 1998 to 2002 were analyzed. Clinical, pathologic, and preoperative imaging findings of FNH all proved surgically and pathologically were retrospectively reviewed.

RESULTSMost of the patients were asymptomatic and serum test was normal. Helical multiphasic liver CT and MR imaging demonstrated characteristic features in 25 patients. Histologically, characteristic scar-like tissues were found in 29 cases. The overall complication rate was 2.1% and there was no death. No recurrence was observed after follow-up of 4 years after operation.

CONCLUSIONThere is little evidence for an increased risk for FNH in people using modern oral contraceptives, while characteristic imaging features exist in helical multiphasic liver CT and MR imaging. Operation is the optimal treatment for FNH, but for proved FNH less than 5 cm and asymptomatic patients, close periodic followed up should be done.

Adolescent ; Adult ; Aged ; Child ; Female ; Focal Nodular Hyperplasia ; diagnosis ; pathology ; surgery ; Follow-Up Studies ; Humans ; Liver ; diagnostic imaging ; pathology ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Tomography, Spiral Computed

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.
Antibodies, Monoclonal, Murine-Derived ; pharmacology ; Antibody-Dependent Cell Cytotoxicity ; Antigens, CD20 ; immunology ; Genes, Reporter ; Humans ; Reproducibility of Results ; Rituximab

Objective To investigate the changes of Th1/Th2 cells and related cytokine levels in chronic hepatitis B (CHB) fibrosis.Methods Forty-six patients with CHB fibrosis underwent liver biopsy during March and October,2011.According to the stage of fibrosis,the patients were divided into S0-1 group (n =15),S2-3 group (n =20) and S4 group (n =11).Ten healthy subjects served as controls.The frequencies of circulating Th1,Th2 cells were detected by flow cytometry.The expressions of interferon-γ (IFN-γ) and interleukin 4 (IL-4) mRNA in peripheral blood mononuclear cells (PBMCs) were detected by real-time quantitative PCR.The serum IFN-γand IL-4 concentrations were determined by enzyme-linked immunosorbent assays.Intrahepatic expressions of IFN-γ and IL-4 were detected by immunohistochemical staining.Differences between groups were analyzed using non-parametric Kruskal-Wallis H test,followed by Mann-Whitney U test for multiple comparisons.Logistic regression was used for multivariate analysis.Results With the degree of liver fibrosis exacerbations,the peripheral Th1/Th2 cells frequencies ratio,IFN-γ/IL-4 mRNA ratio in PBMCs,serum IFN-γ/IL-4 ratio and intrahepatic IFN-γ/IL-4 ratio were declined (x2 =36.259,40.822,26.321 and 31.852,respectively,all P ＜ 0.05).Serum and intrahepatic IFNγ/IL-4 ratio were negatively associated with the stage of liver fibrosis (r =-0.616 and-0.531,P ＜0.01).Logistic regression analysis showed that AST,PT and the serum IFNγ/IL-4 ratio were the risk factors for significant liver fibrosis (S2-4) (OR =5.933,95％ CI:1.324-26.586,P =0.02; OR =12.866,95％CI:1.746-94.788,P =0.01; OR=4.755,95％CI:1.034-21.862,P =0.04).Conclusions The CHB patients has imbalanced Th1/Th2 ratio.With the degree of liver fibrosis exacerbations,Th1/Th2 cytokines drift into Th2 lymphocyte sub-cluster,which suggests that Th1/Th2 imbalance may be involved in the pathogenesis of CHB fibrosis.

Objective To study the effect of curcumin on rat model of N-methylnitrosourea ( MNU) -induced bladder cancer and its mechanism. Methods One hundred SD rats were randomly divided into four groups:control group (n=10), model group (n=10), intervention group (n=40) and treatment group (n=40). Rats in the control group re-ceived intravesical infusion of distilled water. Rats in the other three groups were given MNU (1 mg/mL) in 2 mL saline at 2nd, 4th, 6th and 8th weeks to induce bladder cancer. In the model group, the rats were injected with distilled water in the bladder. The rats in the intervention group received 2 mL curcumin solution (400 μmol/L) at the 1st, 3rd, 5th, 7th and 9th weeks, and were sacrificed at the 11th week. In the model group, the rats were injected with distilled water in the bladder. In the treatment group, the rats had intravesical instillation of curcumin in the bladder (400 μmol/L, 2 mL) at 10, 12, 14, 16, and 18 weeks, and sacrificed at the 19th week. Bladder tissue samples were taken for pathological exami-nation using hematoxylin and eosin ( HE) staining. TUNEL staining assay was used to detect the apoptosis in tumor tissue. The expression of apoptosis-related proteins was detected by Western blot. Results The incidence of bladder cancer was 90% (9/10) in the model group, 12. 5% (5/40) in the intervention group and 92. 5% (37/40) in the treatment group at the 10th week, showing a significant difference between the intervention group and model group (P<0. 05), indicating an obvious interventional effect of curcumin on the bladder cancer. The incidence rate of bladder cancer in the treatment group was 78. 4% (30/37) at the 19th week, and compared with the 10th week before treatment, showing that curcumin can de-lay the recurrence of bladder cancer. TUNEL staining assay confirmed that curcumin significantly promoted the apoptosis in bladder cancer cells and inhibited their proliferation. The Western blot analysis showed that curcumin inhibited the activa-tion of NF-κB and effectively down-regulated the expression of NF-κB-regulated gene product. Conclusions Curcumin has a significant interventional effect on MNU-induced bladder cancer in the rat models. The mechanism may be through inhibi-tion of NF-κB activation and effective down-regulated NF-κB regulation of the gene products, and to regulate the expression of related proteins in bladder cancer, i. e. , inhibition of proliferation, induction of apoptosis, and further play a role of an-ti-cancer intervention and prevention of bladder cancer recurrence.

Objective To investigate the effect of bacillus Calmette-Guérin(BCG)on bladder cancer cells and their metabolites, and to preliminarily explore the possible mechanisms of BCG in the treatment of bladder cancer. Methods The rat model of bladder cancer was induced by intravesical instillation with N-methylnitrosourea(MNU). Bladder cancer cells and normal transitional epithelial cells were isolated and primarily cultured, and were divided into 5 groups according to the different components of the culture medium. The concentration of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10)in the supernatant of each group was detected by enzyme linked immunosorbent assay (ELISA). The concentration of BCG to inhibit the cancer cell growth was determined by MTT assay. Apoptosis of bladder cancer cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). Results Among the 15 rats,2 rats died after 2 times of instillation, and 3 rats died after 3 times of instillation, without obvious tumors found at autopsy. The other 10 rats were killed after completion of the intravesically instillation of MNU, and obvious tumors were found in 8 of them after dissection. The results of MTT assay showed that BCG had an inhibitory effect on the growth of bladder cancer cells,and the inhibitory rate was positively correlated with the concentration of BCG. The results of ELISA showed that the concentrations of TNF-α in the supernatant of groups B and D were(160.654 ± 5.775) ng/L and(124.443 ± 4.972)ng/L, respectively, with significant differences from those of the other three groups. The concentrations of IL-10 in the groups B and E were(16.973 ± 3.428)ng/L and(20.327 ± 2.721)ng/L, significantly higher than those of the other three groups. Apoptosis of cancer cells was not found in all groups. HE staining of the primary bladder cancer cells showed that the volume of cell nucleus was increased, and the nucleo-cytoplasmic ratio was increased. The number of nucleoli in some cells was increased and some nuclei appeared like ink drops with prominent nucleoli. Conclusions BCG has an inhibitory effect on the growth of rat bladder cancer cells. IL-10 and TNF-α secreted by the tumor cells might be involved in this regulatory process. However,apoptosis does not show an obvious effect on this inhibitory process.