AIM: To understand the expression of ecto-5'-nucleotidase (CD73) and explore its effect on the motility and invasion ability of breast cancer cell lines. METHODS: ① CD73 mRNA expression was analyzed by semiquantitative RT-PCR. Surface CD73 activity in breast carcinoma cell lines was measured by HPLC according to the conversion of E-AMP to E-ADO. ② Plasmid pcDNA-NT5E was transfected into human low metastatic breast cancer cell line T47D with lipofectinamine2000. CD73 mRNA expression was detected by semiquantitative RT-PCR and surface CD73 activity was determined by HPLC before and after transfection. The effect of pcDNA-NT5E transfection on motility and invasion ability was observed by in vitro invasion assay. RESULTS: ① The RNA expression and enzyme activity of CD73 were variable in different breast cancer cell lines. ② The transfect-T47D overexpressed CD73 at mRNA level and with high enzyme activity. Constitutively active CD73 drove cells transmembrane and invasion into matrigel. Promotion effects were statistically significant (P

Objective To investigate the level of homocysteinemia in type 2 diabetes mellitus with neuropathy,and observe the effect of medical intervention of hyperhomocysteinemia on diabetic neuropathy.Methods 200 patients with type 2 diabetes mellitus plus neuropathy and 200 control were collected.Patients with diabetic neuropathy plus hyperhomocysteinemia were divided into two groups:medical intervention group(15mg/d folic acid,20mg/d vitamin B6)and control group.The level of homocysteinemia in plasma was measured,and neuropathy score was evaluated by clinical and electrobiological methods before and three months after treatment.Results The morbidity of hyper-homocysteinemia in type 2 diabetes mellitus with neuropathy was 40%,and the levels of homocysteinemia was higher in treatment group than in control(P<0.05).Compared with the control,the levels of homocysteinemia in group of medical intervention was lower(P<0.01),and neuropathy score and nerve conductive velocity showed an obviously improvement after 3-month-treatment(P<0.05).Conclusions Hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in humans.Efficiently intervening the levels of homocysteinemia could improve the electrobiological activity and neuropathy score.

Objective To investigate the clinical application of alpha lipoic acid on early diabetic nephropathy(DN).Methods Seventy patients with early diabetic nephropathy were randomly divided into two groups.Patients in control group were treated with regular glycemic control methods,while patients in treatment group were received alpha lipoic acid 300 mg static drop treatment for two weeks beside regular treatment.High sensitivity C reaction protein,microalbuminuria and urine creatinine were measured before and after the treatment.Microalbuminuria and urine creatinine were re-measured three months later.Results At the end of two weeks treatment,the levels of high sensitivity C reaction protein in the treatment group decreased from (3.92 ± 4.56) mg/L to (2.24 ± 2.49) mg/L,and there was significant difference (t =2.803,P ＜ 0.05).Microalbuminuria decreased from (127.18 ± 76.91) mg/L to (84.50 ± 122.87) mg/L,and there was significant difference (t =2.501,P ＜ 0.05).The ratio of microalbuminuria and urine creatinine in treatment group decreased from(28.95 ± 30.49) mg/mmol to(13.76 ± 20.83) mg/mmol,and there was significant difference (t =3.616,P ＜ 0.05).While there was no significant difference in the control group before and after regular waglycemic control.At three months after treatment,there were no significant difference regarding of microalbuminuria,the ratio of microalbuminuria and urine creatinine between treatment and control groups (P ＞ 0.05).Conclusion Alpha lipoic acid might be with ability of decreasing the levels of microalbuminuria of diabetic nephropathy in the short term,but further study is needed to verify whether alpha lipoic acid can decrease the level of microalbuminuria in the long term.

Objective To investigate the situation of subjective well-being feeling of type 2 diabetes patients and its influential factors.Methods One hundred and twenty-seven patients with type 2 diabetes were recruited as our subjects and all of them were performed the questionnaire investigation about well-being feeling.Influential factors were analyzed through several statistic methods.Results The index of subject wellbeing ranged from 3.70 to 13.70 and the average was 10.67 ± 2.10 of all type 2 diabetes patients.Of which The index of total affectation was 5.11 ± 0.92 and the range was from 2.63 to 6.63.The score of life satisfaction was 5.58 ± 1.22,and the range was from 1.10 to 7.70.Index of subjective well-being significantly associated with education,family income,therapy approach,HbA1 c level,complication,medicine burden.However there was no correlation with gender,age,diabetes duration.Conclusion The main types of subjective well-being feeling of patients with type 2 diabetes were positive effect and positive experience.Many factors could be the influence factor to well-being.Measures should be taken to improve Subjective well-being of type 2 diabetes patients.

AIM: To study the effect of hypoxia on CD73 expression in mouse microvascular endothelial cell line bEnd.3. METHODS: ① bEnd.3 cells were exposed to different periods of hypoxia. ② Concentration of LDH released by bEnd.3 cells into the culture medium was detected. ③ Surface CD73 activity in bEnd.3 cells was measured by HPLC according to the conversion of E-AMP to E-ADO. ④ CD73 mRNA expression were analyzed by semiquantitative RT-PCR. ⑤ Cell surface proteins were biotinylated and CD73 was detected by avidin blots of immunoprecipitation with mAb TY23. RESULTS: ① bEnd.3 cells exposed to hypoxia for 24 h demonstrated a significant increase in LDH release (P

AIM:To study the effect of CD73 on breast cancer cell line MB-MDA-231 adhesion to extracellular matrix.METHODS: ① CD73 siRNA plasmid was constructed and transfected into MB-MDA-231 cells by lipofectamine 2000.② The transfection efficiency was analyzed by flow cytometry using eGFP as a marker gene.Stable transfected MB-MDA-231 cells were selected using G418.③ RT-PCR and Western blotting analysis of CD73 expression in MB-MDA-231 cells was performed.④ The effects of CD73 on MB-MDA-231 cells adhesion to extracellular matrix were assessed by cell adhesion assay.RESULTS: ① Transfection of MB-MDA-231 cells with CD73 siRNA plasmid significantly inhibited CD73 expression at mRNA level.The efficiency was up to 91%.② Transfection of MB-MDA-231 cells with CD73 siRNA plasmid significantly inhibited CD73 expression at a protein level.The efficiency was up to 79.3%.③ Treatment of MB-MDA-231 cells with CD73 siRNA resulted in diminished adhesion to extracellular matrix.CONCLUSION: This study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression in MB-MDA-231 cells leading to adhesion to extracellular matrix suppression.

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.

Objective To compare the density of acetylcholine receptor (AchR) in orbicular muscle of mouth and gastrocnemius muscle and their affmity with rocuronium, trying to elucidate the mechanism for the difference in the sensitivity of the muscles innervated by facial and peripheral nerve respectively to muscle relaxant.Methods Eight pathogen-free adult male SD rats weighing 180-220 g were used in this study. Muscle strips were isolated from orbicular muscle of mouth and gastrocnemius muscle. Each muscle strip was further divided into 6 smaller and slender strips of same size using dissection microscope. One strip was stained with acetylcholinesterase to measure end-plate surface area (ESA). The other 5 strips were exposed to different concentrations of rocuronium (0, 2.5, 5.0, 7.5, 10.0μg/ml). The mean density of AchR at end-plate was obtained by AchR0/ESA. (AchR0 was defined as the number of AchR per end-plate without being exposed to rocuronium. AchRE was defined as the number of free AchR per end-plate after being exposed to different concentrations of rocuronium. ) The degree of saturation of AchR with different concentrations of rocuronium at each neuromuscular junction was calculated by (AchR0 - AchRE)/AchR0 which reflects the affinity of AchR with the rocuronium in orbicular muscle of mouth and gastrocnemius muscle. Results The density of AchR was significantly lower while the affinity with rocuronium was higher in gastrocnemius muscle than in orbicular muscle of mouth ( P ＜ 0.05). Conclusion The density of AchR is lower and the affinity of AchR at end-plate with rocuronium is significantly greater in gastrocnemius muscle innervated by sciatic nerve than in orbicular muscle of mouth innervated by facial nerve. This may explain the mechanism for different sensitivity of the muscles innervated by facial and peripheral nerves to rocuronium.

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133~+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133~+ cells of cord blood MNC was (1.41?1.14)%, and purity was 75%-85% (FACS method). CD133~+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical "cobblestone" morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the (original,) cell markers CD133 and CD34 decreased significantly (77.0%?3.3% to 1.6%?2.2% and 93.1%?4.7% to 37.4%?4.9%, P