Background Establising the culture model of Müller cells for obtaining the highly putified target cells is essential for the study about the physiology and pathology of retinal Müller cells. The exsiting purifing method for culturing Müller cells is dissatisfactory. Objective This study was to establish a method to obtain high purifing Müller cells. Methods The retina from 5 clean newborn SD rats were isolated and digested by 0. 01% trypsin and cultured in DMEM containing 10% fetal bovine serum. The cellular suspension was then prepared,and the target cells were screened using flow cytometry based on the size and the quantity of cells. Cultured and passaged cells were identified by transmission electron microscope and light microscope. Immunocytochemistry was used to detecte the expression of GFAP in cultured cells for the determination of type and purity of the cells. Results The cells showed the similar shape to retinal Müller cells after primarily culture with the large volume, and some small other types of cells could been seen. The growth of cells was quickly 3 weeks later. The fibroblasts were removed using sticking-wall by steps,and neurons were eliminated following passage. Aboundent of cellular organs were seen under the transmission electron microscope. The positive response rate of the cells for CFAP was 100%. Conclution Flow cytometry offer a rapid and feasible approach for purifying Muller cell and it builds the foundation for further study about Müller cells.

Astragalus polysaccharide has been widely used in food and medicinal industry owing to its health-promoting properties. In order to characterize better the relationship among molecular weight, structure-activity and activities, a simple method was used different concentration of ethanol including 30% (PW30), 50% (PW50), 70% (PW70), 75% (PW75), 80% (PW80) and 90% (PW90) to precipitate Astragalus polysaccharides into different molecular weight. As a result, PW90 showed smooth surface and the strongest antioxidant activity among these six fractions (P < 0.05). In conclusion, graded ethanol precipitation was a simple method to separate Astragalus polysaccharides into different molecular weight with different antioxidant activity fractions.
Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Chemical Precipitation ; Ethanol ; chemistry ; Polysaccharides ; chemistry ; pharmacology

16 and the distribution of severe trauma more than 2 anatomic parts.They were randomly divided into two groups:intensive insulin treatment group(n=31)and control group(n=31).Intensive insulin treatment group received insulin with insulin pump in order to maintain blood glucose levels at 4.0-6.1 mol/L,while the control group received routine insulin treatment in order to mmaintain blood glucose levels at 10.0- 11.0 mol/L.Plasma levels of TNF-?,IL-1,IL-6, CRP,APACHEⅡscores and cure rate were analyzed before and after the treatment.Data was expressed as mean?standard deviation.Two- tailed T test and ANOVA were used for comparison in SPSS 10.0,and changes were considered as statistically significant if P value was less than 0.05.Results After the intensive insulin treatment, patient's hemodynamic parameter apparently improved,APACHEⅡscores descended,and the levels of TNF-?, Ib-1,IL-6,CRP all declined,in comparison with control group,there were significant differences. Intensive insulin treatment might improve patient's general condition and decrease complications and mortality of severe multiple trauma.

OBJECTIVETo investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.

METHODSHuman SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.

RESULTSIn vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).

CONCLUSIONThe shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.

Animals ; Cell Proliferation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyaluronan Receptors ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Octamer Transcription Factor-3 ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Smoothened Receptor ; Transcription Factors ; metabolism ; Transfection ; Tumor Burden ; Zinc Finger Protein GLI1

The purpose of the present study was to investigate the effects of advanced glycation end products (AGEs) modified protein on the permeability of endothelium monolayers and morphological changes of actin cytoskeleton. The roles of receptor for AGEs (RAGE), oxidant stress and the activation of p38 MAPK pathway in this pathological procedure were elucidated. Human umbilical vein endothelial cells (HUVECs)-derived cell line (ECV304) were incubated with AGEs modified human serum albumin (AGE-HSA) in concentrations of 12.5, 25, 50, and 100 microg/ml respectively, for 2, 4, 8, 12 and 24 h. As control, HSA of the same concentration was administered to cells. Then TRITC-albumin was added to evaluate Pa value that reflects the permeability of endothelial monolayer. Furthermore, to visualize the morphological changes of actin cytoskeleton, the treated cells were incubated with rhodamine-phalloidin to stain F-actin. The results showed that the trans-endothelial membrane flux of albumin was significantly increased in a concentration- and time-dependent manner upon the stimulation of AGE-HSA, accompanying with actin reorganization. The blockage of AGE and RAGE binding with anti-RAGE IgG and the pharmacological inhibition of NADPH oxidase or p38 MAP kinase greatly attenuated the AGE-induced hyperpermeability response, respectively. These results indicate that RAGE, NADPH oxidase and p38 MAPK are possibly involved in the mediation of AGEs-induced barrier dysfunction and actin cytoskeleton reorganization in endothelial cells.
Actin Cytoskeleton ; physiology ; Capillary Permeability ; physiology ; Cell Line ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Glycation End Products, Advanced ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Oxidative Stress ; physiology ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; physiology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the influence of acupuncture parameters on the acupuncture effect through the cluster analysis in Wistar rat model with middle cerebral artery occlusion (MCAO).

METHODSDuplicated MCAO models by Zea-longa's thread ligation and chose rats with 1-3 scores assessed by Zausinger's six-score method to be grouped. The rats were divided into 6 basic control groups [(including a normal group, a sham group, a model control group, a model group without intervention, a Nimodipine group, a lateral-to-Renzhong (DU6) group] and 6 acupuncture groups [a Neiguan (PC6) group, a Weizhong (BL40) group, a Sanyinjiao (SP6) group, a Chize (LU5) group, a Renzhong (DU6) group and a Feixue (non-acupoint) group]. In the acupuncture groups, for every acupoint or needling site, 9 different parameters [2 factors (frequency and time) and 3 levels (180, 120, and 60 cpm of the frequency and 5, 60, and 180 s of the time)] were set respectively by the orthogonal intersection method, in total 54 groups. The rats were treated by acupuncture with a lifting-thrusting manipulation once every 12 h, in total 6 times. Neurobehavioral scores, cerebral blood flow, infarction rate, microcirculation, light microscopy, etc. were measured. The factor analysis was first applied to get the comprehensive effect scores of the samples in the acupuncture groups and then by which the cluster analysis was made with the statistical software of SPSS17.0.

RESULTSFor the Neiguan (PC6) group, the exceptional results of acupuncture comprehensive effect were parameters 7, 8, 9, 10; the valid results were parameters 2, 3, 4, and the invalid were parameters 5, 6. For the Weizhong (BL40) group, the exceptional results were parameters 2, 4; the valid results were parameters 3, 5, 6, 7, and the invalid were parameters 8, 9, 10. For the Chize (LU5) group, the exceptional results were parameters 7, 8; the valid results were parameters 3, 4, 5, 6, 9, 10; and the invalid was parameter 2. For the Sanyinjiao (SP6) group, the exceptional results were parameters 4, 6; the valid results were parameters 2, 3, 5; and the invalid were parameters 7, 8, 9,10. For the Renzhong (DU6) group, the exceptional results were parameters 3, 4, 6, 7, 9, 10; the valid results were parameters 2, 5; and the invalid was parameter 8. For the Non-acupoint group, the exceptional result was parameter 10; the valid results were parameters 2, 3, 4, 7, 9; and the invalid were parameters 5, 6, 8.

CONCLUSIONSFor each meridian acupoint, different acupuncture parameters could consequently get a different acupuncture effect; each meridian acupoint had the most suitable or optimal acupuncture parameters; acupuncture parameters might be the main factors impacting on acupuncture effect.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Cluster Analysis ; Disease Models, Animal ; Infarction, Middle Cerebral Artery ; therapy ; Male ; Rats ; Rats, Wistar ; Reference Standards

OBJECTIVESTo study the acupoint specificity through the comprehensive evaluation of the acupuncture effect on rat model with the middle cerebral artery occlusion (MCAO).

METHODSDuplicated MCAO model by Zea-longa's thread ligation was assessed by Zausinger's six-point method, and rats with 1-3 scores were chosen to be grouped. According to the randomized and controlled principle, rats were divided into 6 basic control groups (including normal group, sham group, model control group, model group without intervention, Nimodipine group, lateral-to-Renzhong group) and 6 acupuncture groups [including Neiguan (PC6) group, Weizhong (BL40) group, Chize (LU5) group, Sanyinjiao (SP6) group, Renzhong (DU6) group and non-acupoint group]; in each acupoint or non-acupoint 9 different parameter combinations were respectively set by orthogonal intersection method. There were in total 60 groups, and each group had 12 rats. The rats were treated by acupuncture with the lifting-thrusting manipulation every 12 h, in total 6 times. Indices of neurobehavioral scores, cerebral blood flow, infarction rate, microcirculation, and light microscope, etc. were measured. In order to comprehensively evaluate the acupuncture effect to research the specificity of acupoint, a cluster analysis was made with SPSS17.0 for the comprehensive effect scores of the samples computed according to the comprehensive evaluation models, and then sorting and classification were made for the comprehensive effect scores.

RESULTSIn the acupuncture groups every acupoint had different therapeutic effect due to different acupuncture parameter combinations; among the acupuncture groups the orders of curative effect were as followings: Neiguan group was of exceptional result, Weizhong group and Sanyinjiao group were of valid results in upper level, Chize group and Renzhong group were of valid results in lower level, and non-acupoint group was of invalid result; the therapeutic result of acupoint group was superior to non-acupoint group; and the curative effect of acupuncture group was also superior to basic control group.

CONCLUSIONThe acupoint has the specificity of acupuncture effect, and the acupoint specificity is relative.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Cerebral Infarction ; therapy ; Cluster Analysis ; Disease Models, Animal ; Male ; Rats ; Rats, Wistar

Curcumin has a wide spectrum of pharmaceutical properties such as antitumor, antioxidant, antiamyloid, and anti-inflammatory activity. However, poor aqueous solubility and low bioavailability of curcumin are major challenge in its development as a useful drug. To overcome many of these problems, curcumin-loaded long-circulating liposomes (Cur-LCL) were prepared by the ethanol injection method. Morphology of Cur-LCL was observed by transmission electron microscope, mean particle size and Zeta potential were detected by laser particle size analyzer, entrapment efficiency and drug loading were evaluated by ultracentrifugation. The drug release behavior in vitro and pharmacokinetic behavior in rats of Cur-LCL were investigated with curcumin (Cur) and curcumin liposomes (Cur-Lips) as control. The results showed that the mean diameter of Cur-LCL was 110 nm, the Zeta potential was -5.8 mV. The entrapment efficiency and drug loading of Cur-LCL was 80.25%, 2.06%, respectively. The release behavior in vitro studied by dialysis in PBS buffer showed significant sustained release profile that 48.95% Cur were released from Cur-LCL in 7 h, 88.92% in 24 h. The pharmacokinetic parameters showed that compared with Cur and Cur-Lips, the t(1/2beta) of Cur-LCL was extended to 13 and 1.8-fold, respectively. Besides, the AUC values was significantly increased (P < 0.01), and the clearance was evidently decreased (P < 0.01). These results from in vitro and in vivo indicated that Cur-LCL were able to realize controlled drug release and increase circulation time.
Animals ; Curcumin ; chemistry ; pharmacokinetics ; Delayed-Action Preparations ; chemistry ; pharmacokinetics ; Drug Carriers ; chemistry ; Female ; Humans ; Liposomes ; chemistry ; Male ; Particle Size ; Rats ; Rats, Sprague-Dawley ; Solubility

OBJECTIVETo understand the distribution of chronic disease related behavior and lifestyle in adults from Beijing.

METHODS16,658 adult residents from Beijing city were randomly selected with stratified multi-stage cluster sampling method in 2005. Each participant was invited to receive a set of standardized questionnaire, physical examinations and laboratory tests.

RESULTSIn the adults living in Beijing, 33.2% were overweight and 16.4% were obesive. The current smoking rate was 26.2% and the regular smoking rate was 21.4%. 57.7% of the male and 4.6% of the female adults were current smokers. In male adults, 64.3% drank alcoholic beverage at least once per month while 16.1% drank almost everyday, 16.5% drank more alcohol than moderate, and 18.5% were binge drinkers. 46.0% of Beijing adults were in lack of active physical exercise. Unhealthy dietary habits such as:excess consumption of sodium or oil, lower intake of vegetable, milk and soybean productions, skipping breakfast, fond of salted vegetable and fried food intake, as well as eating snacks etc. were quite commonly seen in the adults from Beijing. In addition, most of the risk factors had a higher prevalence in the suburban areas and population at working-age.

CONCLUSIONThe prevalence rate of chronic risk factors was still high in adults of Beijing. Effective interventions should be carried out to prevent further worsening of the situation, especially in the suburban areas and people at working-age.

Adolescent ; Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Alcohol Drinking ; epidemiology ; China ; epidemiology ; Chronic Disease ; epidemiology ; Feeding Behavior ; Female ; Health Behavior ; Humans ; Life Style ; Male ; Middle Aged ; Obesity ; epidemiology ; Overweight ; epidemiology ; Smoking ; epidemiology ; Surveys and Questionnaires ; Young Adult

Adult ; Anti-Bacterial Agents ; administration & dosage ; Bone Transplantation ; Chronic Disease ; Female ; Humans ; Male ; Middle Aged ; Osteomyelitis ; therapy ; Surgical Flaps ; Therapeutic Irrigation