AIM: To evaluate the effects of all-trans retinoic acid (atRA) on the proliferation in cultured mouse cerebral microvascular endothelial cells (bEnd.3) . METHODS: Cultured cells were divided into five groups randomly, one as control group, the other four groups were 10-9, 10-8, 10-7 and 10-6 mol/L group. Effects of atRA on proliferation in bEnd.3 cells were detected by flow cytometry and immunocytochemitry of PCNA and MTT at 24 h, 48 h and 72 h. The effects of atRA (10-6 mol/L group) on the expressions of angiogenic genes in bEnd.3 cells were studied using microarray. RESULTS: The results of MTT and flow cytometry showed that all- trans retinoic acid at concentration of 10-6 mol/L significantly inhibited the proliferation of bEnd.3 cells. Immunocytochemical staining showed the expression of PCNA was markedly decreased in bEnd.3 cells at 24 h after treatment with atRA. Microarray results demonstrated that there were 11 down - regulated angiogenic genes and 2 up - regulated angiogenic genes in 10-6mol/L atRA group. CONCLUSION: All - trans retinoic acid at concentration of 10-6mol/L may significantly inhibit the proliferation of bEnd.3 cells treated for 24 h ire vitro via down-regulation of angiogenic genes and PCNA expression.

Objective To analyze the distribution and antibiotic resistance of pathogens isolated from sputum in early postoperative patients after esophageal surgery, and investigate the risk factors of pulmonary infections after esophageal carcinoma and reasonable application of antibiotics during perioperative period.Methods The clinical data of 414 patients with esophageal carcinoma who were admitted to the Affiliated Hospital of Binzhou Medical University from January 2012 to June 2014 were retrospectively analyzed.The second generation cephalosporins were used as preventive antibiotics for the 181 patients between January and December 2012 and the third generation cephalosporins were used for the 233 patients between January 2013 and June 2014.Sputum samples were collected and inspected before operation and in the first three days after operation for observing distribution and antibiotic resistance of pathogens.The incidence of postoperative pulmonary infection was observed in the two kinds of patients.The risk factors closely related to the occurrence of postoperative pulmonary infections were analyzed.Comparison of rate and univariate analysis were done by chi-square test.Multivariate analysis was done with logistic regression.Results The positive rate of early post-operative sputum culture was 24.64％ (102/414), 32.04％ (58/181) in patients receiving the second generation cephalosporins and 18.88％ (44/233) in patients receiving the third generation cephalosporins, respectively, with a significant difference between the two generations of cephalosporins (x2 =9.502, P ＜ 0.05).A total of 131 strains of pathogens were isolated from early postoperative sputum samples including 12 strains of gram-positive bacteria and 119 strains of gram-negative bacteria.The top three bacteria were Klebsiella pneumoniae subsp pneumoniae (33 trains), Acinetobacter baumannii (20 strains) and Pseudomonas aeruginosa (19 strains).The resistance rate of gram-negative bacteria in early postoperative sputum samples to meropenem, ciprofloxacin and levofloxacin were 5.0％-25.0％, 2.6％-15.2％, 2.6％-20.0％, respectively.Among cephalosporins, the resistance rate to ceftriaxone, cefotaxime, cefepime, ceftazidime, cefuroxime and cefazolin were 10.3％-20.0％ , 5.3％-30.0％ , 12.8％-31.6％, 15.4％-42.1％ , 21.2％-55.0％ and 15.2％-60.0％, respectively.The overall postoperative pulmonary infection rate was 18.60％ (77/414).The pulmonary infection rate of patients receiving the second generation cephalosporins was 26.52％ (48/181), which was higher than 12.45％ (29/233) of patients receiving the third generation cephalosporins (x2=13.326, P ＜ 0.05).The results of univariate analysis showed that smoking, chronic obstructive pulmonary disease, application of the second generation cephalosporins during the perioperative period and early postoperative sputum culture positive were correlated with the postoperative pulmonary infection after esophageal carcinoma surgery (x2 =5.149, 22.765, 13.326, 159.092, P ＜ 0.05).Multivariate statistical analysis with logistic regression demonstrated that application of the third generation cephalosporins during the perioperative period was independent protective factor of postoperative pulmonary infection (OR =2.582, 95％ confidence interval: 1.331-5.009, P ＜ 0.05).Conclusions The pathogens from sputum in early postoperative period after esophageal surgery are complicated with gram-negative bacteria as the main pathogens especially Klebsiella pneumoniae subsp, pneumoniae, and different levels of antibiotic resistance are ubiquitous.The third generation cephalosporins can effectively prevent the postoperative pulmonary infection after esophageal carcinoma surgery.

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Objective To study the relationship between oxidative stress and endothelial cell apoptosis in patients with obstructive sleep apnea-hypoventilation syndrome (OSAHS). Methods Sixth-seven patients definitely diagnosed by potysomnography (PSG) as OSAHS were divided into three groups according to their apnea-hypoventilation index (AHI), 14 in mild group (5 < AHI≤20), 21 in moderate group (20 < AHI≤40) and 32 in severe group (AHI 40). And, 18 healthy persons (AHI <5) were recruited as controls. Blood samples were obtained form all of them after PSG performance for measuring apoptotic endothelial cells (CD146AnnV+) and serum levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Results Serum level of MDA and CD146AnnV+ in moderate and severe OSAHS group were significantly higher than those in control group (all P < 0.05). Serum level of SOD in moderate and severe OSAHS group was significantly lower than that in control group (P < 0.05). CD146AnnV+ correlated positively with AHI, the longest apnea time (LAT) and oxygen desaturation index (ODI) (r = 0.778, 0.609 and 0.689, respectively, all P < 0.05) and correlated reversely with saturation of arterial blood oxygen at night (SaO2min) (r =-0.635, P < 0.01). CD146AAnnV+ correlated positively with serum level of MDA (r = 0. 698, P < 0.01), and correlated reversely with serum level of SOD (r =-0.705, P < 0.01). Results of linear multivariate regression analysis showed that AHI, serum levels of SOD and MDA were independent risk factors for endothelial cells apoptosis in patients with OSAHS. Conclusions There existed oxidative stress due to intermittent hypoxia in patients with OSAHS, which could be one of the major causes in exacerbating endothelial damage.

Objective To investigate the changes in serum level of vascular endothelial growth factor (VEGF), nitric oxide (NO), erythropoietin (EPO) and plasma level of carbon monoxide (CO) in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) and their clinical significance. Methods Sixty-four patients diagnosed as OSAHS by polysomnography (PSG), were divided into two groups according to apnea-hypopnea index (AHI), 20 in mild group [5/h≤AHI≤20/h, with a mean of (12± 5)/hi and 44 in moderate-severe group[AHI > 20/h, with a mean of (63±23)/h]. Blood samples were obtained from all the patients after PSG for measurement of levels of serum VEGF, NO and EPO and plasma CO. Results Levels of serum VEGF [(101±91) ng/L], NO [(10.3±3.3) μmol/L]and plasma CO [(0.56±0. 35) mg/L]in moderate-severe OSAHS group were significantly higher than those in mild group (all P <0.05). There was no significantly difference in serum level of EPO between the two groups (P> 0.05). Levels of serum VEGF, NO and plasma CO in OSAHS patients positively correlated with AHI and percentage of time with percutaneous arterial blood oxygen saturation lower than 0. 9 (SLT90) of all sleep time (all P < 0. 05), but reversely correlated with the lowest arterial blood oxygen saturation (LSaO2) at night (P>0.05). No correlation between EPO and all the indicators was found (P>0.05). Multiple linear regression analysis showed that LSaO2 was an independent risk factor to affect levels of serum VEGF and plasma CO (R2=0.198, P=0.001, and R2=0.210, P=0.000, respectively) and SLT90 was an independent risk factor to affect serum level of NO (R2= 0. 148, P=0.004) in OSAHS patients. Conclusion Hypoxia at night is a main cause leading to increased level of serum VEGF, NO and plasma CO in OSAHS patients.

Objective To asscess cognitive changes of patients with Obstructive Sleep Apnea-Hypopnea Syndrome(OSAHS)by the Expansive Scale of Dementia(ESD),and to compare ESD to Mini Mental Status Examenation(MMSE)in cognitive function.Methods 90 OSAHS patients were investigated in our study.All patients were divided into three groups,according to apnea-hypopnea index(AHI),including mild group,moderate group and severe group.The cognitive function was evaluated by MMSE and ESD.Results The parameters,including AHI,Longest apnea time(LAT)and percentage of time spend when oxygen saturation lower than 0.9(SLT 90%) in severe OSAHS group were significantly higher than those in mild and moderate groups(P0.05).The total score of MMSE and subtest scores of MMSE in memory,attention,calculation and short-and long-term memory were significantly lower in severe OSAHS group than those in mild and moderate groups(P0.05).Conclusion There was the obvious decrease in cognitive function in patients with severe OSAHS,especially in leaning,memory,calculation and constructive function.Both ESD and MMSE could be used to evaluate the cognitive function in OSAHS patients,especially ESD.

AIM: To study the cytological characteristics and gene expression of normal cultured bEnd.3, a mouse brain microvascular endothelial cell strain. METHODS: The morphology of bEnd.3 was studied by light and electronic microscopy, its molecular markers were observed by immunocytochemistry. Cell proliferation kinetics and apoptosis were analyzed by flow cytometry and MTT assay, PGE_2 level was measured by ELISA, and expression of the genes that closely related with vascular endothelial functions was studied by gene micro-array. RESULTS: bEnd.3 had morphological characteristics of microvascular endothelial cells (MVEC) growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli. Furthermore, bEnd.3 showed positive staining for vW factor and CD34 and secreted high level of PGE_2 (644.55?30.24 ng/L). Gene micro-array analysis showed CD31, CD36, CD105 expression, and other genes closely related to microvascular endothelial functions also expressed at relatively high level. In addition, bEnd.3 responsed sensitively to mitogen such as basic fibroblast growth factor. CONCLUSION: bEnd.3 is a kind of MVEC, and it can be utilized to study the mechanisms of some diseases such as cancers and cardio- cerebral vascular diseases.

Objective To evaluate the effects of hyperbaric oxygen (HBO) and cisplatin (DDP) on inhibition of A549/DDP cells in vitro and to explore the impacts of HBO on cell cycle and apoptosis.Methods The cytotoxicity of HBO and DDP was determined by Methyl thiazolyl tetrazolium (MTF) assay; flow cytometry (FCM) was used to investigate the impacts on cell cycle and apoptosis of cell line A549/DDP.Results Hyperbaric oxygen alone significantly suppressed A549/DDP cells proliferation (P ＜ 0.05),compared with control group of normal oxygen,and the inhibition ratio was 2.90％.DDP at concentrations ranging from 3 μg/ml to 24 μg/ml significantly inhibited the proliferation of A549/DDP cells in a dose dependent manner,that was concentrations of 3,6,12 and 24 μg/ml brought inhibition ratio of 1.87％,4.62％,11.15％ and 30.45％ ; Hyperbaric oxygen can enhance the inhibition effect of DDP to A549/DDP cells,when hyperbaric oxygen affiliated with DDP of concentrations above,the inhibition ratios were 4.37％,8.92％,21.88％ and 48.71％,respectively,which has significant difference with the single DDP groups.FCM analyses showed that HBO arrested A549/DDP cells at the S phase,that was cell number of G0/G1 phase reduced significantly and of S phase raised significantly,compared with normal oxygen group(P ＜0.01),that was the percentages of cells in S phase of control group were 9.5％ and HBO group were 20.9％,while the percentages of cells in G0/G1phasc were 82.2％ and 66.3％,respectively.In addition,there were more apoptotic cells in the HBO group with the percentage of 7.9％,while the percentage was 2.8％ in the normal oxygen group.Conclusion Hyperbaric oxygen could play together with DDP to inhibit A549/DDP cells proliferation.HBO could block the cell cycle at S period and induce apoptosis.

AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd. 3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl - 2) and Western blotting (caspase - 3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd. 3 cells in 25 mg/L group was significantly inhibited ( P ＜0.05), but apoptosis in the 100 mg/L group was significantly increased (P ＜ 0.05). In apoptosis inhibited group, the Fas immunocytochemical staining was weaker, the positive cells were significantly decreased ( P ＜ 0.05) and caspase - 3 expression was decreased compared with control group; however, the Bcl - 2 staining was stronger and the positive cells were significantly increased ( P ＜ 0.05). On the other hand, in apoptosis increased group ( 100 mg/L group), the changes were just opposite. CONCLUSIONS: The effect of crenulatin on apoptosis of mouse cerebral microvascular endothelial cells possesses a dual - direction change, inhibitive effect in 25 mg/L and stimulative effect in 100 mg/L group, respectively. The mechanism is related to the alterations of Fas/Bcl - 2 expression and caspase - 3 activity.

] AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd.3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl-2) and Western blotting (caspase-3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd.3 cells in 25 mg/L group was significantly inhibited (P