Objective To investigate the effect of exogenous semaphorin 3A (Sema3A) on apoptosis in primary cultured rat cortical neurons and the roles of phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (Akt) pathway in apoptosis induced by Sema3A.Methods Newborn Sprague-Dawley rat cortical neurons were cultured in vitro and they were identified by microtubule associated protein-2 (MAP-2) staining The cultured cortical neurons were treated with various concentrations of Sema3A (0,500,1 000,and 2 000 μg/ml) for 48hours.Neuronal survival rate was detected with CCK8 assay.Neuronal apoptosis was detected with Hoechst33342 staining and TUNEL staining.The expressions of P-Akt,Akt and Bcl-2 in cortical neurons were determined with Western blotting.Results The purity of cortical neurons culture was more than 95％.CCK8 assay showed that the survival rates of cortical neurons in the groups of 500,1 000and 2 000 μg/ml Sema3A were 80.9％ ± 5.3％,67.5％ ± 3.9％ and 50.2％ ± 4.4％ of the control group,respectively (F =165.042,P =0.000).Hoechst33342 staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 22.4％ ± 1.2％,34.0％ ± 1.2％,39.3％ ± 1.4％ and 47.3％ ±2.3％,respectively (F =103.237,P =0.000).TUNEL staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 23.9％ ± 1.1％,31.9％ ± 1.0％,40.1％ ± 1.5％ and 51.4％ ± 3.4％,respectively (F =103.118,P =0.000).Western blotting showed that the expressions of P-Akt (F =15.959,P =0.001) and Bcl-2 (F=18.776,P =0.001) decreased gradually,while the expression of Akt had no significant changes (F =0.590,P =0.639).Conclusions Sema3A can decrease the survival rate of the cultured cortical neurons,mainly by inducing apoptosis,and the mechanism of which might be related to the down-regulation of expressions of P-Akt and Bcl-2.

Objective To examine cerebrovascular reactivity to CO2 inhalation in mice. Methods In vivo Two-Pho?ton imaging technique was used to record the reaction of cerebral cortical vessels including penetrating artery, surface vein and capillary in 5 male C57 mice after CO2 inhalation under a thinned-skull cranial window. Nitric oxide syntheses inhibitor L-NAME and Prostaglandin syntheses inhibitor Indomethacin were used to block different vasodilator pathways, respectively. Results Different mouse cortical vessels displayed different degrees of dilation to 1-minute 5%CO2 inhala?tion. The penetrating artery exhibited the most obvious dilation (45.01%±4.45%). L-NAME intervention significantly di?minished cerebravascular CO2 reactivity(P<0.05). Indomethacin significantly attenuated the dilation of artery but not capillary comparing with L-NAME intervention(P<0.05). Conclusions Different vessels react differently to CO2 inhala?tion in which postaglandins and NO signal pathways are involved.

Background Quality of life(QOL) refers to the person's subjective appraisal of well-being,life satisfaction,health and functional performance. For those stroke patients who cannot finish the scale themselves,we can try the proxy measurement. However,until recently no QOL scale for proxies were induced to Chinese stroke patients. The goal of this research is to translate and test the SIS 3.0 for proxy,and differences between patient and proxy scores. To translate and test the SIS 3.0 for proxy, and analyzed differences between patient and proxy scores.Methods Ten pairs of patients and their proxies were involved in the primary test. Two hundreds and thirty-three pairs were involved in the formal test. We analyzed the validity, responsiveness, reliability and feasibility of the SIS 3.0 for proxy, as well as the validity in proxy assessment. Results The feasibility was sufficient. Both Split reliability and α coefficient were more than 0.8, demonstrating SIS a reliable instrument. SIS had a good content validity with correlation coefficient more than 0.6. Good criterion validity was established by comparing the scores on various domains to standardized measures with P=0.000. Construct validity was also good as indicated by factor analysis. Proxy scores were significantly different across OHS scales which showed domain responsiveness was good. Comparison of patient and proxy responses resulted in no significant difference. Conclusions SIS for proxy is satisfactory for chinese patients. It is feasible to use a proxy respondent to answer questions on the patient's behalf.

Objective To investigate the effects of insulin on vascular diameter of the peri -infarct region and infarct volume after cerebral infarction in mice. Methods Forty male C57/BL6j mice w ere randomly divided into a control group ( n = 5), a cerebral infarction group ( n = 15), a cerebral insulin resistance group (n = 5), and a cerebral insulin resistance infarction group ( n = 15). A model of cerebral infarction w as induced by the photochemical method. A model of cerebral insulin resistance w as induced by intracerebroventricular injection of streptozocin. Tw o -photon confocal microscope w as used to in vivo evaluate the changes of vascular diameter in the peri-infarct region at 20 min after insulin injection into the cerebelomedulary cistern. After modeling of cerebral infarction, artificial cerebrospinal fluid or insulin (10 ng/ml) w as immediately injected into the cerebelomedulary cistern, and the effect of insulin on cerebral infarct volume w as evaluated at 24 h after infarction. Results Insulin did not have significant effect on various types of cerebral vascular diameters in the normal control group, but it significantly contracted cerebral arteries ( -23.16% ±6.86% and -23.32% ±6.40%, respectively; al P <0.001) and penetrating arteries ( -15.20% ±5.51% and -16.40% ±4.27%, respectively; al P < 0.001) in the cerebral insulin resistance group and the cerebral insulin resistance infarction group, but it did not have any effect on the diameters of the cerebral veins. There w ere no significant differences in the vasoactive effects of insulin betw een the cerebral infarction group and the normal control group, as w el as betw een the cerebral insulin resistance group and the cerebral insulin resistance infarction group. Insulin significantly reduced the volume of cerebral infarction in the cerebral infarction group (9.0 ±1.0 mm3 vs.6.0 ±1.2 mm3; t = 4.294,P =0.002), and it did not have significant effect on the volume of cerebral infarction in the cerebral insulin resistance infarction group ( 12.6 ±2.3 mm3 vs.11.6 ±1.7 mm3; t = 0.782, P = 0.456). Conclusions Insulin can reduce ischemic brain injury in normal mice and can not affect the cerebrovascular diameter of the peri-infarct region. The neuroprotective effect of insulin is not significant in cerebral insulin resistance in mice, and it may be associated w ith the vasoconstrictor effects of insulin in the peri -infarct region.

Objective To investigate the mechanisms underlying neuroprotection of silent information regulation 2 homolog 3 ( SIRT3 ) against hypoxia via preconditioning.Methods PG12 cells were randomly divided into control,hypoxic preconditioning ( Hyp),Hyp with oxygen-glucose deprivation (OGD) and OGD.MTT assay and DAPI staining were used to evaluate cellular viability.MitoSOX Red was used to measure the production of mitochondrial superoxide.The protein expression of SIRT3,PGC-1α and MnSOD were assessed by Western blot.Recombinant SIRT3 was also given to further investigate its roles in hypoxic preconditioning.Results The preconditioned PC12 cells had a higher survival rate.When expressed as a percentage of the control group,MTT values following 6 h OGD were around 51.0% in the OGD group but around 74.7% in the Hyp + OGD group ( F = 56,P ＜ 0.01).Mitochondrial ROS after Hyp was less than the OGD group.Both Hyp + OGD and OGD increased the expression of SIRT3,PGC-1α and MnSOD proteins,and these increases were greater after Hyp + OGD.Similarly,the application of recombinant SIRT3 to OGD also further increased the expression of these proteins.Conclusions Hypoxic preconditioning can protect PC12 cells against hypoxic injury.One possible mechanism of hypoxic preconditioning is via SIRT3 to upregulate PGC-la and,in turn,MnSOD to reduce generation of ROS.

Objective To discuss the relationship between prognosis and different surgical procedures for gallbladder cancer in different stages. Methods The clinical data of 107 patients with gallbladder cancer from January 2001 to May 2007 were retrospectively analyzed. The surgical procedure was chosen according to different stages. Results Eighty-one of the 107 patients (75.6%) were followed up with the median time of 5 years. Of the 10 patients with stage Ⅰ gallbladder cancer who had underwent simple cholecystectomy, 9 survived. Of the 8 patients with stage Ⅱ gallbladder cancer, 3 received palliative cholecystectomy and the median survival time was 12 months, which was significantly shorter than 24 months of the remaining 5 patients who received radical operation (X2= 5.698, P <0.05). Of the 42 patients with stage Ⅲ gallbladder cancer, 18 received radical operation, and the median survival time was 24 months, which was not significantly different from 18 months of the 5 patients who received extended radical operation (X2=0.238, P>0.05). The remaining 19 patients received palliative operation, and the median survival time was 6 months, which was significantly shorter than those of patients received radical operation or extended radical operation (X2=5.772, 6.318, P <0.05). There were 47 patients with stage Ⅳ gallbladder cancer. Seventeen patients received extended radical operation and 30 received palliative operation, and no significant difference upon the median survival time was observed among different surgical procedures (X2=0.001,0.694, P>0.05). The complication recurrence after the extended radical operation was significantly higher than palliative operation (X2=6.039, P<0.05). Conclusions For patients with stage Ⅰ gallbladder cancer, simple cholecystectomy is preferred. Radical operation is good for patients with stage Ⅱ gallbladder cancer. The choose of radical operation or extended radical operation for patients with stage Ⅲ gallbladder cancer should be based on the condition of invasion. Palliative operation could be used to patients with stage Ⅳ gallbladder cancer.

Objective To explore the effects of inducible co-stimulator (ICOS) gene on the cytotoxic activity of cytokine-induced killer (CIK) cells against cholangiocarcinoma cells. Methods CIK-ICOS cells were obtained by stable transfecting ICOS genes into CIK cells through the adenovirus vector whereas untransfected and EGFP-transfected CIK cells were treated as controls. The proliferation and apoptosis of different CIK cells, as well as their cytotoxicity against cholangiocarcinoma cells in the three groups were detected. The expressions of IFN-T, IL-2 and TNF-α in the supernatant of different CIK cells were measured by ELISA. SCID mice with cholangiocarcinoma were randomly divided into CIK group, CIK-EGFP group, CIK-ICOS group and normal saline group. The cytotoxic activity of CIK-ICOS cells against cholangiocarcinoma cells in vivo was observed. Results CIK-ICOS cells displayed better proliferation than CIK cells and CIK-EGFP cells. At day 20 and 23 of culture, the apoptosis rate of CIK-ICOS cells was 0.69% and 0.89%, respectively, while that of the CIK cells was 2.90% and 4.92%. The cytotoxic effect of CIK-ICOS cells at different E: T ratio against cholangiocarcinoma cells was significantly stronger than that of CIK cells and CIK-EGFP cells (F=13.37, 6.46, 25.51, P<0.05). The concentration of IFN-γ in CIK-ICOS cultured supernatant was (49.50±4.73)μg/L, which was significantly higher than that in the cultured supernatant of CIK cells [(30.53±3.73)μg/L] and CIK-EGFP cells [(30.12±2.64)μg/L](F=38.89, P<0.05). The growth of cholangiocarcinoma was significantly slower in CIK-ICOS group than that in CIK group and CIK-EGFP group, whereas the necrosis area of tumor was larger and the CIK cells in CIK-ICOS group was more than those in the other two groups. Conclusions CIK cells had the function of killing cholangiocarcinoma cells in vitro and in vivo. After ICOS genes were transfected into CIK cells, the survival time of CIK cells in vitro was prolonged and the proliferation of CIK cells was enhanced, as well as the secretion of IFN-γ was increased so that the cytotoxicity of CIK cells against cholangiocarcinoma cells in vitro and in vivo was enhanced.

The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The H₂O₂ levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

Objective:To compare the efficacy and safety of Tonghua Dongbao′s insulin aspart injection (Rishulin) and NovoRapid (Novo Nordisk) in the treatment of diabetes.Methods:A 26-week, randomized, open-label, parallel-group, positive control drug and non-inferiority trial was conducted in 23 centers in China. A total of 563 diabetes with poor blood glucose control treated with insulin for at least 3 months before were included. The subjects were randomized(stratified block random method) into those receiving Rishulin or NovoRapid at a ratio of 3∶1. Both groups were combined with basal insulin (Lantus). The primary endpoint was the change in glycosylated hemoglobin (HbA1c) from baseline to the end of 24 weeks of treatment.Results:For full analysis set, after 24 weeks of treatment, HbA1c level of Ruishulin group decreased from (8.66±1.28)% to (7.77±1.09)% ( P<0.001), and that of NovoRapid group decreased from (8.47±1.28) % to (7.65±0.97) % ( P<0.001). Treatment difference in HbA1c (NovoRapid group-Ruishulin group) was -0.061% (95% CI -0.320-0.199). HbA1c<7.0% target reacing rates were 24.26% and 21.21% ( P=0.456), and HbA1c<6.5% target reacing rates were 9.65% and 6.82% ( P=0.310) in Ruishulin group and NovoRapid group, repectively. The standard 2 hours postprandial blood glucose (2hPG) in Ruishulin group decreased from (16.23±5.22) mmol/L to (12.65±4.57) mmol/L ( P<0.001), and 2hPG in NovoRapid group decreased from (16.13±5.37) mmol/L to (11.91)±4.21) mmol/L ( P<0.001). The fingertips blood glucose at 7-point of both groups exhibited varying degrees of reduction compared with those at baseline, repectively. Positive ratios of specific antibodies were 31.68% in Ruishulin group and 36.36% in NovoRapid group ( P=0.320). Ratios of negative to positive were 7.43% and 10.61% ( P=0.360), and ratios of positive to negative were 10.40% and 7.58% ( P=0.360) in Ruishulin group and NovoRapid group, respectively. The incidence of hypoglycemia was 60.05% and 55.40% ( P=0.371), and the incidence of adverse events was 76.60% and 77.70% ( P=0.818) in Ruishulin group and NovoRapid group, respectively. Conclusions:Rishulin is not inferior to NovoRapid, and has shown good efficacy and safety. It can be an ideal choice for clinicians in patients with poor blood glucose control with insulin.

Humans ; Minocycline ; analogs & derivatives ; therapeutic use ; Stem Cell Transplantation ; adverse effects