ObjectiveTo evaluate the effect of naloxone on L-type calcium current (ICa-L) in isolated rat ventricular myocytes.MethodsAdult SD rats of both sexes aged 8 weeks weighing 200-250 g were used in this study.Single cardiac ventricular myocytes were enzymatically isolated from SD rats.ICa-L was measured in ventricular myocytes and recorded using whole cell patch-clamp technique.Different concentrations (20 and 100 μg/ml) of naloxone were added to the cardiomyocytes.The effect of naloxone on ICa-L was evaluated.ResultsThe peak current of ICa-L Was inhibited by naloxone in a concentration-dependent manner.Naloxone had no significant effect on steady-state activation curve.ConclusionNaloxone inhibits the L-type calcium channel of ventricular myocytes and exerts negative effect on ventricular muscle function.

In anesthetic clinical teaching,Dalian Medical University Department of Anesthesiology pays more attention to humanity education and keeps improving teaching methods.It also adopts the methods of “the combination of the virtual and reality”,multiple apprentices,PBL and other ways to enhance students' operational skills development and promote their clinical thinking.

Objective To construct plvx-cyclooxygenase-2(COX-2)-DsRed and establish breast cancer cell line MCF7 which overexpressed COX-2, to explore the effect of COX-2 on breast cancer cell.Methods The full-length COX-2 PCR product was obtained by total COX-2 PCR primers and COX-2 cDNA vector.After the PCR product and lentiviral vector plvx-DsRed-Monomer-N1 were cut simultaneously by restriction enzyme BamH1 and Xholl, they were connected and sequenced, to get lentiviral vector plvx-COX-2-DsRed.After selected by puromycin, overexpressed COX-2 breast cancer cell line MCF7-plvx-COX-2-DsRed was obtained.The stable cell line was verified by real time PCR and Western blot.The differences of proliferation ability between stable cell line and normal one were compared by colony formation test and Western blot.Results The lentiviral vector plvx-COX-2-DsRed and stable cell line MCF7-plvx-COX-2-DsRed after selecting were obtained.COX-2 expression level of the stable cell line was 75.29 times as high as that of MCF7, and 64.91 times as high as that of cell line MCF7-plvx-DsRed-Monomer-N1 by PCR assay (P ＜ 0.05), which was consistent with the results of Western blot and microscope photo.MTT results showed that cell line MCF7-plvx-COX-2-DsRed had grown faster than cell line MCF7 and MCF7-plvx-DsRed-Monomer-N1 from the 2nd day (P ＜ 0.05), which was accordant with colony formation assay.MCF7-plvx-COX-2-DsRed cell line had higher c-myc expression and lower β-catenin expression than MCF7 cell and cell line MCF7-plvx-DsRed-Monomer-N1 detected by Western blot relative quantification (P ＜ 0.05).Conclusion The plvx-COX-2-DsRed lentiviral vector and cell line MCF7-plvx-COX-2-DsRed are successfully constructed.COX-2 can increase proliferation of MCF7 cells through up-regulating the expression of c-myc.

Objective:To analyze the expression of the four mismatch repair genes protein (hMLH1,hMSH2,hMSH6 and hPMS2) of patients with colorectal cancer and its clinical significance.Methods:177 cases of patients with colorectal caner in the Third Affiliated Hospital of Guangzhou Medical University from January 2013 to December 2015 were randomly selected.Tested the expression of the hMLH1,hMSH2,hMSH6 and hPMS2 by immunohistochemistry,the relationship between protein expression and clinical parameters was analyzed.Results:Among 177 cases ofcolorectal cancer tissue,the deletion rate ofhMLH1 protein was 6.2％ (11/177),the deletion rate of hMLH2 protein was 4.0％(7/177),the deletion rate of hMSH6 protein was 1.7％(3/177),the deletion rate of hPMS2 protein was 8.0％(14/177),the sum of the four values accounted for 19.8％(35/177) of all cases of colorectal cancer.The loss of expression of the four mismatch repair genes protein were correlated to tumor location (P＜0.05),besides,the loss of expression of the hMLH1 and hPMS2 protein were correlated to degree of tumor differentiation (P＜0.05),he loss of expression of the hMSH6 protein were correlated to depth of tumor invasion(P＜0.05);But the loss was not correlated to age,sexes,lymph node metastasis and distant metastasis(P＞0.05).Conclusion:The expression of loss phenomenon with mismatch repair protein appears in part of colorectal cancer,the loss phenomenon with mis match repair protein were correlated to tumor location and degree of tumor differentiation.Mutations of four genes in hMLH1,hPMS2,hMSH6 and bMSH2,to provide a reference value for the clinical judgment of prognosis and to develop a treatment plan.

Objective To investigate the change of the neutrophil apoptosis and neutrophil respiratory burst in the patients and the effect of ulinastatin on the apoptosis of neutrophil and respiratory burst of neutrophil during cardiopulmonary bypass (CPB). Methods Sixty-two patients undergoing valve replacement with CPB were randomly divided into two groups: ulinastatin group (U group, 31 cases) and control group (C group, 31 cases). In U group patients received ulinastatin after induction of anesthesia. In C group patients received equal volume of normal saline, instead of ulinastatin. Arterial blood was obtained before operation (T1), 30 min after the start of CPB (T2), 30 min after the termination of CPB (T3). The apoptosis of neutrophil and respiratory burst of neutrophil were measured by flow cytometer. The level of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by kit. Results In C group, compared with T1 [(66.57±5.93)%], the rate of the apoptosis of neutrophil was significantly decreased at T2[(55.37±3.51)%] and T3 [(48.92±4.21)%] (P<0.05). And in U group, compared with T1 [(73.57±7.94)%], the rate of the apoptosis of neutrophil was significantly decreased at T2 [(68.34±4.92)% ] and T3 [(62.13±4.76)%] (P<0.05), And it reached to the minimum at T3. The rate of the neutrephil apeptosis was significantly lower in C group than that in U group (P<0.05). The respiratory burst of neutrophil increased significantly after the start of CPB and reached to the peak at T3[C group (1422.50±89.75) MCF,U group (1156.52±93.20) MCF]. The respiratory burst of neutrophil in U group was significandy lower than that in C group at T2 and T3 (P<0.05). The vitality of SOD decreased significantly after the start of operation in the two groups (P<0.05). The level of MDA increased significantly after the start of operation in the two groups, and reached to the peak at T3. The vitality of SOD in C group was significantly lower than that in U group at T3 (P<0.05). The level of MDA in C group was significantly higher than that in U group at T3 (P<0.05). Conclusions The rate of neutrophil apoptosis decreased and the respiratory burst of neutrophil increased during CPB. By improving the apoptosis of neutrophil and reducing the respiratory burst of neutrophil, ulinastatin can inhibit inflammatory reaction during CPB. Meanwhile, ulinastatin can improve the vitality of SOD and reduce the level of MDA. In conclusion, ulinastatin has a significant protective effect during CPB.

Objective To study the foxml gene and its protective effect on the lung tissue of rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS), and to observe the dexamethason' s (DEX) impacts on foxml gene and the prognosis of ALI. Method Seventy-two healthy mice were randomly(random number) divid-ed into three groups: control group (A group, n = 24), model group (B group, n = 24) and DEX treatment group (C group, n = 24). The observing intervals were respectively set in 24 h, 48 h and 72 hours. At each ob-serving interval, the foxml protein in lung tissue of mice was detected by using immunohistochemistry (IHC), and the expression of foxml gene in lung tissue was detected by using RT-PCR, as well as to observe the pathological changes in lung tissue. Results Comparisons were made between paired groups at 24 h,48 h and 72 h intervals in which the expression of foxml mRNA and the level of foxml protein in lung tissue of mice in C group were signifi-cantly higher than those in B group (P < 0.05), and those in B group were significantly higher than those in A group (P < 0.05). The expression of foxml mRNA and the level of foxml protein in lung tissue of mice in B group at 48 h interval were significantly higher than those both at intervals of 72 h and 24 h (P < 0.05), and the those at 72 interval were significantly higher than those at 24 h interval (P < 0.05). Compared with B group, the pathologi-cal changes in lung tissue of mice in C group were lessened. Conclusions In both model group and dexamethasone treatment group, the expression of foxml mRNA and the level of foxml protein in lung tissue of mice are increased significantly. Dexamethasone lessens the injury of both vascular endothelial cells and alveolar epithelial ceils of lung tissue, and it also significantly increases the expression of foxml mRNA and the level of foxml protein.

Objective To determine whether p38 mitogen-activated protein kinase (p38MAPK) signaling pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.Methods Two hundred and twenty-five male Kunming mice weighing 30-40 g were randomly divided into 4 groups:group control ( group C,n =55 ) ;group fractalkine (group F,n =60); group anti-CX3CR1 + fractalkine (group CF,n =55) and group SB203580 (p38MAPK inhibitor) + fractalkine (group SF,n =55).Fractalkine 100 ng was injected into cerebral lateral ventricle (i.c.v.) in groups F,CF and SF.Anti-CX3CR1 1 μg and SB203580 1 μg were injected i.c.v.at 1 h before fractalkine injection in groups CF and SF respectively.Paw withdrawal latency to a thermal nociceptive stimulus (PWL) was measured at 30 min before the drugs were injected into cerebral lateral ventricle and 30,60,120 and 240 min after fractalkine injection.Five animals were sacrificed after PWL measurement at each time point and their brains were removed for determination of phosphorylated p38MAPK protein expression (by Western blot analysis).Five animals were sacrificed at 30 min before the drugs were injected into cerebral lateral ventricle and 6,12 and 24 h after fractalkine injection for determination of IL-1β and TNF-α contents in the brain (by ELISA) in all the 4 groups.In group F 5 animals were sacrificed at 4 h after fractalkine injection for determination of action of fractalkine on microglia or astrocyte (by immunofluorescence).Results Fractalkine i.c.v.injection significantly reduced PWL and increased phosphorylated 38MAPK,IL-1β and TNF-α levels in group F as compared with group C.Pretreatment with anti-CX3CR1 or SB203580 significantly decreased fractalkine-induced hyperalgesia and phosphorylated-p38MAPK,IL-1β and TNF-α levels in groups CF and SF as compared with group F.Fractalkine was localized at microglia.Conclusion p38MAPK signal transduction pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.

Objective To evaluate the role of inositol triphosphate receptor (IP3 R) in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes (5 ml/dish),50 ml culture flasks (8 ml/flask) or 24-well plates (1 ml/hole) with a density of 1 × 105/ml and randomly divided into 5 groups (n =25 each) ∶ control group (group C),fractalkinegroup (group F),CX3C chemokine receptor 1 (CX3CR1) antibody anti-CX3CR1 + fractalkine group (group CF),IP3R antagonist 2-APB + fractalkine group (group AF) and p38 mitogen-activated protease (p38MAPK) inhibitor SB203580 + fractalkine group (group SF).Fractalkine 10 nmol/L was added to the culture medium in groups F,CF,AF and SF.The anti-CX3CR1 15 μmol/L,2-APB 50 μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF,AF and SF,respectively,1 h before addition of fractalkine.The cells were then cultured for 24 h.The intracellular Ca2+ concentration ([Ca2+]i) was measured during the 10 min incubation with fractalkine.The phosphorylation of p38MAPK was measured at 0,30,60,120 and 240 min of incubation with fractalkine.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in theculture medium were determined at 24 h of incubation with fractalkine.Results Compared with group C,[Ca2+]i,and the phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly increased in groups F,CF,AF and SF (P ＜ 0.05).[Ca2+]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly lower in groups CF,AF and SF than in group F (P ＜ 0.05).Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.

OBJECTIVE To investigate the condition of drug-resistant genes in MRSA and MSSA. METHODS The drug-resistant genes mecA,ermA/B/C,aac(6′)/aph(2″),aph(3′)-Ⅲ,ant(4′,4″) and tetM of MRSA and MSSA were detected by polymerase chain reaction(PCR). RESULTS The 5 kinds of drug-resistant genes,such as mecA,ermA/B/C,aac(6′)/aph(2″),(aph(3′)-Ⅲ) and tetM were positive in MRSA. CONCLUSIONS MRSA is a multi-resistant pathogen.