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Purpose@#Invasive ductal carcinoma (IDC) accounts for 90% of triple-negative breast cancer (TNBC). IDC is mainly derived from the breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves. However, little is known about the contribution of the interactions between sympathetic nerves and breast cancer cells to the malignant progression of TNBC. @*Methods@#The expression levels of the β2 -adrenergic receptor (β2 -AR, encoded by ADRB2 gene), nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA) were determined using immunohistochemistry (IHC). NGF expression levels in the serum were compared by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was assessed using the Cell Counting Kit-8 assay. The β2 -AR, NGF, p-ERK, and p-CERB expression levels were determined using western blotting. TNBC cells and neuronal cells of the dorsal root ganglion (DRG) in 2-day-old Sprague Dawley rats were co-cultured. Using norepinephrine (NE), NGF, and β2 -AR, NGF/TrkA blocker pretreatments, the axon growth of each group of DRG neuron cells was detected by immunofluorescence analysis. @*Results@#The sympathetic adrenergic neurotransmitter NE activated the ERK signaling pathway in TNBC cells. NE/β2 -AR signaling promotes NGF secretion. NGF further facilitates the malignant progression of TNBC by increasing sympathetic neurogenesis. In the coculture assay, the sympathetic adrenergic NE/β2 -AR signal pathway also enhanced NGF secretion. NGF binds TrkA in DRG neurons and promotes axonal growth. @*Conclusion@#These results suggest that NE/β2 -AR pathway promotes cell proliferation and NGF production in triple-negative breast cancer.
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Objective To evaluate the role of miR-146a in bone marrow mesenchymal stem cells ( BMSCs)-induced reduction of acute lung injury ( ALI ) in rats. Methods A total of 105 clean-grade healthy male Wistar rats, weighing 170-190 g, were divided into 5 groups ( n=21 each) using a random number table method: control group (group C), phosphate buffer solution group (group P), group ALI, BMSC group ( group B) and BMSC plus miR-146a inhibitor group ( group BM) . ALI was induced by intra-peritoneally injecting 5 mg∕kg lipopolysaccharide ( LPS) 0. 5 ml in anesthetized rats. Phosphate buffer solu-tion 0. 5 ml was injected via the tail vein in group P. In group B, 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein after establishing the model. In group BM, miR-146a inhibitor 50 mg∕kg was injected via the tail vein after establishing the model, and 2 h later 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein. Group C received no treatment. Blood samples were obtained from the abdominal aorta for blood gas a-nalysis at 6, 24 and 48 h after injection of BMSC ( T1-3 ) , the chest was immediately opened, and the lung tissues were obtained for microscopic examination of pathologic changes and for determination of the wet∕dry lung weight ratio ( W∕D ratio) , expression of IRAK-1, nuclear factor kappa B ( NF-κB) and interleukin-6 ( IL-6) ( by Western blot) and expression of miR-146a and IRAK-1 mRNA ( by quantitative real-time poly-merase chain reaction). Results Compared with group C, the pH value and PO2 were significantly de-creased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggrava-ted in ALI, B and BM groups. Compared with group P, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0. 05), and the pathological changes of lung tissues were aggravated in ALI, B and BM groups. Compared with group ALI, the pH value and PO2 were significantly increased, PCO2 and W∕D ratio were decreased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was down-regulated at each time point ( P<0. 05 ) , and the pathological changes of lung tissues were attenuated in group B. Compared with group B, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggravated in group BM. Conclusion miR-146a is involved in BMSCs-induced reduction of ALI in rats.
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Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on mammalian target of rapamycin (mTOR) signaling pathways in lung tissues of rats with acute lung injury (ALI).Methods Healthy pathogen-free adult male Sprague-Dawley rats were selected,and the BMSCs were obtained and cultured in vitro.One hundred and five healthy clean adult male SpragueDawley rats,weighing 170-190 g,were divided into 5 groups (n=21 each) using a random number table:control group (group C),PBS group,group ALI,ALI plus BMSC group (group ALI+BMSCs),and ALI plus phosphate buffer solution (PBS) group (group ALI+PBS).Group C received no treatment.PBS 0.5 ml was injected via the tail vein in group PBS.Lipopolysaccharide (LPS,0.5 ml) 5 mg/kg was intraperitoneally injected to establish the model of ALI in group ALI.BMSCs (0.5 ml) 1×104 cells/ml were injected via the tail vein after intraperitoneal injection of LPS in group ALI+ BMSCs.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of LPS in group ALI+PBS.Arterial blood samples were collected for blood gas analysis at 6,24 and 48 h after injection of BMSCs.Lungs were then removed for determination of wet/dry weight ratio (W/D ratio) and expression of mTOR,nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-o) in lung tissues (by Western blot) and for examination of the pathologic changes of lungs tissues (using haematoxylin and eosin staining).Results Compared with group C,pH value and PaO2 were significantly decreased,PaCO2 and W/D ratio were increased,and the expression of mTOR,NF-κB and TNF-α was up-regulated at each time point in ALI,ALI+BMSCs and ALI+PBS groups (P＜0.05).Compared with group ALI,pH value and PaO2 were significantly increased,PaCO2 and W/D ratio were decreased,the expression of mTOR,NF-κB and TNF-α was down-regulated at each time point (P＜0.05),and the pathologic changes of lungs tissues were significantly attenuated in group ALI+BMSCs.Conclusion The mechanism by which BMSCs reduce ALI may be associated with inhibiting mTOR signaling pathways in lung tissues of rats.
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Aim To observate the effect of chemokine receptor(CXCR4) gene transfection on biological behavior of bone marrow mesenchymal stem cells in vitro.Methods Firstly, bone marrow mesenchymal stem cells were divided into three groups:GFP(transfected GFP into MSCs), CXCR4+(transfected CXCR4+ into MSCs) and CXCR4-(transfected CXCR4-into MSCs) group.Then, their capacity of proliferation, differentiation and migration ability (in vitro) was assessed with immunofluorescence cytochemistry method, flow cytometry assay and Transwell cell chemotaxis test.Results The high or low expression of CXCR4 had no effect on their ability of proliferation and differentiation into lung tissue.Compared with GFP group, however, CXCR4+-MSCs group significantly increased the number of migrating cells, while CXCR4——MSCs group showed no change in the number of migrating cells.Conclusions The proliferation and differentiation capacities are not affected by the high or low expression of CXCR4.The high expression of CXCR4 can significantly enhance the migration ability of MSCs to inflammatory lesions, and the low one has no effect on the migration of the cells.After the transplantation of MSCs, CXCR4′s high expression will access to the lesion area to participate in tissue repairing rapidly and largely, significantly enhancing the therapeutic efficacy.
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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.
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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.
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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.
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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.
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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.