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Objective:To investigate the influence of α cells and glucagon-like peptide l (GLP-1) in the function of β cells (INS-1 cells) in the rats,and to elucidate the possible mechanism of α cells and INS-1 cells transplantation in influencing hypoglycemia.Methods:The proliferation abilities of INS-1 cells after treated with 10％,20％ and 30％ islet α-cell conditioned medium and 0.03,0.30,3.00,30.0 mg · L-1 of GLP-1 were analyzed by MTT assay.The levels of insulin secretion of INS-1 cells after treated with 10％,20％,30％ α cells,α-cell conditioned medium and different concentrations of GLP-1 were analyzed by enzyme linked immunosorbent assay (ELISA).The concentrations of Ca2+ in INS-1 cells after treated with high glucose and GLP-1 were analyzed by laser confocal microscope.The expression levels of insulin protein after treated with different concentrations of islet α-cell conditioned medium and different concentrations of GLP-1 were detected by Western blotting methed.After the INS-1 cells,the mixture of INS-1 cells and α cells were transplanted into the left renal capsule of the nude mice,the blood glucose levels and the kidney morphology were observed.The levels of insulin/glucagon in the transplanted cells were detected by immunohistochemistry.Results:Compared with control group,both of α-cell conditioned media and GLP-1 promoted the INS-1 cell proliferation and insulin secretion (P ＜ 0.05).The laser confocal microscope results revealed that GLP-1 stimulated the increased intracellular Ca2+ concentration in INS-1 cells (P＜ 0.05).Compared with control group,there was no significant difference in the expression levels of insulin protein in the insulin-1 cells after treated with islet α cell conditioned medium and GLP-1 (P＞0.05).Compared with pre transplantation,the blood glucose level in the transplanted INS-1 cells was significantly decreased at 35 d after renal capsul trasplantation (P＜0.05),and even hypoglycemia presented renal capsular in the diabetic nude mice;the transplantation site was obviously swollen.However,the levels of blood glucose had no change of the diabetic rats after transplated with the mixture of INS-1 and α cells (P＜0.05).The expression of insulin and glucagon in the INS-1 transplanted cells were found by immunohistochemistry staining.Conclusion:Pancreatic islet α cells and their secretions can promote the INS-1 cell proliferation and insulin secretion,and the mixture of INS-1 cells and α cells transplanted under the renal capsule of the diabetic nude mice can reduce the hypoglycemic effect of INS-1 cell transplantation which might be related to the INS-1 cells that can express both of insulin and glucagon genes.
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Objective:To investigate the influence of α cells and glucagon-like peptide l (GLP-1) in the function of β cells (INS-1 cells) in the rats,and to elucidate the possible mechanism of α cells and INS-1 cells transplantation in influencing hypoglycemia.Methods:The proliferation abilities of INS-1 cells after treated with 10％,20％ and 30％ islet α-cell conditioned medium and 0.03,0.30,3.00,30.0 mg · L-1 of GLP-1 were analyzed by MTT assay.The levels of insulin secretion of INS-1 cells after treated with 10％,20％,30％ α cells,α-cell conditioned medium and different concentrations of GLP-1 were analyzed by enzyme linked immunosorbent assay (ELISA).The concentrations of Ca2+ in INS-1 cells after treated with high glucose and GLP-1 were analyzed by laser confocal microscope.The expression levels of insulin protein after treated with different concentrations of islet α-cell conditioned medium and different concentrations of GLP-1 were detected by Western blotting methed.After the INS-1 cells,the mixture of INS-1 cells and α cells were transplanted into the left renal capsule of the nude mice,the blood glucose levels and the kidney morphology were observed.The levels of insulin/glucagon in the transplanted cells were detected by immunohistochemistry.Results:Compared with control group,both of α-cell conditioned media and GLP-1 promoted the INS-1 cell proliferation and insulin secretion (P ＜ 0.05).The laser confocal microscope results revealed that GLP-1 stimulated the increased intracellular Ca2+ concentration in INS-1 cells (P＜ 0.05).Compared with control group,there was no significant difference in the expression levels of insulin protein in the insulin-1 cells after treated with islet α cell conditioned medium and GLP-1 (P＞0.05).Compared with pre transplantation,the blood glucose level in the transplanted INS-1 cells was significantly decreased at 35 d after renal capsul trasplantation (P＜0.05),and even hypoglycemia presented renal capsular in the diabetic nude mice;the transplantation site was obviously swollen.However,the levels of blood glucose had no change of the diabetic rats after transplated with the mixture of INS-1 and α cells (P＜0.05).The expression of insulin and glucagon in the INS-1 transplanted cells were found by immunohistochemistry staining.Conclusion:Pancreatic islet α cells and their secretions can promote the INS-1 cell proliferation and insulin secretion,and the mixture of INS-1 cells and α cells transplanted under the renal capsule of the diabetic nude mice can reduce the hypoglycemic effect of INS-1 cell transplantation which might be related to the INS-1 cells that can express both of insulin and glucagon genes.
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Objective To study the pathological features of two huge spontaneous tumors in Wistar and GK rats. Methods Forty Wistar rats and 40 GK rats were included in this study. Among those rats, two huge spontaneous tumors were observed in a Wistar rat at 14 months of age and in a GK rat at 22 months of age. The growth and survival status of the tumor?bearing rats were recorded. The tumors were surgically removed, and their pathological features were examined using HE and immunohistochemical staining (vimentin, CK19, α?SMA, CD31, CD34, S?100, NF及Ki?67). Results Both the two tumors were completely resected by surgery without much difficulties, and both host rats survived well after the operation. The weight of the two huge tumors was 502 g and 119 g, which corresponding to 64% and 24% of the body weight of their host rats, respectively. The tumors surface had a complete capsule, with a clear boundary separating from the normal surrounding tissues, and no vascular pedicle structure was found. According to the results of immunohistochemical staining, both the two tumors were diagnosed as benign fibroma. Conclusion This type of huge spontaneous tumors is benign fibroma. Besides the impact on the activities of the rats, the tumors have no significant impact on the living conditions in the hosts.
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Objective To determine the influence of P-glycoprotein (P-gp) inhibitor on the blood brain barrier (BBB) transport of amphotericin B (AmB)..Methods An in-vitro BBB model was established with brain capillary endothelia cells (BCEC). AmB was chosen as the test drug and verapamil was chosen as the inhibitor of P-gp.Cellular uptake of AmB at different time points and with series of verapamil concentrations were performed respectively after the determination of appropriate incubation time and drug dosage by the cytotoxicity assay. The AmB concentrations of series of samples were detected using high performance liquid chromatography (HPLC) method. One-way ANOVA analysis and Bonferroni test were used for data analysis.Results The cellular transport of AmB was accumulated as the time prolonged.The inhibitor group had a significant higher cellular uptake levelsof AmBat the time point of 90 min (t=6.753,P=0.001),120 min (t=3.574,P=0.016) and 150 min (t=4.759,P=0.005) as compared with the control group.The AmB cellular uptake level increased significantly when BCEC were incubated with verapamil of 2 μmol/L (P=0.000),5 μmol/L (P=0.014),10 μmol/L (P=0.000),50 μmol/L (P=0.014),75 μmol/L (P=0.000) and 100 tμmol/L (P=0.000),respectively,compared with the control group.Conclusion The P-gp inhibitor verapamil can enhance the cellular uptake of AmB which indicates that P-gp is involved in the BBP transport of AmB.
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Objective To investigate the effect of mitochondrial fission on the function of pancreatic β cells.Methods INS-1 stable cell lines allowing inducible expression of either wild-type dynamin-related protein 1 (Drp-1 WT)or its dominant-negative mutant(Drp-1 K38A)were used.The effect of mitochondrial fission on the function of pancreatic β cells were investigated under different concentrations of glucose.Results There were increased mitochondrial fission and disintegration of the mitochondrial reticulum into multiple punctiform organelles in Drp-1 WT cells induced with doxycycline under high glucose condition.Insulin secretion(P<0.01),mitochondrial membrane potential(P<0.05),and ATP content(P<0.05)were decreased and cytochrome C expression was increased after the expression of Drp-1 WT under high glucose condition while these changes were markedly mild in Drp-1 K38A expression cells.Conclusion The increased mitochondrial fission inhibits pancreatic β cell function.
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Objective To investigate the effect of IH764-3 on the leukocyte-mediated hypoxia-reoxygention injury of human brain microvascular endothelial cell (HBM VEC). Methods MTT assay was used to detect the survival of HBMVEC; gelatin zymography was used to check the activity of MMPs. The level of reactive oxygen species (ROS) in leukocyte was determined via commercially available kit, and the indirect enzyme-linked immunosorbent assay (ELISA) was used to quantify the contents of TNF-α, IL-1α, IL-2 and INF-γ in leukocyte culture medium. Results Survival of HBMVEC was impaired by hypoxia-reoxygenation, which was aggravated by supernatant of activated leukocytes but was attenuated by IH764-3. Leukocytes produced high level of MMP-9, ROS and cytokines (TNF-α, IL-1α, IL-2, IFN-γ) after hypoxia-reoxygenation, the process was inhibited by IH 764-3. Furthermore, IH764-3 could effectively reverse hypoxia-reoxygenation injury of HBMVEC with supernatant of activated leukocytes. Conclusion IH764-3 can protect HBMVEC from leukocyte-involved hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the pathogenic effects of leukocytes products.
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Objective To investigate the effects of lymphangiogenesis on cancer growth and metastasis.Methods Human lymphatic endothelial cells(HLyECs)were isolated and purified from human lymph node by magnetic beads coated with antibody against human VEGFR3.Human breast cancer cell line(MDA-MB-435)or human osteosarcoma cell line(MG-63)was inoculated alone or co-inoculated with HLyECs subcutaneously into nude mice.The weight of tumor and lung surface metastasis were detected;peri-tumor lymphatic vessels were shown by Evans blue,intra-tumor lymphatic vessels were shown by immunohistochemistry for human and mouse PDPN.Conditioned medium of tumor on proliferation of HLyECs was evaluated by MTT assay.Results Compared with inoculating tumor cells alone,the growth and metastasis of MDA-MB-435 were promoted by co-inoculating HLyECs.The peritumoral and intra-tumoral lymphatic vessel density of MDA-MB-435 were increased by co-inoculating HLyECs.Both hPDPN-and mPDPN-positive lymphatic vessels were found.But co-inoculating HLyECs had no effect on lymphatic vessels density of MG-63.Neither intra-nor peri-tumor lymphatic vessels were found.The proliferation of HLyECs was increased by conditioned medium of MDA-MB-435 but not by MG-63.Conclusion Growth and lymphangiogenesis of breast cancer can be enhanced by co-inoculating lymphatic endothelial cells.
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Objective To investigate the effect of IH764-3 on the leukocyte-mediated hypoxia-reoxygention injury of human brain microvascular endothelial cell (HBMVEC). Methods MTT assay was used to detect the survival of HBMVEC; gelatin zymography was used to check the activity of MMPs. The level of reactive oxygen species (ROS) in leukocyte was determined via commercially available kit,and the indirect enzyme-linked immunosorbent assay (ELISA) was used to quantify the contents of TNF-?,IL-1?,IL-2 and INF-? in leukocyte culture medium.Results Survival of HBMVEC was impaired by hypoxia-reoxygenation,which was aggravated by supernatant of activated leukocytes but was attenuated by IH764-3. Leukocytes produced high level of MMP-9,ROS and cytokines (TNF-?,IL-1?,IL-2,IFN-?) after hypoxia-reoxygenation,the process was inhibited by IH 764-3. Furthermore,IH764-3 could effectively reverse hypoxia-reoxygenation injury of HBMVEC with supernatant of activated leukocytes. Conclusion IH764-3 can protect HBMVEC from leukocyte-involved hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the pathogenic effects of leukocytes products.
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Objective To study the dynamic changes of transendothelial electrical resistance (TEER) on our in vitro model of brain blood barrier (BBB) made from primary culture of BALB/c mouse brain microvascular endothelial cells (BMVEC), and to explore the relation between TEER and BBB permeability, and search for the best culture condition. Methods BMVEC were isolated from BALB/c mouse and cultured on a transwell insert with special micro-pore. The cells were identified with immunohistochemical methods and electron microscope. TEER over BMVEC was measured after BBB model establishment for determining the 3H-Glucose permeability of BBB in vitro. Results BMVEC cultured in the transwell insert exhibited typical "flagstone" appearance and in a tight monolayer structure under electron microscope. Immunohistochemical detection of ZO-1 protein, a marker antigen of tight junction, showed smooth, continuous and tight junctions between confluent BMVEC. TEER over BMVEC monolayer increased to (346?10) ?/cm2 when the permeability for 3H-Glucose was decreased to the minimum. Conclusion BBB model in vitro made from primary culture of BMVEC in transwell has the basic characteristics of BBB in morphology, electrical resistance and permeability.
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Objective: To establish an in vitro model of brain blood barrier (BBB) using cultured mouse brain microvascular endothelial cells (BMVEC). Methods: Mouse BMVEC were seeded on micro pore membrane of gelatin coated cell culture insert and cultured to confluence. The establishment of BBB was preliminary judged by a 4 h water leaking test. The tight junctions between BMVEC were demonstrated by scanning and transmission electron microscope. The transendothelial electrical resistance(TEER) over BMVEC was measured. The permeability of Horseradish peroxidase (HRP) through the BBB was analyzed and the effect of RMP 7 on permeability of the BBB was investigated. Results: The 4 h water leaking test became positive when BMVEC were cultured to confluence. By scanning and transmission electron microscope, the tight junctions were demonstrated on confluent BMVEC. The TEER over BMVEC monolayer increased 3.2 and 7.68 times and the permeability rates for HRP were 13.4% and 6.7% respectively, as compared with sub confluent BMVEC and human umbilical vein endothelial cell monolayer(HUVEC). The HRP permeability rate in the model of BBB increased 2.7 times after treatment with RMP 7. Conclusion: The established in vitro model of BBB has basic characteristics of BBB in vivo , and is suitable for central nervous system (CNS) drug research over BBB.
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In INS-1 cells, the insulin secretion was investigated by radioimmunoassay (RIA) after 4 h incubation in medium containing different concentrations of glucose and recombinant human glucagon-like peptide-1 (rhGLP-1) (7-36). Insulin mRNA level in INS-1 cells was assessed by a semi-quantitative RT-PCR method. rhGLP-1 (7-36) is not only a powerful insulin secretagogue, but also can increase insulin gene expression in INS-1 cells.
