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??OBJECTIVE To explore the effects of luteolin on the proliferation and migration of the endothelial cells co-cultured with cancer cells and its molecular mechanism. METHODS Human umbilical vein endothelial cells (HUVECs) were primarily isolated and identified by the expression of VE-cadherin. Cancer-endothelial cell co-culture model was established using the Transwell system. The effects of luteolin at different concentrations (0 [Co-culture control], 20 and 50 ??mol??L^-1) on the proliferation and migration of HUVECs in the co-culture system were determined. A HUVECs control group removed of prostate cancer PC3 cells was also included. Human angiogenesis antibody array kit was used to assay the secretion levels of various protein factors in each group. RESULTS VE-cadherin was expressed on all the cultured HUVECs. Increased proliferation ability was found in the HUVECs co-cultured with PC3 cells compared with that in HUVECs control group (P<0.01). Treatment with 20 or 50 ??mol??L^-1 luteolin significantly inhibited the proliferation ability of the HUVECs in co-culture system (both P<0.01). Increased migration ability was found in the HUVECs co-cultured with PC3 cells compared with that in HUVECs control group (P<0.01). Treatment with 20 or 50 ??mol??L^-1 luteolin significantly inhibited the migration ability of the HUVECs in co-culture system (both P<0.01). Secretion levels of multiple angiogenesis-related proteins in the cultural supernatant of co-culture system were significantly increased compared with those in HUVECs control group. Treatment with 20 ??mol??L^-1 luteolin significantly inhibited the secretion levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1(MCP-1) in the cultural supernatant of co-culture system (all P<0.01). CONCLUSION Luteolin may inhibit the proliferation and migration of endothelial cells via suppressing the secretions of IL-8, VEGF and MCP-1 in cancer-endothelial co-culture system.     

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??OBJECTIVE To investigate the apoptosis effect of human hepatocellular carcinoma cell line SMMC-7721 induced by dihydroartemisinin in vitro and the possible mechanism. METHODS After treatment with 25, 50, 100, 200, and 400 ??mol??L^-1 dihydroartemisinin for 24 h. The proliferation inhibitory effect of dihydroartemisinin on SMMC-7721 cell was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The change of apoptotic morphology was detected by confocal laser scanning microscopy. Rho 123 staining method was used to detect the changes of mitochondrial membrane potential. Western blot was used to detect expression of Bcl-2, Bax, Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C. RESULTS MTT results showed that 25-400 ??mol??L^-1 dihydroartemisinin can inhibit the proliferation of SMMC-7721 cells obviously. The cell cycle detection results of flow cytometry showed that dihydroartemisinin could block SMMC-7721 cell cycle in G₂/M phase. The results of Hochest 333258 staining showed that the nuclei were heterogeneous, condensed and fragmented in the DHA treatment group. The cell apoptosis detection results of flow cytometry showed that the apoptosis rate of dihydroartemisinin treated groups were increased obviously (P<0.01). The results of Rho 123 staining showed that the mitochondrial membrane potential was decreased significantly (P<0.01). Western blot results showed that the expression of Bcl-2 was down-regulated, expression of Bax was up-regulated, the ration of Bax/Bcl-2 was increased and the expression of Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C were up-regulated. CONCLUSION Dihydroartemisinin can induce apoptosis of SMMC-7721 cells, on the mechanism of apoptosis may be related to mitochondrial pathway.     

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??OBJECTIVE To explore the protective effect of palmitic acid against glutamate-induced apoptosis in PC12 cells. METHODS PC12 cells were randomly divided into six groups: normal group, solvent group(DMSO 0.1%), model group (Glu 25 mmol??L^-1), nimodipine(NMP,7 ??mol??L^-1)-treated group or MK801(10 ??mol??L^-1) -treated group set as positive control group, palmitic acid groups of 100, 10, 1 ??mol??L^-1 dose. Cell viability and membrane permeability were examined by MTT assay and LDH kit. Morphologic change of apoptosis were detected by AO/EB and Hoechst staining with fluorescence microscope. The apoptosis rates were measured by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The expression of Bcl-2, Bax and GluR1 protein was assayed by Western blot methods. RESULTS Compared with the model group, the results showed that palmitic acid could significantly improve the viability of PC12 cells damaged by glutamate, and reduce the permeability of cell membranes. After AO/EB staining, nuclear chromatin of the cells in model group were stained strongly into orange by EB. In palmitic acid ?Ctreated group, only few cells were stained into orange. Compared with model group, after the treatment of palmitic acid , slight blue fluroscence stained by Hoechst in nucleus had been emitted. Palmitic acid could increase the expression of Bcl-2 and inhibit the level of the expression of Bax protein and GluR1 protein. CONCLUSION In summary, palmitic acid had protective effect on glutamate-induced apoptosis in PC12 cells through increasing the expression of Bcl-2 and inhibiting the expression of Bax and GluR1.
     

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??OBJECTIVE To investigate the inhibitory effects of bispecific antibody-drug conjugate Fv-LDM-NGR targeting EGFR and CD13 on human tumor cells and endothelial cells,and possible mechanisms. METHODS Human breast cancer cells MCF-7, human lung adenocarcinoma cells A549 and human microvascular endothelial cells HMEC-1, were studied. MTT assay was applied to measure proliferative activity of tumor cells. The influence of Fv-LDM-NGR on tube formation of HMEC-1 was observed. Transwell assay was applied to measure migration and invasion capacity in tumor cells. Western blot was applied for analyzing intracellular signaling transduction pathways. Flow cytometry, Hochest stain and Annexin ??-FITC/PI were used to detect cell cycle and apoptosis. RESULTS Fv-LDM-NGR could inhibit the proliferation of tumor cells and microvascular endothelial cells with IC₅₀ values of 10^-14-10^-12mol??L^-1. Fv-LDM-NGR prevented tube formation in microvascular endothelial cells, and suppressed migration and invasion in tumor cells. Fv-LDM-NGR interfered with the intracellular signaling transduction pathways, then caused G₂/M and S phase arrest and induced apoptosis. CONCLUSION Bispecific antibody-drug conjugate Fv-LDM-NGR could prevent cell invasion in tumor cells and tube formation in microvascular endothelial cells through blocking activity of CD13. And it could down-regulate the expression and the phosphorylation of EGFR, interfere with cellular signal pathways, induce cell cycle arrest and cell apoptosis, and inhibit cell proliferation and migration.     

白杨素调控YAP介导的EMT进程抑制人结肠癌HCT-116细胞转移的机制研究

目的 探讨白杨素在体内外对人结肠癌HCT-116细胞侵袭与转移作用及相关机制。方法 本实验应用CCK-8法检测白杨素对HCT-116细胞增殖能力的抑制作用。利用划痕和Transwell法检测白杨素干预HCT-116细胞后对细胞迁移和侵袭能力的作用。通过Western blot检测白杨素干预后对HCT-116细胞中上皮间质转化(EMT)相关蛋白E-cadherin、N-cadherin、Vimentin和Snail表达水平的影响,并检测白杨素对HCT-116细胞内Yes相关蛋白(YAP)蛋白表达的作用。利用siRNA干扰实验考察YAP低表达对白杨素在HCT-116细胞侵袭作用和EMT进程中的影响。构建体内实验性肺转移模型考察白杨素对HCT-116细胞体内转移的作用。结果 白杨素从8 μmol·L^-1开始能够显著抑制HCT-116细胞的增殖,8 μmol·L^-1以下对HCT-116细胞增殖没有显著抑制作用。划痕和Transwell实验显示,白杨素(1、2和4 μmol·L^-1)能够剂量依赖性的抑制HCT-116细胞的迁移与侵袭。Western blot结果显示,白杨素能够上调E-cadherin的蛋白表达,下调N-cadherin,Vimentin及转录因子Snail的蛋白表达。同时还发现白杨素能够下调YAP蛋白表达水平,而利用siRNA下调YAP后能显著逆转白杨素对HCT-116细胞迁移与侵袭的抑制作用,并能逆转白杨素对HCT-116细胞EMT过程的促进作用。体内肺转移实验显示,白杨素显著抑制了HCT-116在肺上转移结节。结论 本实验表明,白杨素能够在体内外抑制人结肠癌HCT-116细胞的侵袭与转移能力,其作用机制与抑制YAP的蛋白表达,下调EMT过程相关。     

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??OBJECTIVE To investigate the effect and mechanism of juglone on the apoptosis of human gastric cancer SGC-7901 cells. METHODS The antiproliferative effect of juglone on SGC-7901 cells was tested by the MTT assay. The apoptosis rate and intracellular reactive oxygen species(ROS) level were detected by flow cytometry (FCM). The expression of JNK, p-JNK, p38, and p-p38 proteins were examined by Western blot. In order to clarify the role of ROS in the apoptosis induced by juglone on SGC-7901 cells, the combination of the juglone and ROS inhibitor NAC groups were set up in each experiment above. RESULTS Juglone could effectively inhibit the proliferation of SGC-7901 cells (IC₅₀ for 72 h was 24.16 ??mol??L^-1). When combination juglone with NAC, the IC₅₀ raised to 36.91 ??mol??L^-1. After 72 h of exposure to 5-20 ??mol??L^-1 of juglone, the cell apoptosis rate increased gradually with the increase of juglone concentration. After adding NAC, the apoptosis rates declined and the apoptosis rate of 20 ??mol??L^-1 group decreased from 32.06% to 11.56%. After SGC-7901 cells were treated with juglone for 24 h, the ROS level increased and mitochondrial transmembrane potential decreased which were inhibited by the pretreatment of NAC. After 48 h of exposure to different concentration of juglone, the expressions of p-p38 and p-JNK proteins were up-regulated which could also be inhibited by the adding of NAC. Meanwhile, there were no significant changes in p38 and JNK protein expression in all groups. CONCLUSION Juglone can induce apoptosis of human gastric cancer SGC-7901 cells by JNK and P38 pathway mediated by reactive oxygen species.     

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??OBJECTIVE To investigate the effect and the mechanism of baicalin in alleviating human umbilical vein endothelial cell (HUVEC) injury induced by lipocalin-2. METHODS Lipocalin-2 at different concentration (0, 5, 10, 20 ??mol??L^-1) and different time gradient (0, 24, 48 and 72 h) were added in HUVEC, the proliferation and apoptosis of HUVEC, contents of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in HUVEC supernatant, lactic dehydrogenase (LDH) activities and the expressions of p-JNK, Bax and Bcl-2 in HUVEC were measured by MTT assay, flow cytometry, enzyme-linked immuno-sorbent assay (ELISA), colorimetry and Western blot, respectively. Different concentration of baicalin (15??30??60 mg??L^-1) or 10 ??mol??L^-1 SP600125, the specific inhibitor of JNK pathway, was added into HUVEC to detect the effect of baicalin. RESULTS Compared with the control group, 10 ??mol??L^-1 lipocalin-2 could significantly increase the apoptosis, IL-6, MCP-1 contents and LDH activities, p-JNK expression and Bax/Bcl-2 rate but restrain the proliferation in HUVEC (P<0.05). Compared with lipocalin-2 group, 30 mg??L^-1 baicalin could significantly restrain the apoptosis, IL-6, MCP-1 contents and LDH activities, p-JNK expression and Bax/Bcl-2 rate but increase the proliferation in HUVEC (P<0.05). CONCLUSION Our findings indicate that baicalin could alleviate HUVEC injury induced by lipocalin-2, the protective mechanism is related to the inhibition of JNK pathway.     

细柱五加叶中凹脉鹅掌柴酸通过抑制NF-κB-iNOS-NO信号保护胰岛β细胞研究

目的 基于通过抑制NF-κB-iNOS-NO信号通路,研究细柱五加叶(Acanthopanax gracilistylus W. W. Smith)中凹脉鹅掌柴酸(impressic acid)的胰岛β细胞保护作用。方法 运用炎症细胞因子IL-1β和IFN-γ诱导RIN-m5F细胞建立2型糖尿病细胞模型;采用MTT 法对细胞毒性进行评价;采用ELISA 法测胰岛素分泌评价胰岛细胞功能;采用Griess法测定导致胰岛β细胞的凋亡的关键物质一氧化氮(NO)水平;采用Caspase-3活性检测法检测Caspase-3水平;检测细胞内活性氧物质(reactive oxygen species,ROS)水平;并采用 Western blot法检测相关蛋白(iNOS、NF-κB)的表达。结果 细柱五加叶中提取的凹脉鹅掌柴酸(5~20 μmol·L^-1) 无细胞毒性,并且能够通过抑制NF-κB-iNOS-NO信号通路以浓度依赖的方式恢复细胞因子IL-1β和IFN-γ诱导RIN-m5F细胞凋亡、下调NF-κB表达,下调iNOS表达,降低NO水平,抑制细胞凋亡剪切酶Caspase-3活性,下调活性氧物质ROS水平保护胰岛素β细胞。结论 凹脉鹅掌柴酸(5~20 μmol·L^-1)能够通过抑制NF-κB-iNOS-NO信号通路对胰岛β细胞起到保护作用,从而抗2型糖尿病进展。     

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??OBJECTIVE To investigate the effects of interference of riboflavin (RIB) metabolic pathways on adhesion and migration to cisplatin (DDP) in ovarian cancer HO8910 cells. METHODS Intervention RIB metabolic pathways by lentiviral vector harboring shRNA of riboflavin transporter 2 (RFT2) and chlorpromazine (CHL), a competitive inhibitor of RIB. HO8910 ovarian cancer cell line was divided into normal control group, shRNA control group, RFT2 shRNA group, CHL (50 ??mol??L^-1) group, DDP (20 ??mol??L^-1) group, RFT2 shRNA+DDP group, CHL + DDP group and DDP + RIB (20 ??mol??L^-1) group. Each group cells were collected after treatment 48 h according to the design. And then cell adhesive abilities were detected by adhesion experiment, the cells invasive abilities were observed by transwell method, the protein expressions of VEGF, MMP9 and MMP2 were detected by Western blot, and the expressions of TNF-??, NF-??B/p65 were assayed with laser confocal microscopy. RESULTS Compared with the sole DDP treatment group, RFT2 shRNA or CHL combination with DDP had great advantages in reducing cell adhesion and migration viabilities, deceasing expressions of MM9 and MMP2, reducing cell expressions of TNF-?? and NF-??B/p65. However, the RIB could weaken the effects of DDP on HO8910 cell. CONCLUSION Interference metabolic pathway of RIB can enhance DDP effects on adhesion and migration viabilities of ovarian cancer HO8910 cell lines, and the effects are associated with blocking the pathway of TNF-??/NF-??B; However, RIB could attenuate the anti-tumor effects of cisplatin on HO8910 cell.