目的观察小檗碱对高糖环境下人肝癌细胞株HCCLM3细胞增殖的抑制、自噬作用。方法将传至3—4代HCCLM3细胞随机分为空白对照组和低糖组、中糖组、高糖组(各组分别加入含有1000、2000、4000、8000 mg·L^-1的葡萄糖培养液)及小檗碱A组...目的观察小檗碱对高糖环境下人肝癌细胞株HCCLM3细胞增殖的抑制、自噬作用。方法将传至3—4代HCCLM3细胞随机分为空白对照组和低糖组、中糖组、高糖组(各组分别加入含有1000、2000、4000、8000 mg·L^-1的葡萄糖培养液)及小檗碱A组、小檗碱B组、小檗碱C组、二甲双胍(MET)组(高糖组不给予任何干预措施,小檗碱A、B、C 3组分别加入小檗碱含药培养液5、10、20 μmol·L^-1,MET组加入MET含药培养液 10 μmol·L^-1)。CCK-8检测HCCLM3细胞的增殖率、增殖抑制率,酶联免疫吸附试验法(ELISA)检测HCCLM3细胞上清液IL-6、TNF-α的水平,实时荧光定量PCR (qPCR)检测HCCLM3细胞NF-κB mRNA、Beclin1 mRNA的表达水平。结果中糖、高糖2组24 h时HCCLM3细胞增殖率均高于空白对照组(均 P <0.05)。小檗碱A、B、C 3组和MET组24、48、72 h时HCCLM3细胞增殖抑制率均高于高糖组(均 P <0.05),小檗碱A、B、C 3组24、48、72 h时HCCLM3细胞增殖抑制率均高于MET组(均 P <0.05)。小檗碱A、B、C 3组和MET组HCCLM3细胞上清液IL-6、TNF-α水平及HCCLM3细胞NF-κB mRNA表达水平均低于高糖组(均 P <0.01),小檗碱A、B 2组HCCLM3细胞上清液IL-6、TNF-α水平均高于MET组(均 P <0.05),小檗碱A组HCCLM3细胞NF-κB mRNA表达水平高于MET组、Beclin1 mRNA表达水平低于MET组(均 P <0.01),小檗碱B、C 2组HCCLM3细胞Beclin1 mRNA表达水平均低于MET组、小檗碱C组 HCCLM3细胞上清液TNF-α水平低于MET组( P <0.05或 P <0.01)。结论 BBR对高糖环境下HCCLM3细胞增殖具有抑制作用,它可能是通过调控核因子NF-κB降低IL-6、TNF-α的表达,进而上调Beclin1基因表达水平,促进自噬产生抗肿瘤作用。展开更多
AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were f...AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h of in vitro culture (FastDC).METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/1 L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1 μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/1 polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritornas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS).Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy.RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas.CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.展开更多
文摘目的观察小檗碱对高糖环境下人肝癌细胞株HCCLM3细胞增殖的抑制、自噬作用。方法将传至3—4代HCCLM3细胞随机分为空白对照组和低糖组、中糖组、高糖组(各组分别加入含有1000、2000、4000、8000 mg·L^-1的葡萄糖培养液)及小檗碱A组、小檗碱B组、小檗碱C组、二甲双胍(MET)组(高糖组不给予任何干预措施,小檗碱A、B、C 3组分别加入小檗碱含药培养液5、10、20 μmol·L^-1,MET组加入MET含药培养液 10 μmol·L^-1)。CCK-8检测HCCLM3细胞的增殖率、增殖抑制率,酶联免疫吸附试验法(ELISA)检测HCCLM3细胞上清液IL-6、TNF-α的水平,实时荧光定量PCR (qPCR)检测HCCLM3细胞NF-κB mRNA、Beclin1 mRNA的表达水平。结果中糖、高糖2组24 h时HCCLM3细胞增殖率均高于空白对照组(均 P <0.05)。小檗碱A、B、C 3组和MET组24、48、72 h时HCCLM3细胞增殖抑制率均高于高糖组(均 P <0.05),小檗碱A、B、C 3组24、48、72 h时HCCLM3细胞增殖抑制率均高于MET组(均 P <0.05)。小檗碱A、B、C 3组和MET组HCCLM3细胞上清液IL-6、TNF-α水平及HCCLM3细胞NF-κB mRNA表达水平均低于高糖组(均 P <0.01),小檗碱A、B 2组HCCLM3细胞上清液IL-6、TNF-α水平均高于MET组(均 P <0.05),小檗碱A组HCCLM3细胞NF-κB mRNA表达水平高于MET组、Beclin1 mRNA表达水平低于MET组(均 P <0.01),小檗碱B、C 2组HCCLM3细胞Beclin1 mRNA表达水平均低于MET组、小檗碱C组 HCCLM3细胞上清液TNF-α水平低于MET组( P <0.05或 P <0.01)。结论 BBR对高糖环境下HCCLM3细胞增殖具有抑制作用,它可能是通过调控核因子NF-κB降低IL-6、TNF-α的表达,进而上调Beclin1基因表达水平,促进自噬产生抗肿瘤作用。
基金Supported by the Key Program Foundation for Clinical Subject of the Ministry of Public Health,China (2001)
文摘AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h of in vitro culture (FastDC).METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/1 L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1 μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/1 polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritornas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS).Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy.RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas.CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.
