目的研究线粒体转录因子A(TFAM)对宫颈癌HeLa细胞及骨肉瘤U2OS细胞的线粒体功能、自噬、增殖、侵袭、迁移的影响。方法HeLa、U2OS细胞转染TFAM小干扰片段(si-TFAM)下调TFAM表达,Mito-Tracker Red CMXRos染色结合激光共聚焦显微镜检测线...目的研究线粒体转录因子A(TFAM)对宫颈癌HeLa细胞及骨肉瘤U2OS细胞的线粒体功能、自噬、增殖、侵袭、迁移的影响。方法HeLa、U2OS细胞转染TFAM小干扰片段(si-TFAM)下调TFAM表达,Mito-Tracker Red CMXRos染色结合激光共聚焦显微镜检测线粒体膜电位(MMP)、MitoSOX^(TM)Red标记法检测线粒体活性氧(mtROS)水平、实时定量PCR检测线粒体DNA(mtDNA)的表达,免疫荧光细胞化学染色检测自噬体数量的变化。Western blot法检测TFAM、微管相关蛋白1轻链3A/B(LC3A/B)、自噬相关基因2A(ATG2A)、ATG2B、ATG9A、锌指转录因子Snail、基质金属蛋白酶2(MMP2)和MMP9的表达。CCK-8法、平板集落形成实验检测细胞增殖,Transwell^(TM)实验、划痕愈合实验检测细胞侵袭、迁移的变化。结果下调TFAM表达导致HeLa及U2OS细胞MMP减少,mtDNA拷贝数减少,mtROS产生量增加。LC3A/B蛋白含量较对照组明显下降,胞质内自噬体数量明显减少,自噬早期阶段蛋白ATG2B、ATG9A表达量明显减少。HeLa及U2OS细胞Snail、MMP2和MMP9蛋白表达均减少。干扰TFAM表达,抑制HeLa及U2OS细胞的增殖、侵袭、迁移能力。结论下调TFAM表达抑制线粒体功能,延缓自噬进程,降低宫颈癌细胞及骨肉瘤细胞的增殖、侵袭、迁移能力。展开更多
[目的]本试验旨在探究PPARGC1A/NRF1/TFAM通路核心基因在湖羊性成熟前后的表达变化。[方法]选取体况良好和系谱清楚的雄性湖羊18只(3和9月龄,各9只),颈静脉采血后进行阉割处理。ELISA检测血样中睾酮(T)、瘦素、胰岛素及胰岛素样生长因子...[目的]本试验旨在探究PPARGC1A/NRF1/TFAM通路核心基因在湖羊性成熟前后的表达变化。[方法]选取体况良好和系谱清楚的雄性湖羊18只(3和9月龄,各9只),颈静脉采血后进行阉割处理。ELISA检测血样中睾酮(T)、瘦素、胰岛素及胰岛素样生长因子Ⅰ(IGF-Ⅰ)的浓度;免疫组化法检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关蛋白的表达定位; RT-qPCR和Western blot检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关基因与蛋白的表达变化。[结果]与3月龄湖羊相比,9月龄湖羊外周血中T、瘦素、胰岛素及IGF-Ⅰ的浓度显著升高(P<0.05)。免疫组化法检测发现PPARGC1A、NRF1和TFAM蛋白在3月龄和9月龄湖羊睾丸组织的多种细胞内均呈时空特异性表达。此外,与3月龄湖羊相比,9月龄湖羊睾丸组织中PPARGC1A、NRF1和TFAM m RNA和蛋白表达水平均显著升高(P<0.05)。[结论]PPARGC1A/RNF1/TFAM通路相关基因可能与湖羊睾丸发育和性成熟过程密切相关。展开更多
ATP depletion is one of the pathological bases in cerebral ischemia.Electro-acupuncture(EA)is widely used in clinical practice for ischemia.However,the mechanism of EA remains unclear.The purpose of this study was to ...ATP depletion is one of the pathological bases in cerebral ischemia.Electro-acupuncture(EA)is widely used in clinical practice for ischemia.However,the mechanism of EA remains unclear.The purpose of this study was to investigate whether EA could activate the AMPK/PGC-1α/TFAM signaling pathway and,consequently,increase the preservation of ATP in rats with ischemia.In this study,48 rats were randomly divided into four groups as a sham-operation control group(sham group),a middle cerebral artery occlusion group(MCAO group),an EA group,and an EA group blocked by the AMPK inhibitor compound C(EA+CC group)(N=12/group).The rats of the EA group and EA+CC group received the EA treatment for 7 days.The rats that belonged in the two remaining groups were only grasped in the same condition.Then,their brain tissues were collected for further detection.When compared with other groups,EA significantly reduced neurological deficits score and increased motor function.The cerebral infarction volume was significantly reduced in the EA group according to TTC staining.With western blot,we found that EA improved the ratio of p-AMPKα/AMPKα(P<0.05),however,there is no difference between the MCAO group and sham group(P>0.05).In addition,EA also increased the expression of PGC-1αand TFAM(all P<0.05).By Elisa,we observed that EA increased the preservation of ATP(P<0.05)and mitochondrial respiratory enzymes,including Complex I(P<0.05),Complex IV(P<0.05),but not Complex III(P>0.05).In summary,we conclude that EA may protect against ischemic damage in MCAO rats,improve the preservation of ATP and mitochondrial respiratory enzymes.This effect may be positively regulated by the activation of the PGC-1α/TFAM signaling pathway.展开更多
文摘目的研究线粒体转录因子A(TFAM)对宫颈癌HeLa细胞及骨肉瘤U2OS细胞的线粒体功能、自噬、增殖、侵袭、迁移的影响。方法HeLa、U2OS细胞转染TFAM小干扰片段(si-TFAM)下调TFAM表达,Mito-Tracker Red CMXRos染色结合激光共聚焦显微镜检测线粒体膜电位(MMP)、MitoSOX^(TM)Red标记法检测线粒体活性氧(mtROS)水平、实时定量PCR检测线粒体DNA(mtDNA)的表达,免疫荧光细胞化学染色检测自噬体数量的变化。Western blot法检测TFAM、微管相关蛋白1轻链3A/B(LC3A/B)、自噬相关基因2A(ATG2A)、ATG2B、ATG9A、锌指转录因子Snail、基质金属蛋白酶2(MMP2)和MMP9的表达。CCK-8法、平板集落形成实验检测细胞增殖,Transwell^(TM)实验、划痕愈合实验检测细胞侵袭、迁移的变化。结果下调TFAM表达导致HeLa及U2OS细胞MMP减少,mtDNA拷贝数减少,mtROS产生量增加。LC3A/B蛋白含量较对照组明显下降,胞质内自噬体数量明显减少,自噬早期阶段蛋白ATG2B、ATG9A表达量明显减少。HeLa及U2OS细胞Snail、MMP2和MMP9蛋白表达均减少。干扰TFAM表达,抑制HeLa及U2OS细胞的增殖、侵袭、迁移能力。结论下调TFAM表达抑制线粒体功能,延缓自噬进程,降低宫颈癌细胞及骨肉瘤细胞的增殖、侵袭、迁移能力。
文摘[目的]本试验旨在探究PPARGC1A/NRF1/TFAM通路核心基因在湖羊性成熟前后的表达变化。[方法]选取体况良好和系谱清楚的雄性湖羊18只(3和9月龄,各9只),颈静脉采血后进行阉割处理。ELISA检测血样中睾酮(T)、瘦素、胰岛素及胰岛素样生长因子Ⅰ(IGF-Ⅰ)的浓度;免疫组化法检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关蛋白的表达定位; RT-qPCR和Western blot检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关基因与蛋白的表达变化。[结果]与3月龄湖羊相比,9月龄湖羊外周血中T、瘦素、胰岛素及IGF-Ⅰ的浓度显著升高(P<0.05)。免疫组化法检测发现PPARGC1A、NRF1和TFAM蛋白在3月龄和9月龄湖羊睾丸组织的多种细胞内均呈时空特异性表达。此外,与3月龄湖羊相比,9月龄湖羊睾丸组织中PPARGC1A、NRF1和TFAM m RNA和蛋白表达水平均显著升高(P<0.05)。[结论]PPARGC1A/RNF1/TFAM通路相关基因可能与湖羊睾丸发育和性成熟过程密切相关。
基金supported by the National Natural Science Foundation of China(No.81574048 and No.81904268)Key personnel training project of Health Care in middle-aged and young people in Fujian province(No.2019-ZQN-81).
文摘ATP depletion is one of the pathological bases in cerebral ischemia.Electro-acupuncture(EA)is widely used in clinical practice for ischemia.However,the mechanism of EA remains unclear.The purpose of this study was to investigate whether EA could activate the AMPK/PGC-1α/TFAM signaling pathway and,consequently,increase the preservation of ATP in rats with ischemia.In this study,48 rats were randomly divided into four groups as a sham-operation control group(sham group),a middle cerebral artery occlusion group(MCAO group),an EA group,and an EA group blocked by the AMPK inhibitor compound C(EA+CC group)(N=12/group).The rats of the EA group and EA+CC group received the EA treatment for 7 days.The rats that belonged in the two remaining groups were only grasped in the same condition.Then,their brain tissues were collected for further detection.When compared with other groups,EA significantly reduced neurological deficits score and increased motor function.The cerebral infarction volume was significantly reduced in the EA group according to TTC staining.With western blot,we found that EA improved the ratio of p-AMPKα/AMPKα(P<0.05),however,there is no difference between the MCAO group and sham group(P>0.05).In addition,EA also increased the expression of PGC-1αand TFAM(all P<0.05).By Elisa,we observed that EA increased the preservation of ATP(P<0.05)and mitochondrial respiratory enzymes,including Complex I(P<0.05),Complex IV(P<0.05),but not Complex III(P>0.05).In summary,we conclude that EA may protect against ischemic damage in MCAO rats,improve the preservation of ATP and mitochondrial respiratory enzymes.This effect may be positively regulated by the activation of the PGC-1α/TFAM signaling pathway.
