AIM: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD).METHODS: WD copper transporting properties in some organelles of the cultu...AIM: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD).METHODS: WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls. These cultured hepatocytes were incubated in the media of copper 15mg.L-1 only, copper 15 mg. L-1 with vincristine (agonist of P-type ArPase) 0.5mg. L-1, or copper 15 mg. L-1 withvanadate (antagonist of P-type ATPase) 18.39 mg. L-1separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls.WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls.RESULTS: The specific WD proteins (Mr155 000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls,and the addtion of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate,the copper concentrations in microsome were obviously decreased. The results indicated that there were Wdproteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase.CONCLUSION: Copper transportion P-type ATPase plays an important role in hepatocytic copper metabolism.Dysfunction of hepatocytic WD protein copper transportion might be one of the most important factors for WD.展开更多
目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂...目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂溶液（0.5mg/ml）的明胶海绵;Ⅲ组为双耳圆窗龛放置顺铂溶液（1mg/ml）的明胶海绵;Ⅳ组双耳圆窗龛放置硫酸铜溶液（0.02mg/kg）的明胶海绵。采用免疫组织化学技术,对各组耳蜗冰冻切片行CTR1蛋白的免疫组织化学染色,光学显微镜下观察耳蜗组织中CTR1的表达情况;提取各组耳蜗组织总蛋白,应用Western-blot技术检测各组CTR1蛋白表达水平;提取耳蜗组织的总RNA,应用RT-PCR技术检测各组CTR1mRNA的表达水平。结果 Ⅰ-Ⅳ组大鼠的耳蜗各回Corti器、螺旋神经节、血管纹的细胞质和细胞膜上均有CTR1表达,CTR1的平均光密度值呈降低趋势;各组耳蜗组织均有明显的CTR1表达,Ⅰ-Ⅳ组CTR1蛋白的光密度值分别为0.532±0.031、0.394±0.024、0.234±0.030和0.191±0.015,呈下降趋势,各组间比较差异有统计学意义（P〈0.05）;各组耳蜗组织均有明显的CTR1mRNA表达,Ⅰ-Ⅳ组光密度值分别为0.508±0.035、0.391±0.022、0.240±0.023和0.186±0.021,呈下降趋势,各组间比较差异均有统计学意义（P〈0.05）。结论 CTR1在大鼠内耳有丰富的表达,其表达量可随鼓室内顺铂浓度增高而下降,且鼓室内给予硫酸酮其表达量下降。展开更多
OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these differen...OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.展开更多
基金Supported by Key Clinical Program of Ministry of Ministry of Health(No.37091)"211 Project"of SUMS sponsored by Ministry of Health and Guangdong Provincial Natural Science Foundation,No.990064
文摘AIM: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD).METHODS: WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls. These cultured hepatocytes were incubated in the media of copper 15mg.L-1 only, copper 15 mg. L-1 with vincristine (agonist of P-type ArPase) 0.5mg. L-1, or copper 15 mg. L-1 withvanadate (antagonist of P-type ATPase) 18.39 mg. L-1separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls.WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls.RESULTS: The specific WD proteins (Mr155 000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls,and the addtion of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate,the copper concentrations in microsome were obviously decreased. The results indicated that there were Wdproteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase.CONCLUSION: Copper transportion P-type ATPase plays an important role in hepatocytic copper metabolism.Dysfunction of hepatocytic WD protein copper transportion might be one of the most important factors for WD.
文摘目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂溶液（0.5mg/ml）的明胶海绵;Ⅲ组为双耳圆窗龛放置顺铂溶液（1mg/ml）的明胶海绵;Ⅳ组双耳圆窗龛放置硫酸铜溶液（0.02mg/kg）的明胶海绵。采用免疫组织化学技术,对各组耳蜗冰冻切片行CTR1蛋白的免疫组织化学染色,光学显微镜下观察耳蜗组织中CTR1的表达情况;提取各组耳蜗组织总蛋白,应用Western-blot技术检测各组CTR1蛋白表达水平;提取耳蜗组织的总RNA,应用RT-PCR技术检测各组CTR1mRNA的表达水平。结果 Ⅰ-Ⅳ组大鼠的耳蜗各回Corti器、螺旋神经节、血管纹的细胞质和细胞膜上均有CTR1表达,CTR1的平均光密度值呈降低趋势;各组耳蜗组织均有明显的CTR1表达,Ⅰ-Ⅳ组CTR1蛋白的光密度值分别为0.532±0.031、0.394±0.024、0.234±0.030和0.191±0.015,呈下降趋势,各组间比较差异有统计学意义（P〈0.05）;各组耳蜗组织均有明显的CTR1mRNA表达,Ⅰ-Ⅳ组光密度值分别为0.508±0.035、0.391±0.022、0.240±0.023和0.186±0.021,呈下降趋势,各组间比较差异均有统计学意义（P〈0.05）。结论 CTR1在大鼠内耳有丰富的表达,其表达量可随鼓室内顺铂浓度增高而下降,且鼓室内给予硫酸酮其表达量下降。
基金The project supported by National Natural Science Foundation of China(81373439,81473234 and U1303221)
文摘OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.
