【作者】 张蕾；

【导师】 朱俐；

【作者基本信息】 南通大学 ， 航空、航天与航海医学， 2006， 硕士

【摘要】 目的：研究不同的缺氧方法对培养细胞二价金属转运体（Divalent metal transporter 1，DMTl）表达的影响，初步探讨低氧诱导因子1（Hypoxia inducible factor-1,HIF-1）与DMTl两种异构体（+IRE DMTl和-IRE DMTl）之间的关系。方法：用CoCl₂诱导PC12细胞及HepG2细胞缺氧，检测DMTl的表达，确定对COCl₂缺氧敏感的细胞株。在此基础上用Western Blot法检测低氧（1％O₂）及呼吸链抑制剂叠氮钠（NaN₃）和鱼藤酮（Rotenone）对该细胞株HIF-1α及DMTl表达的影响，用免疫细胞化学法检测低氧（1％O₂）对HIF-1α及DMTl在该细胞内分布的影响。结果：1．用不同浓度的COCl₂（0.025mM、0.05mM、0.125mM、0.25mM、0.5mM、1mM）处理PC12细胞4小时后，其+IRE DMTl的表达量与未用COCl₂处理时相比，均无明显改变。2．HepG2细胞在经过不同浓度的COCl₂处理4小时后，随着COCl₂浓度的增加，HIF-1α及DMTl的两种异构体的表达均逐渐增加。当COCl₂浓度为1mM时HIF-1α表达量最大，在COCl₂浓度为0.5mM时+IRE DMTl和-IRE DMTl的表达量达到最高值。3．HepG2细胞HIF-1α在低氧（1％O₂）处理1小时开始即有表达，3小时至6小时其表达量达到高峰。+IRE DMTl在低氧处理1小时也有增加，6小时达到最高峰。-IRE DMTl在低氧3小时开始表达有明显增加，6小时达到最高并持续至12小时处。4．HepG2细胞HIF-1α在低氧6小时有明显表达，复氧24小时后，HIF-1α被降解。复氧24小时后，+IRE DMTl的表达也明显少于单纯低氧6小时的表达量，而-IRE DMTl的表达量却无明显改变。5．HepG2细胞用NaN₃处理1小时后，DMTl的两种异构体在NaN₃浓度为10mM及20mM时，其表达量均比对照组明显增加；用rotenone处理24小时后，+IRE DMTl在rotenone浓度为20μM时，其表达量与对照组相比有明显的增加，-IRE DMTl在rotenone浓度为1μM及20μM时，其表达量与对照组相比均无明显改变；NaN₃和rotenone都不能使HepG2细胞HIF-1α明显表达。6．在常氧环境中，DMTl两种异构体在HepG2细胞胞浆及胞核中均匀分布，HIF-1α无明显表达。低氧（1％O₂）可使+IREDMTl聚集于胞浆及核膜上，而原本位于胞浆中的-IRE DMTl更趋向于胞膜，胞核中的-IRE DMTl也有向核膜集中的趋势，但不如+IRE DMTl明显，HIF-1α则在细胞核中大量表达。结论：1．CoCl₂和低氧（1％O₂）可使HepG2细胞的HIF-1α蓄积和DMTl两种异构体的表达增加，并且三者的变化趋势基本一致，提示DMTl的表达可能受到HIF-1的调控，DMTl可能是HIF-1的下游基因。2．NaN₃和rotenone虽不能使HepG2细胞HIF-1α产生蓄积，但却能使DMTl的表达发生变化，提示DMTl表达的调控可能除HIF-1之外还有其它的调控途径。3．低氧（1％O₂）可使HepG2细胞+IRE DMTl和-IRE DMTl在细胞内的分布发生不同的改变，提示低氧对DMTl两种异构体在细胞内分布的调节不同，这可能与两种异构体的功能不完全一致有关。更多还原

【Abstract】 Objective: To study the effects of hypoxic conditions on the expression of DMT1 in cultured cells and the relationship between HIF-1αand DMT1.Methods: Exposing PC12 cells and HepG2 cells to CoCl₂, the expression of +IRE DMT1 were detected by Western Blot for choosing the sensitive cell line to chemical hypoxia. Then we exposed the sensitive cell line to NaN₃, rotenone or hypoxia（1% O₂）. The expression of +IRE DMT1, -IRE DMT1 and HIF-1αwere detected by Western Blot. The effect of hypoxia on the two isoforms of DMT1 and HIF-1αsubcellular distribution was detected by immunocytochemistry.Results:1. The expression of +IRE DMT1 changed little in PC12 cells which were exposed to different concentrations of CoCl₂ （ 0. 025mM、0. 05 mM、0. 125mM、0. 25 mM、0. 5mM、1 mM） for 4 hours.2. CoCl₂ could promote the expression of HIF-1αand the two isoforms of DMT1 in HepG2 cells. When HepG2 cells were exposed to 1mM CoCl₂ for 4 hours, the expression of HIF-1αreached the peak. The levels of +IRE DMT1 and - IRE DMT1 reached the highest point in HepG2 cells which were exposed to 0. 5mM CoCl₂ for 4 hours.3. Under hypoxia, HIF-1αin HepG2 cells expressed starting at 1 h hypoxia and reached the highest point at 3 to 6 h hypoxia. The level of +IRE DMT1 increased starting at 1 hour hypoxia and reached the peak at 6 h hypoxia. The expression of - IRE DMT1 increased starting at 3 h hypoxia and reached the peak at 6 h hypoxia, which lasted to 12 h.4. HIF-1αand the two isoforms of DMT1 expressed obviously in HepG2 cells which were exposed to hypoxia for 6 h. When the cells were exposed to reoxidation for 24 h, the level of +IRE DMT1 decreased while - IRE DMT1 changed little.5. When HepG2 cells were exposed to 10mM or 20mM NaN₃ for 1 h, the expression of the two isoforms of DMT1 increased significantly. The level of +IRE DMT1 in the cells also increased which were exposed to 20μM rotenone for 24 h, while -IRE DMT1 changed little. Additionally, HIF-1αcould not accumulate whenever HepG2 cells were exposed to NaN₃ or rotenone.6. In normoxia, HIF-1αexpressed little and the two isoforms of DMT1 localized in both cytoplasm and nucleus uniformly in HepG2 cells. When in hypoxia, HIF-1αexpressed significantly in the nucleus, +IRE DMT1 mainly located in cytoplasm and nuclear membrane, while the majority of - IRE DMT1 in the cytoplasm clustered closer to the cellular membrane and changed little in the nucleus.Conclusions:1. HIF-1αaccumulated and the expression of the two isoforms of DMT1 increased in HepG2 cells when they were exposed to CoCl₂ and hypoxia. The spontaneous increasing in the expression of HIF-1αand DMT1 prompt that DMT1 expression may be regulated by HIF-1. 2. When HepG2 cells were exposed to NaN₃ or rotenone, HIF-1αcould not accumulate while the expression of DMT1 increased. These phenomena demonstrate that the expression of DMT1 may be regulated through another pathway except for HIF-1.3. The different changes between the distribution of +IRE DMT1 and - IRE DMT1 in HepG2 cells after being exposed to hypoxia may be related to the different functions of +IRE DMT1 and - IRE DMT1.更多还原