低剂量辐射对人肺上皮细胞和肺癌细胞Nrf2表达差异的研究
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摘要
肺癌是世界范围内最为常见的恶性肿瘤之一，我国肺癌的发病率和死亡率占城市恶性肿瘤的首位，其发病率和死亡率逐年升高，越来越引起研究者的关注。
肺癌常规治疗包括手术，化疗、放疗、生物治疗、分子靶向治疗和中药治疗等。据临床统计，非小细胞肺癌确诊时仅有20%病例能进行根治性手术，在手术切除的病例中5年生存率仅为30%~40%。术后放疗能在一定程度上提高肺癌患者局部控制率和生存率，因此被广泛的应用。放疗是治疗肺癌的常用方法。但放疗在破坏和杀灭癌细胞同时，也不可避免地对常组织细胞造成一定损伤，这已成为增加放疗剂量以提高疗效的一大障碍。在增加杀伤肿瘤作用的同时，如何减少对正常组织的损伤，一直是肿瘤工作者希望得到的理想结果。低剂量辐射具有对正常细胞的兴奋效应和适应性反应，而同时对肿瘤细胞可能产生与大剂量放疗的协同杀伤作用，这对低剂量辐射与大剂量照射联合应用于临床，加强放疗疗效、减轻放疗副作用具有重要意义。
近年来，对核因子E2相关因子2（Nrf2）的研究成为氧化损伤领域的热点，Nrf2已被证实是调节细胞内众多抗氧化物表达的关键性因子，具有维持细胞氧化-抗氧化平衡、抑制凋亡、抗炎的多重生物活性。
本实验对低剂量辐射后正常肺上皮细胞及肺癌细胞的生物学效应差异进行了研究，检测了氧化应激相关基因变化，并对其机制进行探讨。
本实验应用MTT检测低剂量辐射（75mGy）后3、6、12、24和48h肺上皮细胞（HBE）和肺癌细胞（A549）的增殖情况；应用流式细胞术检测细胞周期各阶段的细胞比例，对比HBE细胞及A549细胞低剂量辐射后的细胞周期变化；应用Western blot检测两种细胞低剂量辐射后6、24h Nrf2蛋白的表达量；应用ELISA检测两种细胞内Nrf2调控的下游蛋白（谷胱甘肽）的浓度水平。
本实验的主要结果是：
（1）LDR后正常肺上皮细胞增殖明显加快，S期细胞比例明显增加；而肺癌细胞无明显增殖，S期细胞比例也无明显变化。表明LDR可诱导正常肺上皮细胞产生兴奋效应，而在肺癌细胞则不能诱导兴奋效应。
（2）LDR后正常肺上皮细胞Nrf2表达水平明显增加，而肺癌细胞Nrf2表达水平无明显变化。
（3）LDR后正常肺上皮细胞谷胱甘肽浓度水平明显增加，而在肺癌细胞未发现谷胱甘肽浓度明显变化。
这些结果表明，低剂量辐射在正常肺上皮细胞和肺癌细胞引起不同的抗氧化应激反应，这可能是低剂量辐射在两类细胞引起生物学效应差异的机制之一。
Lung cancer is one of the most common malignant tumors worldwide. In our country,incidence and mortality of lung cancer are situated the first place of malignant tumors incities, the incidence and mortality are increasing in the recent years, and more and moreresearchers are attracted by these situations.
The conventional treatments for lung cancer include surgery, chemotherapy,radiotherapy, biological therapy, molecular targeted therapy and Traditional Chinesemedicine and so on. According to clinical statistics, only20%of patients with non-small celllung cancer have chance to accept radical surgery at the time of diagnosis. The5-yearsurvival rate of the cases after surgery is from30%to40%. Postoperative radiotherapy canimprove local control and survival rate of patients with lung cancer to a certain extent, and isused widely in clinical treatment. However, the radiotherapy will unavoidably cause damagein the normal tissues meanwhile killing cancer cells, that has become a major obstacle ofincreasing radiation dose for improving radiation efficacy. The decreased in the damage tonormal tissues when radiation treatment of tumors is an ideal result for tumor researchers.Low-dose radiation has hormesis and adaptive response in the normal cells. Low-doseradiation in combined with high-dose radiation in the clinic may cause synergistic killing effectson tumor cells, that have an important significance for the enhancement of radiotherapyefficacy and alleviation of the side effects of radiotherapy.
In the recent years, the studies on the nuclear factor E2-related factor2(Nrf2) havebecome hot spots in the field of oxidative damage. Nrf2has been proved to be a key factor inadjustment of expression of many intracellular antioxidants. Nrf2possesses multiplebiological activities, such as keeping the oxidation-antioxidation balance in cells, inhibitingapoptosis, anti-inflammatory effect.
In this study, the differential biological effects induced by low-dose radiation in humanepithelial cells and lung cancer cells has been studied, the changes of gene expressioninvolved to oxidative stress were detected, as well as the mechanisms were explored.
In this study, the proliferation of normal lung epithelial cells(HBE) and lung cancercells(A549) at3,6,12,24and48h after low-dose radiation was detected by MTT assay; the proportion of cells in cell cycle stages was evaluated using flow cytometry, and the changesin the cell cycle in HBE cells and A549cells after low-dose radiation ware studied; Westernblot was used to detect expression of Nrf2in two kinds of cells at6h and24h after radiation;glutathione concentrations regulated by Nrf2in two kinds of cells were determined byELISA.
Main results of the present study:
(1) Proliferation of normal lung epithelial cells was noticeably accelerated after LDR,the proportion of S-phase cells was significantly increased; while the proliferation of lungcancer cells and the proportion of S-phase cells had no significant change, indicating thatLDR was able to induce hormesis, but LDR failed to induce hormesis in lung cancer cells.
(2) Expression levels of Nrf2in normal lung epithelial cells were significantly increasedafter LDR, while expression levels of Nrf2in lung cancer cells did not markedly change.
(3) After LDR, the glutathione concentrations of normal lung epithelial cells weresignificantly increased, while changes in glutathione concentrations in lung cancer cells werenot found.
These results suggested that low-dose radiation induced differential oxidative stressresponse in normal lung epithelial cells and in lung cancer cells, and that may be one of themechanisms of low-dose radiation resulted in the differential biological effects in two kindsof cells.
肺癌常规治疗包括手术，化疗、放疗、生物治疗、分子靶向治疗和中药治疗等。据临床统计，非小细胞肺癌确诊时仅有20%病例能进行根治性手术，在手术切除的病例中5年生存率仅为30%~40%。术后放疗能在一定程度上提高肺癌患者局部控制率和生存率，因此被广泛的应用。放疗是治疗肺癌的常用方法。但放疗在破坏和杀灭癌细胞同时，也不可避免地对常组织细胞造成一定损伤，这已成为增加放疗剂量以提高疗效的一大障碍。在增加杀伤肿瘤作用的同时，如何减少对正常组织的损伤，一直是肿瘤工作者希望得到的理想结果。低剂量辐射具有对正常细胞的兴奋效应和适应性反应，而同时对肿瘤细胞可能产生与大剂量放疗的协同杀伤作用，这对低剂量辐射与大剂量照射联合应用于临床，加强放疗疗效、减轻放疗副作用具有重要意义。
近年来，对核因子E2相关因子2（Nrf2）的研究成为氧化损伤领域的热点，Nrf2已被证实是调节细胞内众多抗氧化物表达的关键性因子，具有维持细胞氧化-抗氧化平衡、抑制凋亡、抗炎的多重生物活性。
本实验对低剂量辐射后正常肺上皮细胞及肺癌细胞的生物学效应差异进行了研究，检测了氧化应激相关基因变化，并对其机制进行探讨。
本实验应用MTT检测低剂量辐射（75mGy）后3、6、12、24和48h肺上皮细胞（HBE）和肺癌细胞（A549）的增殖情况；应用流式细胞术检测细胞周期各阶段的细胞比例，对比HBE细胞及A549细胞低剂量辐射后的细胞周期变化；应用Western blot检测两种细胞低剂量辐射后6、24h Nrf2蛋白的表达量；应用ELISA检测两种细胞内Nrf2调控的下游蛋白（谷胱甘肽）的浓度水平。
本实验的主要结果是：
（1）LDR后正常肺上皮细胞增殖明显加快，S期细胞比例明显增加；而肺癌细胞无明显增殖，S期细胞比例也无明显变化。表明LDR可诱导正常肺上皮细胞产生兴奋效应，而在肺癌细胞则不能诱导兴奋效应。
（2）LDR后正常肺上皮细胞Nrf2表达水平明显增加，而肺癌细胞Nrf2表达水平无明显变化。
（3）LDR后正常肺上皮细胞谷胱甘肽浓度水平明显增加，而在肺癌细胞未发现谷胱甘肽浓度明显变化。
这些结果表明，低剂量辐射在正常肺上皮细胞和肺癌细胞引起不同的抗氧化应激反应，这可能是低剂量辐射在两类细胞引起生物学效应差异的机制之一。
Lung cancer is one of the most common malignant tumors worldwide. In our country,incidence and mortality of lung cancer are situated the first place of malignant tumors incities, the incidence and mortality are increasing in the recent years, and more and moreresearchers are attracted by these situations.
The conventional treatments for lung cancer include surgery, chemotherapy,radiotherapy, biological therapy, molecular targeted therapy and Traditional Chinesemedicine and so on. According to clinical statistics, only20%of patients with non-small celllung cancer have chance to accept radical surgery at the time of diagnosis. The5-yearsurvival rate of the cases after surgery is from30%to40%. Postoperative radiotherapy canimprove local control and survival rate of patients with lung cancer to a certain extent, and isused widely in clinical treatment. However, the radiotherapy will unavoidably cause damagein the normal tissues meanwhile killing cancer cells, that has become a major obstacle ofincreasing radiation dose for improving radiation efficacy. The decreased in the damage tonormal tissues when radiation treatment of tumors is an ideal result for tumor researchers.Low-dose radiation has hormesis and adaptive response in the normal cells. Low-doseradiation in combined with high-dose radiation in the clinic may cause synergistic killing effectson tumor cells, that have an important significance for the enhancement of radiotherapyefficacy and alleviation of the side effects of radiotherapy.
In the recent years, the studies on the nuclear factor E2-related factor2(Nrf2) havebecome hot spots in the field of oxidative damage. Nrf2has been proved to be a key factor inadjustment of expression of many intracellular antioxidants. Nrf2possesses multiplebiological activities, such as keeping the oxidation-antioxidation balance in cells, inhibitingapoptosis, anti-inflammatory effect.
In this study, the differential biological effects induced by low-dose radiation in humanepithelial cells and lung cancer cells has been studied, the changes of gene expressioninvolved to oxidative stress were detected, as well as the mechanisms were explored.
In this study, the proliferation of normal lung epithelial cells(HBE) and lung cancercells(A549) at3,6,12,24and48h after low-dose radiation was detected by MTT assay; the proportion of cells in cell cycle stages was evaluated using flow cytometry, and the changesin the cell cycle in HBE cells and A549cells after low-dose radiation ware studied; Westernblot was used to detect expression of Nrf2in two kinds of cells at6h and24h after radiation;glutathione concentrations regulated by Nrf2in two kinds of cells were determined byELISA.
Main results of the present study:
(1) Proliferation of normal lung epithelial cells was noticeably accelerated after LDR,the proportion of S-phase cells was significantly increased; while the proliferation of lungcancer cells and the proportion of S-phase cells had no significant change, indicating thatLDR was able to induce hormesis, but LDR failed to induce hormesis in lung cancer cells.
(2) Expression levels of Nrf2in normal lung epithelial cells were significantly increasedafter LDR, while expression levels of Nrf2in lung cancer cells did not markedly change.
(3) After LDR, the glutathione concentrations of normal lung epithelial cells weresignificantly increased, while changes in glutathione concentrations in lung cancer cells werenot found.
These results suggested that low-dose radiation induced differential oxidative stressresponse in normal lung epithelial cells and in lung cancer cells, and that may be one of themechanisms of low-dose radiation resulted in the differential biological effects in two kindsof cells.
引文
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