利用蛋白质芯片技术探索肺纤维化早期标志物及硒代金属硫蛋白的防护作用
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摘要
金属硫蛋白(Metallothionein，简称MT)是指一类低分子量，高巯基含量，能结合大量金属离子的细胞内非酶蛋白质。它广泛存在于生物界并具有非常保守的结构特征，在体内参与必需金属离子的代谢，重金属解毒和清除自由基抗电离辐射等重要过程，并与细胞凋亡与增生、机体能量代谢等密切相关。
蛋白质芯片指纹图谱(SELDI)技术，是20世纪末继基因芯片之后发展起来的新技术。在后基因组时代的今天，人们已经认识到蛋白质组“全景式”分析，是认识重大疾病的基础，判断一个未知基因的功能，最终必须通过在蛋白质水平上的研究才能确定。SELDI技术将蛋白质芯片与飞行质谱相结合，使它既具有芯片的高通量、高效率和重复性好的特点，又具有飞行质谱的高灵敏度飞级(fg，10~(-15))水平检测的功能，可直接对血清及组织粗样品中的各种蛋白质进行检测，其理论和技术的发展为各种疾病的研究提供了新的方法。
本课题的研究目的主要是：建立两种肺纤维化的动物模型，利用蛋白质芯片
中文摘要
指纹图谱技术，建立肺组织和血清的蛋白质芯片指纹图谱标准及损伤模型，从中
寻找与之相关性高的特异性生物标志物，找到并确立肺纤维化特异性强的生物学
动态观察指标。同时对肺组织液中Se一MT进行鉴定并评价Se一MT生物学效应，为
J晦床进行肺纤维化早期无创性诊断以及防治药物的药效学评估奠定基础。
本课题在第一部分根据MT具有金属鳌合特性，特别是与铜有非常强的亲和
力，因此我们选用了蛋白质芯片中的IMAC一Cu芯片对Se一MT进行鉴定。结果发
现:根据Sigma公司MT标准品的蛋白质表达指纹图谱模型，我们在给有机硒诱
导小鼠的肺组织液中MT的表达明显高于正常对照组，证明了有机硒对MT确实
具有高效诱导性，也证明了硒能诱导肺产生MT，为以后的研究提供了可靠的依据。
本课题在第二部分建立了小鼠放射性肺损伤模型，并对预先给与有机硒诱导
Se一MT产生的防护作用进行评价。结果表明:预先给与有机硒组小鼠的存活时间
明显长于单纯损伤组(P<0.05)。而使用蛋白质指纹图谱方法得到小鼠血清和肺组
织指纹图谱在照射14天和7天后，分别有2个和1个标志性蛋白能将正常对照组
和损伤组区别开来，而在有机硒预先给药组中这些蛋白的表达与正常组相似。这
样我们初步确立了肺放射性损伤的蛋白质生物标记，有可能在今后的研究中作为
一种新的方法被利用。
本课题在第三部分建立了化学物质百草枯致小鼠肺纤维化的动物模型，并研
究了Se一MT对肺纤维化产生的防护作用。病理结果表明，Se预先诱导MT组小鼠
肺脏的组织结构基本接近正常小鼠，而损伤组小鼠均已出现肺纤维化病理改变;
经SELD工一TOF一MASS测定蛋白质指纹图谱结果显示有4个能将正常对照组和损伤组
区别开来的标志性蛋白。其中肺组织和血清中各有2个，而且这些具有损伤意义
的蛋白随时间的变化呈现出一定的剂量依赖关系。而在Se保护组，可见这些蛋白
的表达与对照组相比无明显变化。这些标志性蛋白的发现，不仅为今后进一步的
研究工作提供了依据，而且极有希望捕获肺纤维化早期具有特异性变化的蛋白质
或蛋白质群标志物，从而建立起肺纤维化无创伤性早期诊断方法。
Metallothionein (MT) is a group of cysteine-rich, low molecular weight, metal-binding intracellular protein which are widely distributed in nature. It has been proved that MT is closely correlative with essential metal metabolism, heavy metal detoxification, anti-radiation, anti-oxidation, cleaning free radical, apoptosis and proliferation of tumor cells and energy metabolism et al.
SELDI is a new technology which developed after genechip in the end of 20th century.In the era of post-genomics,scientists have taken cognizance that the panoramic analysis of proteimics is the basic for people to understand a gravedisease, and estimating the function of an unknowen gene must be confirmed on the level of protein. SELDI can combine proteinchip technology with Mass Spectrometry and has some merits including not only high quantum,high efficiency,good repeation of chip but also high sensitivity of Mass. The development of knowledge and technology in proteomics has provided a new way to study many kind of diseases.
The aim of this study is to prove:(1)establishing two kinds of animal model of pulmonary fibrosis; (2) establishing lung tissue and serum proteinchip Spectrometry including standard and injure models by SELDI technology, exploring the early biomarker, eatablishing strong especially biological observing marker closely relatated to the pulmonary fibrosis . (3) evaluating and indentifing the biological effect of Se-MT in order to diagnose forepart pulmonary fibrosis and evaluate the effect of some medicines on clinic.
In the first part, we select the kind of IMAC-Cu proteinchip to indentify the Se-MT on the basic that MT has a metal chelated character especially strong power to combine with copper. The results showed that: Compared with the proteinchip Spectrometry of MT standard sample from Sigma Co., the expression of MT in mice lung tissues
induced by organic selenium is higher than that in control groups. The results prove that organic selenium has strong ability to induce MT and lung can be induced to produce MT. These results provide a good reference for the further study.
In the second part, we establish the radiate lung injure model of mice , and evaluate the protective effects of MT induced by organic selenium (Se-malt). The results showed that: the living time of mice induced by Se-malt is obviosely longer than the radiate injure groups (P<0.05) .Meanwhile,we indentify 2 and 1 biomarker respectively in 14 and 7 days after radiation of mice serum and lung tissue that can distinguish the control and injure groups by SELDI ,moreover the expression of these protein in Se treated group is similar with control group.In this way ,we ensure the protein biomarker of lung radiate injure model.This way maybe used as a new method in the future study.
In the third part, we establish mice pulmonary fibrosis model by chemistry substance PQ and study the protection of Se-MT. The pathology results show that the lung tissue structure of mice in Se treated group is similar with control group ,while the mice in the injure group show pulmonary fibrosis pathology changes. The proteinchip spectrometry by SELDI-TOF-MASS show that there are 4 protein marker that can distinguish the control and injure group and the change depends on the dose. There are no significant changes of protein expression between in Se treated group and in control group, in this study the protein fingerprinting pattern of serum and liver tissue lyses established The discovery of these marker protein not only provided a novel powerful proteomic model ,but also hopely captured the early marker in forepart pulmonary fibrosis . This non-injury method could be widely used in both clinic trial and pre-clinic study in the future.
蛋白质芯片指纹图谱(SELDI)技术，是20世纪末继基因芯片之后发展起来的新技术。在后基因组时代的今天，人们已经认识到蛋白质组“全景式”分析，是认识重大疾病的基础，判断一个未知基因的功能，最终必须通过在蛋白质水平上的研究才能确定。SELDI技术将蛋白质芯片与飞行质谱相结合，使它既具有芯片的高通量、高效率和重复性好的特点，又具有飞行质谱的高灵敏度飞级(fg，10~(-15))水平检测的功能，可直接对血清及组织粗样品中的各种蛋白质进行检测，其理论和技术的发展为各种疾病的研究提供了新的方法。
本课题的研究目的主要是：建立两种肺纤维化的动物模型，利用蛋白质芯片
中文摘要
指纹图谱技术，建立肺组织和血清的蛋白质芯片指纹图谱标准及损伤模型，从中
寻找与之相关性高的特异性生物标志物，找到并确立肺纤维化特异性强的生物学
动态观察指标。同时对肺组织液中Se一MT进行鉴定并评价Se一MT生物学效应，为
J晦床进行肺纤维化早期无创性诊断以及防治药物的药效学评估奠定基础。
本课题在第一部分根据MT具有金属鳌合特性，特别是与铜有非常强的亲和
力，因此我们选用了蛋白质芯片中的IMAC一Cu芯片对Se一MT进行鉴定。结果发
现:根据Sigma公司MT标准品的蛋白质表达指纹图谱模型，我们在给有机硒诱
导小鼠的肺组织液中MT的表达明显高于正常对照组，证明了有机硒对MT确实
具有高效诱导性，也证明了硒能诱导肺产生MT，为以后的研究提供了可靠的依据。
本课题在第二部分建立了小鼠放射性肺损伤模型，并对预先给与有机硒诱导
Se一MT产生的防护作用进行评价。结果表明:预先给与有机硒组小鼠的存活时间
明显长于单纯损伤组(P<0.05)。而使用蛋白质指纹图谱方法得到小鼠血清和肺组
织指纹图谱在照射14天和7天后，分别有2个和1个标志性蛋白能将正常对照组
和损伤组区别开来，而在有机硒预先给药组中这些蛋白的表达与正常组相似。这
样我们初步确立了肺放射性损伤的蛋白质生物标记，有可能在今后的研究中作为
一种新的方法被利用。
本课题在第三部分建立了化学物质百草枯致小鼠肺纤维化的动物模型，并研
究了Se一MT对肺纤维化产生的防护作用。病理结果表明，Se预先诱导MT组小鼠
肺脏的组织结构基本接近正常小鼠，而损伤组小鼠均已出现肺纤维化病理改变;
经SELD工一TOF一MASS测定蛋白质指纹图谱结果显示有4个能将正常对照组和损伤组
区别开来的标志性蛋白。其中肺组织和血清中各有2个，而且这些具有损伤意义
的蛋白随时间的变化呈现出一定的剂量依赖关系。而在Se保护组，可见这些蛋白
的表达与对照组相比无明显变化。这些标志性蛋白的发现，不仅为今后进一步的
研究工作提供了依据，而且极有希望捕获肺纤维化早期具有特异性变化的蛋白质
或蛋白质群标志物，从而建立起肺纤维化无创伤性早期诊断方法。
Metallothionein (MT) is a group of cysteine-rich, low molecular weight, metal-binding intracellular protein which are widely distributed in nature. It has been proved that MT is closely correlative with essential metal metabolism, heavy metal detoxification, anti-radiation, anti-oxidation, cleaning free radical, apoptosis and proliferation of tumor cells and energy metabolism et al.
SELDI is a new technology which developed after genechip in the end of 20th century.In the era of post-genomics,scientists have taken cognizance that the panoramic analysis of proteimics is the basic for people to understand a gravedisease, and estimating the function of an unknowen gene must be confirmed on the level of protein. SELDI can combine proteinchip technology with Mass Spectrometry and has some merits including not only high quantum,high efficiency,good repeation of chip but also high sensitivity of Mass. The development of knowledge and technology in proteomics has provided a new way to study many kind of diseases.
The aim of this study is to prove:(1)establishing two kinds of animal model of pulmonary fibrosis; (2) establishing lung tissue and serum proteinchip Spectrometry including standard and injure models by SELDI technology, exploring the early biomarker, eatablishing strong especially biological observing marker closely relatated to the pulmonary fibrosis . (3) evaluating and indentifing the biological effect of Se-MT in order to diagnose forepart pulmonary fibrosis and evaluate the effect of some medicines on clinic.
In the first part, we select the kind of IMAC-Cu proteinchip to indentify the Se-MT on the basic that MT has a metal chelated character especially strong power to combine with copper. The results showed that: Compared with the proteinchip Spectrometry of MT standard sample from Sigma Co., the expression of MT in mice lung tissues
induced by organic selenium is higher than that in control groups. The results prove that organic selenium has strong ability to induce MT and lung can be induced to produce MT. These results provide a good reference for the further study.
In the second part, we establish the radiate lung injure model of mice , and evaluate the protective effects of MT induced by organic selenium (Se-malt). The results showed that: the living time of mice induced by Se-malt is obviosely longer than the radiate injure groups (P<0.05) .Meanwhile,we indentify 2 and 1 biomarker respectively in 14 and 7 days after radiation of mice serum and lung tissue that can distinguish the control and injure groups by SELDI ,moreover the expression of these protein in Se treated group is similar with control group.In this way ,we ensure the protein biomarker of lung radiate injure model.This way maybe used as a new method in the future study.
In the third part, we establish mice pulmonary fibrosis model by chemistry substance PQ and study the protection of Se-MT. The pathology results show that the lung tissue structure of mice in Se treated group is similar with control group ,while the mice in the injure group show pulmonary fibrosis pathology changes. The proteinchip spectrometry by SELDI-TOF-MASS show that there are 4 protein marker that can distinguish the control and injure group and the change depends on the dose. There are no significant changes of protein expression between in Se treated group and in control group, in this study the protein fingerprinting pattern of serum and liver tissue lyses established The discovery of these marker protein not only provided a novel powerful proteomic model ,but also hopely captured the early marker in forepart pulmonary fibrosis . This non-injury method could be widely used in both clinic trial and pre-clinic study in the future.
引文
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