生物標(biāo)志物在電離輻射中的應(yīng)用與研究進(jìn)展

劉紫薇,任伯緒，江獻(xiàn)旺 (長(zhǎng)江大學(xué)醫(yī)學(xué)院，湖北 荊州 434023)

作為一種公認(rèn)的致癌物，高劑量或低劑量/劑量率的電離輻射(ionizing radiation，IR)暴露增加了各種疾病發(fā)生的風(fēng)險(xiǎn)。電離輻射對(duì)人體生理的影響從上個(gè)世紀(jì)開(kāi)始就已被陸續(xù)報(bào)道，主要是來(lái)自環(huán)境、醫(yī)療或職業(yè)方面的輻射暴露。譬如，近年來(lái)核事故及核污染仍有發(fā)生，給人類生活造成了嚴(yán)重的后果[1～3]。除了來(lái)自環(huán)境的輻射暴露，在醫(yī)療設(shè)備中也逐漸體現(xiàn)出輻射暴露的負(fù)面危害，例如直接數(shù)字平板X線成像系統(tǒng)(digital radiography，DR)、電子計(jì)算機(jī)斷層掃描設(shè)備(computed tomography，CT)以及正電子發(fā)射型計(jì)算機(jī)斷層顯像設(shè)備(positron emission computed tomography，PET)等。目前，來(lái)源于放射診斷和治療的輻射暴露構(gòu)成了醫(yī)療工作者大部分的暴露風(fēng)險(xiǎn)。相對(duì)地，接受放射相關(guān)診療的患者也遭遇了不可避免的輻射暴露，甚至引起了嚴(yán)重的損傷和病變[4, 5]。另外,日常生活中也存在易被忽視的風(fēng)險(xiǎn)因素，例如長(zhǎng)時(shí)間地使用移動(dòng)電話也將對(duì)人體造成一定的危害[6, 7]。

隨著人們?cè)絹?lái)越多地關(guān)注輻射所造成的損傷效應(yīng)，有必要不斷更新與改進(jìn)現(xiàn)有的理論依據(jù)和實(shí)驗(yàn)技術(shù)，估量高劑量或低劑量/劑量率帶來(lái)的輻射風(fēng)險(xiǎn)。在這篇論述里，主要從分子和細(xì)胞水平上列舉多種與IR相關(guān)的潛在生物標(biāo)志物，旨在為評(píng)估高劑量或低劑量/劑量率的輻射暴露提供新思路，并加強(qiáng)對(duì)生物標(biāo)記物與電離輻射之間相關(guān)性的理解。

1 生物標(biāo)志物的功能與特性

生物標(biāo)志物是指一類可以用來(lái)反映生物系統(tǒng)和環(huán)境介質(zhì)之間相互作用的物質(zhì)，可以是化學(xué)、物理或生物來(lái)源等等[8]。在過(guò)去的幾十年里，盡管不同類型生物標(biāo)志物的定義和分類存在細(xì)微的變化[9～12], 我們?nèi)匀豢梢愿鶕?jù)時(shí)效性對(duì)其功能進(jìn)行總結(jié)：在輻射暴露后的某一時(shí)刻可用于檢測(cè)吸收劑量；在輻射暴露之前、期間或之后可用于預(yù)測(cè)輻射效應(yīng)增加的風(fēng)險(xiǎn)；在臨床發(fā)現(xiàn)輻射誘發(fā)疾病或死亡之前，評(píng)估輻射暴露后對(duì)健康的影響；評(píng)估暴露很長(zhǎng)一段時(shí)間后長(zhǎng)期的輻射效應(yīng)等。因而生物標(biāo)志物的深入研究具有重要的臨床意義，除了評(píng)估輻射暴露-效應(yīng)之間的關(guān)系以及這些聯(lián)系如何隨個(gè)體的敏感性而變化，還能有助于探知疾病機(jī)制或潛在的病理途徑。

生物標(biāo)志物的定義、特性以及應(yīng)用雖然復(fù)雜且繁多，考慮到不同的檢測(cè)樣品以及其他干擾因素，對(duì)于一種生物標(biāo)志物是否良好的判定存在一定的困難性，但理想型生物標(biāo)志物的共同特征可以被逐一總結(jié)和列舉，例如敏感性、特異性、再現(xiàn)性等等[13]。評(píng)定標(biāo)準(zhǔn)可大致歸納為：能夠有效地分析檢測(cè)，確保盡量避免系統(tǒng)誤差和隨機(jī)誤差；生物標(biāo)志物自身具有良好的特性，例如敏感性、特異性、重復(fù)性以及生物學(xué)可信性；在分析研究中標(biāo)記物與檢測(cè)方法的適用性；在生物樣品、采集方法等方面的可行性。

而關(guān)于生物樣品的采集，并不僅僅限于常規(guī)的血液收集。例如，唾液可作為一種生物樣本被采集，應(yīng)用于輻射暴露的標(biāo)志物研究中[14]。當(dāng)大量的樣本被需求時(shí)，這將給采集帶來(lái)一定的便捷。手指或腳趾的指甲作為生物樣本，也將極大簡(jiǎn)化提取樣本的過(guò)程，相比于其他生物來(lái)源，從這些組織中提取的DNA可更長(zhǎng)時(shí)間地保存[15]。此外，人體呼出的氣體也可以用來(lái)估量輻射暴露[16]。

2 生物標(biāo)志物的分類

確切地說(shuō)，許多生物標(biāo)志物都可以分到多個(gè)交叉類別，例如γH2AX是磷酸化H2A組蛋白家族成員之一，同時(shí)也與DNA損傷存在密切聯(lián)系。鑒于分類的復(fù)雜性，我們將重點(diǎn)關(guān)注于以下類別：與細(xì)胞遺傳學(xué)相關(guān)的生物標(biāo)志物、與核苷酸或DNA損傷有關(guān)的生物標(biāo)記物。

2.1 細(xì)胞遺傳學(xué)相關(guān)的生物標(biāo)志物

關(guān)于細(xì)胞遺傳學(xué)的研究主要是指染色體水平，尤其是染色體異常。其中，一部分具有高敏感性和特異性的細(xì)胞遺傳學(xué)端點(diǎn)可以作為輻射暴露的生物標(biāo)記物，而另外一部分作為生物標(biāo)志物，則可用來(lái)檢測(cè)輻射暴露的延遲效應(yīng)。

1)雙著絲粒染色體 雙著絲粒染色體(dicentric)是染色體異常類型之一，指具有兩個(gè)著絲粒的結(jié)構(gòu)異常的染色體。這種變異除少數(shù)例外，幾乎完全由IR誘導(dǎo)引起畸變。由于雙著絲?；儗?duì)輻射的特異性以及幾乎不存在干擾因素，已作為生物標(biāo)志物的選擇之一，應(yīng)用于輻射暴露中。通過(guò)對(duì)淋巴細(xì)胞中雙著絲?；兊挠?jì)數(shù)，可對(duì)高劑量的輻射暴露進(jìn)行生物劑量學(xué)評(píng)估[17]。對(duì)于意外暴露于電離輻射的全身或局部的急性病例，自動(dòng)檢測(cè)雙著絲粒仍是一種可靠的選擇[18]。因此，雙著絲粒分析作為一種研究工具，已被應(yīng)用于大規(guī)模的輻射事故中來(lái)進(jìn)行生物劑量測(cè)定[19, 20]。值得注意的是，為了更加準(zhǔn)確地估計(jì)輻射劑量，校準(zhǔn)曲線在任何時(shí)候都是十分必要的[21]。通過(guò)對(duì)檢測(cè)技術(shù)的改善與融合，目前已實(shí)現(xiàn)對(duì)檢測(cè)過(guò)程的自動(dòng)化以及劑量-效應(yīng)曲線的描繪，更加直觀地反映了輻射劑量與生物效應(yīng)之間的聯(lián)系[22]。

2)染色體易位 相比于雙著絲?；?，染色體易位(translocations)同樣屬于一種染色體變異類型，甚至可以劃分為復(fù)雜染色體重組(complex chromosomal rearrangements，CCRs)的一部分。染色體易位是一個(gè)公認(rèn)的生物標(biāo)志物，用于測(cè)定來(lái)自環(huán)境、職業(yè)以及醫(yī)療的輻射暴露。采用熒光原位雜交分析(fluorescent in situ hybridization analysis，F(xiàn)ISH)技術(shù)，對(duì)于長(zhǎng)期慢性暴露于放射性同位素鍶的居民進(jìn)行染色體易位測(cè)定，可有效地評(píng)估輻射劑量并分析劑量與生物效應(yīng)之間的線性關(guān)系[23]。此外，染色體易位還可以應(yīng)用于測(cè)定急性輻射暴露之后各個(gè)不同時(shí)間段的延遲效應(yīng)[24]，從而應(yīng)對(duì)不能及時(shí)檢測(cè)的輻射事件。在放射醫(yī)療檢查所產(chǎn)生的低劑量輻射暴露中，染色體易位在一定程度上也反映了生物損傷與輻射劑量之間的關(guān)系[25]。同時(shí)，易位分析也可作為一種對(duì)職業(yè)暴露人群(如核電站工作者、放射工業(yè)工人)的回顧性生物劑量測(cè)定的有效方法[26, 27]。而染色體易位作為一種生物標(biāo)志物，仍然存在一些檢測(cè)時(shí)的干擾因素，其中年齡和抽煙可能是較為主要的混雜因素[28, 29]。

3)復(fù)雜染色體重組 復(fù)雜染色體重組(complex chromosomal rearrangements，CCRs)涉及到兩條或更多染色體至少三處或以上的畸變，可以被認(rèn)為是若干個(gè)簡(jiǎn)單變異如易位、雙著絲粒的組合等等[30]。有研究顯示，CCRs可作為高傳能線密度(linear energy transfer，LET)和重離子暴露的標(biāo)志，主要用來(lái)評(píng)估急性照射產(chǎn)生的生物效應(yīng)[31, 32]。在放射治療誘導(dǎo)的并發(fā)癥中，高度復(fù)雜的染色體畸變也可被觀察到[33]。由于暴露于低劑量輻射和高強(qiáng)度輻射后，分別誘導(dǎo)產(chǎn)生了不同的復(fù)雜的變異[34]，因此，這一潛在生物標(biāo)志物在低劑量暴露以及未來(lái)的研究仍存在挑戰(zhàn)性。

4)染色體超前凝聚 在成熟促進(jìn)因子(maturation promoting factor，MPF)的作用下，間期細(xì)胞的染色體提前凝聚成分裂期可見(jiàn)的凝聚染色體的這一過(guò)程，即為染色體超前凝聚(premature chromosome condensation，PCC)。正如多數(shù)研究所闡明，PCC在評(píng)估高劑量急性照射時(shí)最為有效[35, 36]，主要用于測(cè)量輻射引起的染色體損傷。相比于雙著絲粒測(cè)定，在高劑量范圍的大規(guī)模傷亡事故中，PCC對(duì)暴露劑量的評(píng)估也是可行的[37]。而PCC試驗(yàn)技術(shù)也在不斷地改進(jìn)與優(yōu)化[38]，其中，細(xì)胞周期進(jìn)展指數(shù)(cell-cycle progression index，CPI)已被證實(shí)為一種新的有效的評(píng)估參數(shù)，在輻射事件發(fā)生后的早期，用于檢測(cè)劑量從0到10Gy范圍的輻射暴露[39]。

5)端粒長(zhǎng)度 端粒(telomeres)是指真核細(xì)胞染色體末端的一小段由簡(jiǎn)單重復(fù)的DNA序列和特殊蛋白質(zhì)組成的復(fù)合體。端粒重復(fù)的長(zhǎng)度以及結(jié)合蛋白的完整性，在保護(hù)染色體的末端不退化、避免與相鄰的染色體融合等作用中都是十分重要的，端粒結(jié)構(gòu)的破壞將導(dǎo)致疾病風(fēng)險(xiǎn)的增加[40]。多項(xiàng)研究表明，端粒的縮短對(duì)受輻照后的細(xì)胞基因組不穩(wěn)定性、細(xì)胞凋亡和輻射敏感性增強(qiáng)有促進(jìn)作用[41, 42]。Lustig等對(duì)核事故后幸存者的白細(xì)胞端粒長(zhǎng)度進(jìn)行了分析，發(fā)現(xiàn)端粒長(zhǎng)度與IR的劑量呈負(fù)相關(guān)，結(jié)果顯示電離輻射的長(zhǎng)期效應(yīng)與端粒長(zhǎng)度密切相關(guān)[43]。端粒長(zhǎng)度的測(cè)定也被應(yīng)用于長(zhǎng)期暴露于低劑量電離輻射而誘導(dǎo)的疾病中，并可對(duì)病程進(jìn)行早期評(píng)估[44]。此外，對(duì)腫瘤患者端粒長(zhǎng)度的量化，也可作為生物標(biāo)志物來(lái)預(yù)示潛在的二次惡性腫瘤及晚期并發(fā)癥風(fēng)險(xiǎn)[45]。同樣，在端粒長(zhǎng)度分析時(shí)也應(yīng)該考慮到可能的混雜因子包括年齡、性別等[46]。

6)微核 微核(micronuclei，MN)是指在有絲分裂期間，由整條染色體或染色體片段畸變所產(chǎn)生的小的核外體，通常是基因毒性事件或染色體不穩(wěn)定性的標(biāo)志。利用微核分析可對(duì)暴露于LET的小鼠固體腫瘤細(xì)胞進(jìn)行生物學(xué)效應(yīng)的測(cè)定[47]。核事故暴露或空間輻射環(huán)境實(shí)際上多數(shù)是由復(fù)合射線輻射組成，當(dāng)暴露于這種復(fù)合輻射場(chǎng)或高輻射情況下，含有微核的細(xì)胞所占的比例也可作為輻射誘導(dǎo)損傷的一種生物標(biāo)志物[48]。Ahmadi等利用昆蟲(chóng)進(jìn)行實(shí)驗(yàn)，發(fā)現(xiàn)微核現(xiàn)象與輻射劑量的大小和強(qiáng)度有關(guān)[49]，從一定程度上可間接反映輻射所造成的生物學(xué)效應(yīng)。此外，有研究數(shù)據(jù)表明，MN也可作為職業(yè)暴露預(yù)防醫(yī)學(xué)篩查的重要標(biāo)志[50]。微核測(cè)定的混雜因素同樣也包括年齡和性別等方面。

2.2 DNA損傷相關(guān)的生物標(biāo)志物

DNA損傷主要有兩種形式：一種是內(nèi)源性損傷，由正常代謝產(chǎn)物產(chǎn)生的活性氧(reactive oxygen species，ROS)等誘發(fā)；另一種是外源性損傷，主要來(lái)源于一系列的外部因素，例如紫外線、電離輻射、生物毒素以及病毒等。IR可以直接或間接地誘導(dǎo)DNA損傷，這使DNA損傷相關(guān)的多種表現(xiàn)形式，如DNA鏈斷裂、γH2AX等為輻射暴露的潛在生物標(biāo)志物。

1)DNA SSB/DSB DNA單鏈斷裂(single strand breaks，SSB)或雙鏈斷裂(double strand breaks，DSB)是暴露于IR后的DNA損傷的高度表現(xiàn)特征，檢測(cè)它們的形成可作為輻射暴露或個(gè)人輻射敏感性的生物標(biāo)記物。過(guò)去有研究者采用正常的人外周胸腺細(xì)胞和幼鼠胸腺細(xì)胞接受伽馬射線輻照，檢測(cè)到了輻射先后誘導(dǎo)的SSB和DSB[51]。而放射診療環(huán)境產(chǎn)生的低劑量輻射暴露也可誘發(fā)DSB形成或者DSB修復(fù)的延遲[52]。隨后也有實(shí)驗(yàn)數(shù)據(jù)證明，DNA DSBs及其后的凋亡DNA片段測(cè)定，有可能作為評(píng)估人體暴露在輻射生物劑量學(xué)中的生物標(biāo)記物[53]。

2)γH2AX γH2AX是磷酸化的H2A組蛋白家族成員之一(phosphorylated H2A histone family member X，γH2AX)，和DNA雙鏈斷裂的早期細(xì)胞效應(yīng)密切相關(guān)。在DNA損傷后的短時(shí)間內(nèi)，細(xì)胞核內(nèi)的γH2AX 焦點(diǎn)(γH2AX foci)將形成并逐漸積累。γH2AX作為電離輻射誘導(dǎo)DSB形成的一個(gè)特定的損傷標(biāo)志，可以預(yù)測(cè)并評(píng)估多種癌癥發(fā)病的風(fēng)險(xiǎn)[54, 55]。在放射醫(yī)療檢查中， 即使是低劑量的X射線仍可致兒科患者的DNA損傷，γH2AX foci作為一種在體的效應(yīng)標(biāo)志物，量化了患者接受的射線劑量，為優(yōu)化檢查方案可提供一定的指征[56]。 在檢測(cè)低劑量輻射暴露的應(yīng)用中,γH2AX也顯示了相對(duì)較高的敏感性[57]。而且，在對(duì)比并優(yōu)化了檢測(cè)技術(shù)以及參數(shù)之后，可對(duì)意外發(fā)生的大規(guī)模輻射暴露事故進(jìn)行快速的γH2AX 檢測(cè)[58～60]。

3)細(xì)胞外8-oxo-dG 8-羥基-2-脫氧鳥(niǎo)苷(8-Oxo-2’-deoxyguanosine，8-oxo-dG)是DNA氧化的主要產(chǎn)物之一，組織中聚集增加的8-oxo-dG可以作為氧化應(yīng)激的生物標(biāo)志物[61]。當(dāng)活性氧(reactive oxygen species，ROS)的生成超過(guò)細(xì)胞抗氧化能力時(shí)，氧化應(yīng)激就會(huì)發(fā)生，而8-oxo-dG可在DNA和核苷酸池中通過(guò)ROS形成誘變損傷[62]。在早期的研究中，通過(guò)檢測(cè)腫瘤患者尿液中的8-oxo-dG含量，可對(duì)個(gè)體的放射敏感性進(jìn)行預(yù)測(cè)，從而將8-oxo-dG 納入了檢測(cè)急性放射敏感性的潛在標(biāo)志物[63]。尿液中8-oxo-dG也可用于診斷心導(dǎo)管插入術(shù)中輻射誘導(dǎo)細(xì)胞的DNA損傷[64]，同時(shí)在放射治療中作為患者生存的預(yù)測(cè)因子[65]。另外，8-oxo-dG 在生態(tài)環(huán)境以及動(dòng)物實(shí)驗(yàn)?zāi)M的移動(dòng)電話的輻射暴露中也可進(jìn)行相應(yīng)的評(píng)估[66, 67]。目前，對(duì)于8-oxo-dG的分析及檢測(cè)技術(shù)得到了一定程度的優(yōu)化[68]。

3 結(jié)語(yǔ)

生物標(biāo)志物是否合適，必須考慮到標(biāo)志物自身的敏感性、特異性、再現(xiàn)性、生物學(xué)合理性以及采樣可行性等等。此外，生物樣品的收集、加工和儲(chǔ)存，生物測(cè)定方法的選擇以及潛在的混雜因素也至關(guān)重要。目前，并不存在最為理想的生物標(biāo)志物來(lái)評(píng)估輻射暴露，特別是低劑量或低劑量率的電離輻射。就特異性而言，急性輻射暴存在若干合適的生物標(biāo)志物，例如雙著絲粒變異，但在低劑量輻射方面卻較為缺乏。正如諸多文獻(xiàn)中所提及，大多數(shù)潛在的生物標(biāo)志物仍停留在探索階段?？梢钥隙ǖ氖?，新穎的想法與思考以及逐步增加對(duì)高劑量/低劑量輻射生物效應(yīng)機(jī)制的理解，將有助于生物標(biāo)志物的深入研究和開(kāi)發(fā)。

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