甲胎蛋白的研究文獻(xiàn)綜述1.1甲胎蛋白的研究進(jìn)展甲胎蛋白于上世紀(jì)60年代首次被蘇聯(lián)科學(xué)家加里·阿貝列夫(GarryAbelev)發(fā)現(xiàn)。在產(chǎn)科上，甲胎蛋白可用于孕期胎兒的缺陷性檢測(cè)如是否罹患唐氏綜合征或神經(jīng)管缺陷等染色體畸變的疾?。辉趦?nèi)科上，AFP作為HCC的一類特異性腫瘤標(biāo)志物，是檢測(cè)原發(fā)性肝細(xì)胞癌的重要工具，為提高檢測(cè)的靈敏度，常與AFP異質(zhì)體AFP-L3，AFP-mRNA等進(jìn)行聯(lián)合檢測(cè)。1.2AFP的生物學(xué)功能促進(jìn)肝癌細(xì)胞的轉(zhuǎn)移參與腫瘤細(xì)胞轉(zhuǎn)移的蛋白質(zhì)有CXCR4、EpCAM、MMPs與K19ADDINEN.CITEADDINEN.CITE.DATA[3]。PTEN屬于抑癌基因，其經(jīng)由調(diào)控細(xì)胞遷移、侵襲和血管生成來實(shí)現(xiàn)對(duì)腫瘤的抑制。PTEN蛋白的作用機(jī)制是通過去磷酸化實(shí)現(xiàn)PIP3向PI-（4,5）-P2的轉(zhuǎn)化，由此對(duì)PIP3施以有效負(fù)調(diào)節(jié)，使得Akt/PKB信號(hào)途徑受阻，對(duì)癌癥達(dá)成有效抑制目的。AFP與PTEN蛋白結(jié)合導(dǎo)致PI3K/Akt/mTOR信號(hào)通路被激活，促進(jìn)CXCR4的表達(dá)——細(xì)胞質(zhì)AFP可導(dǎo)致Akt的磷酸化并促進(jìn)p-Akt和p-mTOR的產(chǎn)生，p-mTOR可與CXCR4基因啟動(dòng)子結(jié)合，從而導(dǎo)致細(xì)胞內(nèi)CXCR4含量上升ADDINEN.CITE<EndNote><Cite><Author>Dubrovska</Author><Year>2012</Year><RecNum>40</RecNum><DisplayText><styleface="superscript">[4]</style></DisplayText><record><rec-number>40</rec-number><foreign-keys><keyapp="EN"db-id="re9dfzas8rpfrpeavs9paazhsew59x9fpa9t"timestamp="1621388180">40</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Dubrovska,Anna</author><author>Elliott,Jimmy</author><author>Salamone,RichardJ</author><author>Telegeev,GennadyD</author><author>Stakhovsky,AlexanderE</author><author>Schepotin,IhorB</author><author>Yan,Feng</author><author>Wang,Yan</author><author>Bouchez,LaureC</author><author>Kularatne,SumithA%JPloSone</author></authors></contributors><titles><title>CXCR4expressioninprostatecancerprogenitorcells</title></titles><pages>e31226</pages><volume>7</volume><number>2</number><dates><year>2012</year></dates><isbn>1932-6203</isbn><urls></urls></record></Cite></EndNote>[4]。此外，AFP也通過調(diào)節(jié)PTEN基因的轉(zhuǎn)錄后修飾來促進(jìn)HCC的惡化。刺激肝癌細(xì)胞生長(zhǎng)細(xì)胞質(zhì)AFP通過與caspase‐3形成復(fù)合物并阻礙caspase‐8傳遞信號(hào)，敲除AFP可以觸發(fā)caspase‐3信號(hào)傳遞，提升HCC細(xì)胞對(duì)ATRA/TRAIL的敏感性ADDINEN.CITEADDINEN.CITE.DATA[5]。細(xì)胞質(zhì)AFP也與維甲酸受體（RAR）發(fā)生競(jìng)爭(zhēng)性反應(yīng)并且抑制RAR從細(xì)胞質(zhì)到細(xì)胞核的易位，該結(jié)果表明細(xì)胞質(zhì)AFP可能會(huì)在生長(zhǎng)和/或細(xì)胞凋亡調(diào)控網(wǎng)絡(luò)中起到一個(gè)輔阻遏物類似角色ADDINEN.CITEADDINEN.CITE.DATA[6]。此外，AFP與AFPR的結(jié)合激活cAMP-PKA途徑ADDINEN.CITEADDINEN.CITE.DATA[7]，促進(jìn)細(xì)胞對(duì)AFP的內(nèi)吞，導(dǎo)致AFP進(jìn)入細(xì)胞中促進(jìn)c-fos，c-jun和Ras基因的表達(dá)并對(duì)肝癌細(xì)胞的生長(zhǎng)具促進(jìn)作用ADDINEN.CITEADDINEN.CITE.DATA[8]。肝癌細(xì)胞的免疫逃逸AFP結(jié)合PTEN，能夠使信號(hào)通路PI3K/Akt/mTOR活化，從而上調(diào)自噬相關(guān)蛋白p-mTOR水平，實(shí)現(xiàn)對(duì)細(xì)胞自噬的有效抑制ADDINEN.CITE<EndNote><Cite><Author>Wang</Author><Year>2018</Year><RecNum>41</RecNum><DisplayText><styleface="superscript">[9]</style></DisplayText><record><rec-number>41</rec-number><foreign-keys><keyapp="EN"db-id="re9dfzas8rpfrpeavs9paazhsew59x9fpa9t"timestamp="1621391017">41</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Wang,Shanshan</author><author>Zhu,Mingyue</author><author>Wang,Qiaoyun</author><author>Hou,Yuli</author><author>Li,Lei</author><author>Weng,Honglei</author><author>Zhao,Yan</author><author>Chen,Dexi</author><author>Ding,Huiguo</author><author>Guo,Junli</author><author>Li,Mengsen</author></authors></contributors><titles><title>Alpha-fetoproteininhibitsautophagytopromotemalignantbehaviourinhepatocellularcarcinomacellsbyactivatingPI3K/AKT/mTORsignalling</title><secondary-title>CellDeath&Disease</secondary-title></titles><periodical><full-title>CellDeath&Disease</full-title></periodical><pages>1027</pages><volume>9</volume><number>10</number><dates><year>2018</year><pub-dates><date>2018/10/09</date></pub-dates></dates><isbn>2041-4889</isbn><urls><related-urls><url>/10.1038/s41419-018-1036-5</url></related-urls></urls><electronic-resource-num>10.1038/s41419-018-1036-5</electronic-resource-num></record></Cite></EndNote>[9]。AFP可以促進(jìn)HCC細(xì)胞中Fas配體的表達(dá)并抑制淋巴細(xì)胞中Fas的表達(dá)ADDINEN.CITEADDINEN.CITE.DATA[10]，F(xiàn)as/FasL系統(tǒng)會(huì)誘使細(xì)胞程序性凋亡，因此AFP可導(dǎo)致HCC細(xì)胞免于凋亡。CD8+細(xì)胞毒性淋巴細(xì)胞（CTLs）在腫瘤清除中起著重要角色，然而調(diào)節(jié)T細(xì)胞可抑制CTLs的免疫攻擊，AFP有潛在增強(qiáng)調(diào)節(jié)T細(xì)胞免疫抑制效果的可能ADDINEN.CITE<EndNote><Cite><Author>Laderoute</Author><Year>2015</Year><RecNum>43</RecNum><DisplayText><styleface="superscript">[11]</style></DisplayText><record><rec-number>43</rec-number><foreign-keys><keyapp="EN"db-id="re9dfzas8rpfrpeavs9paazhsew59x9fpa9t"timestamp="1621391163">43</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Laderoute,M.P.</author></authors></contributors><auth-address>ImmuneSystemManagementClinicandLab,Ottawa,Ontario,K1S5R5,Canada.</auth-address><titles><title>AnewparadigmaboutHERV-K102particleproductionandblockedreleasetoexplaincortisolmediatedimmunosenescenceandage-associatedriskofchronicdisease</title><secondary-title>DiscovMed</secondary-title><alt-title>Discoverymedicine</alt-title></titles><periodical><full-title>DiscovMed</full-title><abbr-1>Discoverymedicine</abbr-1></periodical><alt-periodical><full-title>DiscovMed</full-title><abbr-1>Discoverymedicine</abbr-1></alt-periodical><pages>379-91</pages><volume>20</volume><number>112</number><edition>2016/01/14</edition><keywords><keyword>Aging/*pathology</keyword><keyword>*ChronicDisease</keyword><keyword>EndogenousRetroviruses/*metabolism</keyword><keyword>Humans</keyword><keyword>Hydrocortisone/*metabolism</keyword><keyword>*Immunosenescence</keyword><keyword>RiskFactors</keyword><keyword>Virion/*metabolism</keyword></keywords><dates><year>2015</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>1539-6509</isbn><accession-num>26760982</accession-num><urls></urls><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[11]。此外，細(xì)胞質(zhì)AFP會(huì)影響HIF-1α的水平從而刺激PD-L1的表達(dá)ADDINEN.CITEADDINEN.CITE.DATA[12]，PD-L1對(duì)肝癌細(xì)胞的免疫逃逸和抗藥性均有聯(lián)系。樹突狀細(xì)胞（DC）是抗原呈遞細(xì)胞，可直接激活初始T細(xì)胞。AFP通過誘導(dǎo)DC的凋亡，引起DC功能障礙導(dǎo)致免疫抑制和腫瘤發(fā)展。在臨床上，AFP水平高的HCC患者通常比健康受試者的血清TNF-α含量要低得多ADDINEN.CITE<EndNote><Cite><Author>Um</Author><Year>2004</Year><RecNum>37</RecNum><DisplayText><styleface="superscript">[13]</style></DisplayText><record><rec-number>37</rec-number><foreign-keys><keyapp="EN"db-id="re9dfzas8rpfrpeavs9paazhsew59x9fpa9t"timestamp="1621348218">37</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Um,SoonHo</author><author>Mulhall,Catherine</author><author>Alisa,Akeel</author><author>Ives,AnnetteRobyn</author><author>Karani,John</author><author>Williams,Roger</author><author>Bertoletti,Antonio</author><author>Behboudi,Shahriar</author></authors></contributors><titles><title>α-FetoproteinImpairsAPCFunctionandInducesTheirApoptosis</title><secondary-title>TheJournalofImmunology</secondary-title></titles><periodical><full-title>TheJournalofImmunology</full-title></periodical><pages>1772</pages><volume>173</volume><number>3</number><dates><year>2004</year></dates><urls><related-urls><url>/content/173/3/1772.abstract</url></related-urls></urls><electronic-resource-num>10.4049/jimmunol.173.3.1772</electronic-resource-num></record></Cite></EndNote>[13]?？鼓[瘤藥物的研發(fā)AFP與細(xì)胞膜表面的受體和細(xì)胞質(zhì)蛋白結(jié)合來促進(jìn)惡性腫瘤生長(zhǎng)，故可以使用AFP衍生肽，AFP片段和重組AFP（AFP抑制片段，AIFs）來阻止AFP與信號(hào)傳導(dǎo)分子或AFP受體結(jié)合，從而抑制由AFP介導(dǎo)的惡性腫瘤生長(zhǎng)行為ADDINEN.CITEADDINEN.CITE.DATA[14]。肽AIF包括源于AFP的生長(zhǎng)抑制肽段（GIP）和其類似物組成，GIP不與AFP受體（AFPR）結(jié)合，但可通過胞吞作用進(jìn)入腫瘤細(xì)胞中影響相關(guān)酶的活性ADDINEN.CITEADDINEN.CITE.DATA[15]。其類似物是來源于AFP的3域，包括AFP-3BC，rAFP3D，r3dAFP，它們可以與AFPR和信號(hào)傳導(dǎo)分子結(jié)合，起到抑制HCC惡化的作用。腫瘤細(xì)胞可通過產(chǎn)生髓樣抑制細(xì)胞（MDSC）來偽裝自己，逃離免疫監(jiān)視ADDINEN.CITEADDINEN.CITE.DATA[16]。MDSC表達(dá)的蛋白可與免疫細(xì)胞結(jié)合，“關(guān)閉”其免疫功能ADDINEN.CITEADDINEN.CITE.DATA[17]。AIF與抗腫瘤藥物結(jié)合，不僅可以破壞腫瘤細(xì)胞，還可以刺激免疫細(xì)胞回復(fù)正常功能并殺死癌細(xì)胞。肽AIF可設(shè)計(jì)與PD-1/PD-L1抑制劑結(jié)合，AIF結(jié)合MDSC/腫瘤細(xì)胞，PD-1/PD-L1抑制劑能夠使T細(xì)胞功能活化，將NK細(xì)胞激活，發(fā)揮免疫監(jiān)視和清除功能。1.3甲胎蛋白的檢測(cè)方法化學(xué)發(fā)光免疫分析（CLIA）CLIA有效結(jié)合了化學(xué)發(fā)光法（CL）與免疫分析法（IA）。基于免疫原理，有效結(jié)合抗原與抗體，借助CL來判斷含量，通過發(fā)光反應(yīng)總釋放的光來明確抗體/抗原水平，同時(shí)借助相對(duì)光單位（RLU）實(shí)施測(cè)量。具有線性范圍較寬，更高的回收率和需要樣本量較少，準(zhǔn)確度高等優(yōu)勢(shì)，但需要特定的儀器進(jìn)行檢驗(yàn)，檢測(cè)成本高，需要較高的成本，適合有條件的實(shí)驗(yàn)室進(jìn)行使用ADDINEN.CITEADDINEN.CITE.DATA[18]。圖1-1化學(xué)發(fā)光免疫分析（CLIA）示意圖放射免疫分析（RIA）此方法是通過放射標(biāo)記藥物（一般為125I）進(jìn)行檢測(cè)的技術(shù)，基于標(biāo)記與未標(biāo)記抗原雙方競(jìng)爭(zhēng)特定抗體位點(diǎn)行為，由此得到抗原-抗體復(fù)合物，同時(shí)借助伽馬儀開展測(cè)定。靈敏度和準(zhǔn)確度較高，檢測(cè)成本適中，可以滿足臨床檢驗(yàn)需求。但試劑中存有放射性物質(zhì)，易對(duì)環(huán)境造成污染對(duì)檢測(cè)者造成輻射，已經(jīng)逐漸被其他方法取代。圖1-2放射免疫分析（RIA）示意圖酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）抗體（或抗原）經(jīng)由非共價(jià)相互作用于固定96孔板上吸附，再對(duì)固定的抗體以及內(nèi)有目標(biāo)分析物分布的測(cè)試溶液實(shí)施聯(lián)合孵育。經(jīng)過一段時(shí)間的孵育和洗滌后，通過添加與抗原上剩余抗原位點(diǎn)結(jié)合的酶聯(lián)抗體來檢測(cè)結(jié)合的抗原，并通過顏色的深度判斷待測(cè)物含量。優(yōu)點(diǎn)是操作簡(jiǎn)單，靈敏度較高，檢測(cè)費(fèi)用低，適合自然人群的早期篩查。不足之處是重復(fù)性不高，容易收到外來物質(zhì)的干擾，檢測(cè)范圍較窄，定性檢驗(yàn)容易受到人為因素的影響。圖1-3酶聯(lián)吸附免疫分析（ELISA）示意圖膠體金法（GICA）膠體金法以膠體金作為指示標(biāo)記，以醋酸纖維膜為載體，AFP先與包被在膜上的AFP單克隆抗體反應(yīng)，然后與膠體金納米粒子反應(yīng)，最后呈現(xiàn)顏色，是現(xiàn)今全球最具理想性、簡(jiǎn)便性的AFP測(cè)定技術(shù)。GICA屬于一類定性測(cè)定技術(shù)，費(fèi)用少，容易操作，便攜，無需依賴儀器，能夠直觀呈現(xiàn)測(cè)定結(jié)果，廣泛用于體檢篩查，但無法進(jìn)行定量檢測(cè)ADDINEN.CITEADDINEN.CITE.DATA[19]。熒光免疫分析（FIA）FIA屬于一類借助光學(xué)顯微鏡（OM）與熒光顯微鏡（FM）進(jìn)行微生物樣本分析的技術(shù)。將特異性抗原/抗體分子進(jìn)行熒光標(biāo)記，通過對(duì)熒光分子進(jìn)行檢測(cè)實(shí)現(xiàn)對(duì)標(biāo)志物的定量分析。已經(jīng)廣泛應(yīng)用于檢測(cè)不同的標(biāo)志物-金屬離子，蛋白質(zhì)，小分子物質(zhì)，核酸和一些特定的與疾病相關(guān)的目標(biāo)標(biāo)志物。在檢測(cè)生物分子的眾多方法中，熒光檢測(cè)因?yàn)樗锌梢姽饩€，易于操作，迅速閱讀，低成本，簡(jiǎn)單而得到了廣泛關(guān)注。但熒光分子穩(wěn)定性較差，不適合長(zhǎng)期保存。五種甲胎蛋白檢測(cè)方法檢測(cè)優(yōu)缺點(diǎn)如下表1-1所示。圖1-4免疫熒光分析（FIA）示意圖表1-1甲胎蛋白檢測(cè)方法AFP檢測(cè)方法捕獲/檢測(cè)元件優(yōu)點(diǎn)缺點(diǎn)化學(xué)放光免疫分析抗體/單鏈抗體/納米抗體操作簡(jiǎn)單，靈敏度較高，試劑較穩(wěn)定工作曲線隨時(shí)間漂移放射免疫分析放射性核素標(biāo)記抗體試劑成本較低，靈敏度較高操作復(fù)雜，放射性污染，有效期短酶聯(lián)吸附免疫分析抗體/單鏈抗體/納米抗體試劑成本較低，操作簡(jiǎn)單靈敏度低，適用于定性和半定量測(cè)定膠體金法抗體操作簡(jiǎn)易快速，無需儀器設(shè)備靈敏度低，只適合初篩熒光免疫分析抗體/單鏈抗體/納米抗體靈敏度高儀器昂貴，對(duì)技術(shù)要求高，不適合長(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