紅細(xì)胞在腫瘤免疫中的作用與地位

1、紅細(xì)胞在腫瘤免疫中的作用與地位蔡小勇林進(jìn)令文明星在腫瘤的發(fā)生發(fā)展過(guò)程中機(jī)體的免疫功能起著非常重要的作用，過(guò)去的研究多只涉及白細(xì)胞免疫系統(tǒng)，而紅細(xì)胞在其中所起的作用一直受到忽視。1981年siegel1提出了紅細(xì)胞免疫系統(tǒng)（redcellimmunesystem）的新概念，開辟了機(jī)體免疫系統(tǒng)的新領(lǐng)域。目前對(duì)紅細(xì)胞免疫功能的表現(xiàn)、機(jī)理、調(diào)節(jié)機(jī)制與疾病關(guān)系等的研究都取得了一定的進(jìn)展，紅細(xì)胞具有免疫功能已是不容質(zhì)疑的事實(shí)，大量研究表明紅細(xì)胞也具有一定的免疫功能，因而對(duì)紅細(xì)胞的作用與地位有必要加以重新認(rèn)識(shí)，本文將該方面研究進(jìn)展綜合總結(jié)如下。1腫瘤免疫中紅細(xì)胞的作用1.1促吞噬作用forslid等2發(fā)現(xiàn)c

2、3b致敏的酵母菌，中性粒細(xì)胞對(duì)其吞噬率為15，當(dāng)加入紅細(xì)胞后，吞噬率增加一倍，紅細(xì)胞碎片亦有相同作用。作者推測(cè)紅細(xì)胞所含抗氧化劑類物質(zhì)能保護(hù)中性粒細(xì)胞吞噬過(guò)程中釋放的氧自由基對(duì)中性粒細(xì)胞的自身細(xì)胞毒作用，從而促進(jìn)吞噬。徐瑛等3亦證明人紅細(xì)胞能明顯促進(jìn)外周血多形核白細(xì)胞（pmn）吞噬功能，在紅細(xì)胞存在下對(duì)酵母菌的吞噬率增加70左右，促吞噬作用與紅細(xì)胞補(bǔ)體1型受體（c3breceptor，cr1）數(shù)量不同有關(guān)。將肺癌患者的紅細(xì)胞和淋巴細(xì)胞按一定步驟與經(jīng)血清致敏的癌細(xì)胞作用，觀察肺癌患者紅細(xì)胞與淋巴細(xì)胞圍攻癌細(xì)胞情況。結(jié)果顯示：肺癌患者紅細(xì)胞與淋巴細(xì)胞不僅可各自單獨(dú)圍攻癌細(xì)胞，且具有協(xié)同抗腫瘤免疫作

3、用，肺癌患者紅細(xì)胞對(duì)淋巴細(xì)胞免疫粘附癌細(xì)胞的促進(jìn)作用較正常人降低4。徐瑛3檢測(cè)了惡性腫瘤患者紅細(xì)胞促pmn吞噬能力，發(fā)現(xiàn)患者紅細(xì)胞促吞噬率明顯低于正常人，認(rèn)為癌瘤患者紅細(xì)胞cr1減少是紅細(xì)胞促pmn吞噬減弱的原因之一。郭峰等5發(fā)現(xiàn)紅細(xì)胞能直接粘附腫瘤細(xì)胞，腫瘤細(xì)胞經(jīng)與補(bǔ)體作用后粘附紅細(xì)胞能力明顯增強(qiáng)，并能促進(jìn)淋巴細(xì)胞、粒細(xì)胞對(duì)腫瘤細(xì)胞的粘附，而cr1單克隆抗體能抑制紅細(xì)胞粘附腫瘤細(xì)胞的活性，說(shuō)明在腫瘤免疫中紅細(xì)胞有免疫調(diào)控，增強(qiáng)其它細(xì)胞功能的作用，這種作用是紅細(xì)胞膜上的cr1介導(dǎo)的。siegel推測(cè)紅細(xì)胞能阻止癌細(xì)胞在血循環(huán)中擴(kuò)散，因?yàn)榘┘?xì)胞在外周血中遇到紅細(xì)胞的機(jī)會(huì)比白細(xì)胞大1000倍，癌細(xì)

4、胞表面覆蓋有抗體補(bǔ)體，易被紅細(xì)胞粘附而被捕捉吞噬。1.2清除循環(huán)免疫復(fù)合物腫瘤產(chǎn)生的大量抗原與血中抗體形成的免疫復(fù)合物（ic）被認(rèn)為是一腫瘤免疫抑制因子，是造成腫瘤免疫逃逸的原因之一。ic沉著于組織某些部位，激活補(bǔ)體系統(tǒng)，亦可造成組織損害。紅細(xì)胞膜具有cr1，通過(guò)cr1，紅細(xì)胞與抗原抗體補(bǔ)體復(fù)合物結(jié)合，并將其運(yùn)送至肝脾固定吞噬系統(tǒng)，ic從紅細(xì)胞上解離，被吞噬細(xì)胞吞噬清除，釋放ic后的紅細(xì)胞可再回到血循環(huán)中，仍具有結(jié)合ic的能力8。cr1為分子量205000的糖蛋白，存在于紅細(xì)胞、b細(xì)胞、pmn及單核細(xì)胞上，每種細(xì)胞所含cr1數(shù)量不同，紅細(xì)胞為950，b細(xì)胞為2100，pmn為57000，單核細(xì)

5、胞為48000，從數(shù)字上看每個(gè)紅細(xì)胞所含cr1數(shù)僅為有核細(xì)胞的1/201/50，但由于血循環(huán)中紅細(xì)胞數(shù)為有核細(xì)胞數(shù)的1000倍，而血循環(huán)中95的cr1是分布于紅細(xì)胞上的，清除ic主要是紅細(xì)胞而非白細(xì)胞。medof6等的體外試驗(yàn)結(jié)果支持上述推測(cè)，他們將抗原抗體補(bǔ)體的復(fù)合物與人血細(xì)胞混合孵育，然后測(cè)定各類細(xì)胞結(jié)合復(fù)合物的數(shù)量，結(jié)果發(fā)現(xiàn)紅細(xì)胞結(jié)合了82.884.8的復(fù)合物，而中性粒細(xì)胞與單核細(xì)胞分別結(jié)合了8.315.2和1.65.8的復(fù)合物。最近發(fā)現(xiàn)紅細(xì)胞cr1與有核細(xì)胞cr1在功能和結(jié)構(gòu)上不同，紅細(xì)胞cr1在細(xì)系姆植汲蝕匭?cluster)。paccaud等7比較了pmn與紅細(xì)胞結(jié)合ic的能力，發(fā)

6、現(xiàn)在相同細(xì)胞濃度下，pmn結(jié)合ic能力與紅細(xì)胞結(jié)合ic能力相同，盡管pmncr1數(shù)量是紅細(xì)胞cr1數(shù)量的4倍，在相同cr1數(shù)量條件下，靜止的或激活的pmn結(jié)合ic的能力始終低于紅細(xì)胞。電鏡下發(fā)現(xiàn)紅細(xì)胞上50的cr1呈簇性分布，而pmn小于15，激活的pmn雖cr1數(shù)量增加，但簇性cr1的數(shù)量并不增加，因而認(rèn)為pmn的功能是組織吞噬，清除ic為紅細(xì)胞的功能。1.3效應(yīng)細(xì)胞樣作用紅細(xì)胞表面有過(guò)氧化物酶，能使紅細(xì)胞直接銷毀粘附的抗原物質(zhì)，從而起效應(yīng)細(xì)胞樣作用。郭峰等6發(fā)現(xiàn)紅細(xì)胞能與多種癌細(xì)胞發(fā)生粘附包括血清致敏的肝癌原代、傳代細(xì)胞株，鼠淋巴母細(xì)胞瘤，艾氏腹水癌細(xì)胞，各種腫瘤細(xì)胞與紅細(xì)胞形成花環(huán)的百分

7、率平均值大1660.97。電鏡下可見(jiàn)紅細(xì)胞發(fā)生變形運(yùn)動(dòng)以順應(yīng)腫瘤細(xì)胞的表面形態(tài)，甚至還可發(fā)生阿米巴樣運(yùn)動(dòng)，包繞壞死的腫瘤細(xì)胞碎片，粘附處的紅細(xì)胞膜與腫瘤細(xì)胞膜粘附、融合。腫瘤細(xì)胞與紅細(xì)胞結(jié)合處有破損現(xiàn)象，在紅細(xì)胞中可見(jiàn)癌細(xì)胞碎片，這種粘附作用可被cr1單抗或c3多抗阻斷。1.4紅細(xì)胞對(duì)淋巴細(xì)胞和細(xì)胞因子的調(diào)控作用yaelli8等觀察了紅細(xì)胞對(duì)lak細(xì)胞殺傷活性的影響，作者采用51cr釋放微量細(xì)胞毒法檢測(cè)了12例癌癥患者lak細(xì)胞對(duì)daudi腫瘤細(xì)胞的殺傷活性，發(fā)現(xiàn)在培養(yǎng)時(shí)未用ficollhypaque液離心除去紅細(xì)胞者其lak細(xì)胞活性較除去紅細(xì)胞者增強(qiáng)13倍，最大1例達(dá)20倍，進(jìn)一步發(fā)現(xiàn)紅白細(xì)

8、胞比從3100:1時(shí)，溶解瘤細(xì)胞活性逐漸增強(qiáng)，在100:1時(shí)至少增加2倍，并證明紅細(xì)胞的增強(qiáng)作用是在lak細(xì)胞培養(yǎng)的誘導(dǎo)期且需要細(xì)胞間的接觸。因而認(rèn)為制備lak細(xì)胞時(shí)不需要分離除去紅細(xì)胞，相反加入適量的紅細(xì)胞對(duì)增強(qiáng)lak細(xì)胞毒活性更為有益。nk細(xì)胞(naturekillercell)在體內(nèi)擔(dān)負(fù)著重要的免疫監(jiān)視功能。紅細(xì)胞能直接增強(qiáng)nk細(xì)胞的抗腫瘤活性，shou等9檢測(cè)了在紅細(xì)胞存在下nk細(xì)胞毒活性，發(fā)現(xiàn)紅細(xì)胞與效應(yīng)細(xì)胞比值為1.3:1時(shí)nk細(xì)胞毒活性開始增強(qiáng)，在2.520:1時(shí)增加最明顯，不論自體，同種異體或異種紅細(xì)胞都能使nk細(xì)胞活性增強(qiáng)，但破碎的紅細(xì)胞無(wú)增強(qiáng)作用，增強(qiáng)作用可能與nk細(xì)胞上補(bǔ)

9、體受體有關(guān)。郭峰6亦發(fā)現(xiàn)，當(dāng)在效：靶：rbc比為10:1:25的條件下時(shí)加rbc組nk細(xì)胞活性明顯高于未加rbc組。腫瘤患者紅細(xì)胞對(duì)nk細(xì)胞活性的正性效應(yīng)降低。shou10最近發(fā)現(xiàn)在rbc的胞漿內(nèi)存在著一種自然殺傷細(xì)胞增強(qiáng)因子(naturekillerenhancingfactor,nkef)能增強(qiáng)nk細(xì)胞活性，并對(duì)其理化特性做了初步分析，認(rèn)為rbc在調(diào)節(jié)nk細(xì)胞方面可能起著重要的作用。sigfuon等11發(fā)現(xiàn)，用美州商陸絲原刺激淋巴細(xì)胞轉(zhuǎn)化，加自身紅細(xì)胞可增加淋巴細(xì)胞轉(zhuǎn)化率和igg、igm、iga的合成。virella等12進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn)，在培養(yǎng)前紅細(xì)胞與抗lfa3單克隆抗體作用或淋巴細(xì)胞與

10、抗cd2的單克隆抗體作用后培養(yǎng)時(shí)不增加b細(xì)胞反應(yīng)，說(shuō)明淋巴細(xì)胞轉(zhuǎn)化合成抗體與紅細(xì)胞lfa3和淋巴細(xì)胞cd2相互作用有關(guān)。紅細(xì)胞能使人t細(xì)胞增殖加強(qiáng)，促進(jìn)t細(xì)胞il2受體表達(dá)以及腫瘤壞死因子13和干擾素產(chǎn)生14。用pha刺激人外周血單個(gè)核細(xì)胞可誘導(dǎo)干擾素產(chǎn)生，keyes等14發(fā)現(xiàn)在培養(yǎng)時(shí)加入紅細(xì)胞可使干擾素產(chǎn)量增加410倍，干擾素產(chǎn)量隨紅細(xì)胞與淋巴細(xì)胞比值關(guān)系而變化，最適宜濃度為1050:1。紅細(xì)胞的促進(jìn)作用與血型無(wú)關(guān)，紅細(xì)胞碎片亦有相同刺激作用，抗cd2的單克隆抗體可抑制紅細(xì)胞對(duì)t細(xì)胞產(chǎn)生干擾素的促進(jìn)作用。kalechman等15亦發(fā)現(xiàn)在培養(yǎng)時(shí)加入自身rbc可使人單核細(xì)胞或鼠脾細(xì)胞對(duì)亞適合劑量

11、的絲裂原的反應(yīng)增強(qiáng)，包括細(xì)胞增殖，il2、il3、il6、克隆刺激因子及干擾素的分泌增加，這種增強(qiáng)效應(yīng)為劑量依賴性的。還發(fā)現(xiàn)在無(wú)絲裂原存在下rbc能增強(qiáng)人單核細(xì)胞及鼠脾細(xì)胞il2r的表達(dá)，這種增強(qiáng)效應(yīng)與紅細(xì)胞膜與t細(xì)胞上的cd2分子相互作用有關(guān)。2紅細(xì)胞免疫功能的調(diào)控及意義siegel等1在兔血清中發(fā)現(xiàn)了抑制紅細(xì)胞免疫粘附的因子，該因子為一不耐熱物質(zhì)，5830分鐘可除去其活性，推測(cè)該因子為一大分子物質(zhì)，機(jī)體通過(guò)控制該因子的合成來(lái)調(diào)節(jié)紅細(xì)胞免疫功能。yin等16發(fā)現(xiàn)該因子為一種球蛋白，對(duì)熱不穩(wěn)定，有與賴氨酸結(jié)合的位點(diǎn)，該因子可阻止ic粘附到紅細(xì)胞上，降低紅細(xì)胞粘附攜帶ic的能力。郭峰等17,18

12、還發(fā)現(xiàn)血清中除存在紅細(xì)胞免疫抑制因子外，還存在著紅細(xì)胞免疫粘附促進(jìn)因子，該因子耐熱，58不能滅活。在正常人血清中促進(jìn)因子活性明顯大于抑制因子，腫瘤患者抑制因子活性上升，促進(jìn)因子活性下降，因而認(rèn)為機(jī)體內(nèi)存在著紅細(xì)胞免疫正負(fù)調(diào)節(jié)機(jī)制，該調(diào)節(jié)機(jī)制紊亂可能是某些疾病發(fā)生發(fā)展的原因。最近還發(fā)現(xiàn)內(nèi)啡肽、胸腺素、轉(zhuǎn)移因子6等對(duì)紅細(xì)胞免疫功能有促進(jìn)作用，說(shuō)明神經(jīng)內(nèi)分泌系統(tǒng)、白細(xì)胞系統(tǒng)亦參加紅細(xì)胞免疫功能的調(diào)控。臨床上已發(fā)現(xiàn)腫瘤患者血清中紅細(xì)胞免疫抑制因子活性增強(qiáng)。3腫瘤紅細(xì)胞免疫功能改變章岳山等19發(fā)現(xiàn)，近交系615小鼠在接種可移植性組織細(xì)胞淋巴瘤lii后，小鼠紅細(xì)胞免疫功能隨著腫瘤的發(fā)展呈下降趨勢(shì)，在腫瘤侵

13、襲早期和中期下降最為迅速，而腫瘤轉(zhuǎn)移開始至腫瘤轉(zhuǎn)移晚期以后，其下降速度減慢，認(rèn)為這一改變可作為評(píng)估腫瘤發(fā)展程度的參考指標(biāo)之一。currie等20采用抗cr1單克隆抗體檢測(cè)腫瘤患者紅細(xì)胞cr1受體數(shù)，發(fā)現(xiàn)在hodgki病、小細(xì)胞肺癌、鱗狀細(xì)胞肺癌和淋巴瘤，其cr1受體數(shù)較正常人減少將近一半，而在緩解期均有不同程度的恢復(fù)，因而認(rèn)為紅細(xì)胞cr1減少為獲得性的，對(duì)紅細(xì)胞cr1檢測(cè)可能對(duì)判定腫瘤患者病情轉(zhuǎn)歸有意義。已發(fā)現(xiàn)在乳腺、胃、大腸、肝、卵巢、血液系統(tǒng)等多種腫瘤患者cr1活性降低。紅細(xì)胞cr1減少，一方面使紅細(xì)胞對(duì)腫瘤細(xì)胞的調(diào)理促吞噬作用功能降低，另一方面導(dǎo)致ic清除障礙，循環(huán)中ic增高。在腫瘤患者

14、血清中存在著促進(jìn)腫瘤生長(zhǎng)的因子，稱封閉因子，目前認(rèn)為該因子為腫瘤抗原與抗體形成的免疫復(fù)合物，ic增高加重破壞了宿主抗腫瘤免疫能力，造成惡性循環(huán)，腫瘤細(xì)胞逃逸宿主免疫系統(tǒng)的攻擊得以生長(zhǎng)繁殖。siegel1推測(cè)紅細(xì)胞能阻止癌細(xì)胞在血循環(huán)中的擴(kuò)散，但缺乏證據(jù)。郭峰等6證明紅細(xì)胞與癌細(xì)胞能發(fā)生粘附，在艾氏腹水癌腹水中發(fā)現(xiàn)了紅細(xì)胞包繞癌細(xì)胞形成花環(huán)的現(xiàn)象即為一例證，從而在形態(tài)學(xué)上有力地支持了siegel1的這一推測(cè)，更重要的是紅細(xì)胞與癌細(xì)胞發(fā)生粘附后除可促進(jìn)吞噬細(xì)胞吞噬外，紅細(xì)胞本身還表現(xiàn)出效應(yīng)細(xì)胞樣作用，這一新發(fā)現(xiàn)對(duì)探討紅細(xì)胞在腫瘤免疫中的作用很有意義。已發(fā)現(xiàn)荷瘤小鼠紅細(xì)胞粘附腫瘤細(xì)胞能力明顯低于正常

15、鼠；臨床上，已發(fā)現(xiàn)在乳腺癌、胃癌、食管癌等患者，紅細(xì)胞粘附腫瘤細(xì)胞能力降低，同時(shí)還發(fā)現(xiàn)在消化道癌患者腫瘤紅細(xì)胞花環(huán)率高低于手術(shù)后證實(shí)腫瘤發(fā)生轉(zhuǎn)移與否有相關(guān)性，手術(shù)切除腫瘤可使患者的紅細(xì)胞免疫功能改善。因而，檢測(cè)紅細(xì)胞免疫功能可能對(duì)判斷腫瘤轉(zhuǎn)移，療效估計(jì)及預(yù)后有一定價(jià)值。nieha等21最近在多種癌細(xì)胞上檢測(cè)到補(bǔ)體抑制蛋白cd46(mcp)，cd55(daf)及cd59(protectin)的表達(dá)，cd46可協(xié)助i因子加速對(duì)c3b的滅活，避免c3b沉積在癌細(xì)胞表面，從而使紅細(xì)胞不能通過(guò)其cr1受體與癌細(xì)胞發(fā)生免疫粘附，這可能是腫瘤免疫逃逸的機(jī)制之一。紅細(xì)胞免疫功能提出至今已取得了很大進(jìn)展，但許多

16、問(wèn)題尚待澄清。腫瘤患者紅細(xì)胞cr1減少是腫瘤發(fā)展過(guò)程產(chǎn)生的還是引起腫瘤的原因之一？抑制因子及促進(jìn)因子的來(lái)源性質(zhì)，相互作用機(jī)制及在腫瘤發(fā)病中的作用，紅細(xì)胞粘附腫瘤細(xì)胞的意義等仍需做進(jìn)一步的研究。另外能否通過(guò)藥物調(diào)節(jié)紅細(xì)胞免疫功能來(lái)治療某些疾病亦是今后需進(jìn)一步研究的方向qcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka

17、1dkagtgkqcwa3p tgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgk tgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn

18、3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2 tgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i3

19、0ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawger4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1

20、dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls

21、50clmtwn60eo8wgqv7xav2 tgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawger4

22、i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3p

23、tgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2tgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydia

24、wgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawger4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkq

25、cwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa

26、3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2eo8wgqv7xav2ohum32wgeauwydiawger4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dka

27、ghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50c

28、lmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkaggeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1

29、dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawger4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls5

30、0clmtwn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav hhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmt

31、wn60eo8wgqv7xav2ohum32wgeauwydiawgmer4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwydiawger4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgz7r4i30ka1dkagtgkqcwa3ptgz7r4i30ka1dkaghn3xtkknbycudxqa7fhyi2chhi92tgkqcwa3ptgshls50clmtwn60eo8wgqv7xav2ohum32wgeauwy