王秋蘭，盧育洪，李盛璞，王牡，蔡繼業(yè)

1 暨南大學(xué)化學(xué)系，廣州 510632

2 暨南大學(xué)附屬第一臨床醫(yī)院血液科，廣州 510632

B細(xì)胞膜CD20抗原的分布與單分子力譜探測(cè)

王秋蘭1，盧育洪2，李盛璞1，王牡1，蔡繼業(yè)1

1 暨南大學(xué)化學(xué)系，廣州 510632

2 暨南大學(xué)附屬第一臨床醫(yī)院血液科，廣州 510632

CD20抗原分子在B細(xì)胞上表達(dá)下降是慢性B淋巴細(xì)胞白血病 (B-CLL) 的標(biāo)志性特征。采用激光掃描共聚焦顯微鏡 (LSCM) 和量子點(diǎn)標(biāo)記相結(jié)合的方法對(duì)正常和B-CLL外周血CD20+B淋巴細(xì)胞膜表面CD20抗原分子的表達(dá)及分布進(jìn)行了熒光成像。同時(shí)，采用原子力顯微鏡 (AFM) 對(duì) CD20+B細(xì)胞的形貌及超微結(jié)構(gòu)特征進(jìn)行了表征，并且將AFM針尖用生物素化的單克隆抗體進(jìn)行修飾，對(duì)CD20+B細(xì)胞表面的CD20抗原-抗體之間的單分子力譜進(jìn)行了探測(cè)。LSCM熒光圖像顯示，B-CLL CD20+B淋巴細(xì)胞上CD20分子的表達(dá)量比正常CD20+B淋巴細(xì)胞顯著降低。AFM結(jié)果顯示，B-CLL CD20+B淋巴細(xì)胞超微結(jié)構(gòu)比正常的粗糙。力譜結(jié)果顯示，CD20抗原-抗體的相互作用力大約是非特異性黏附力的5倍，CD20分子在正常CD20+B淋巴細(xì)胞膜上分布比較均勻，小部分有聚集現(xiàn)象，反之，在B-CLL CD20+B淋巴細(xì)胞膜表面分布稀疏。利用以上兩種方法能進(jìn)一步觀察到B-CLL外周血B淋巴細(xì)胞的異常，并在一定程度上解釋臨床上B-CLL病人對(duì)利妥昔的低反應(yīng)現(xiàn)象，為針對(duì)抗原CD20的治療用藥選擇提供參考。

B-CLL，外周血CD20+B淋巴細(xì)胞，CD20分子，LSCM，AFM

Abstract:The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFMtips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical.Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.

Keywords:B-chronic lymphocytic leukemia (B-CLL), peripheral blood CD20+B lymphocytes, CD20 molecule, laser scanning confocal microscopy (LSCM), atomic force microscope (AFM)

CD20分子是一種在細(xì)胞周期起始和細(xì)胞分化過(guò)程中起重要作用的膜蛋白，它在正常B淋巴細(xì)胞及大部分B細(xì)胞惡性淋巴瘤膜表面均有明顯表達(dá)，這為抗 CD20抗體的免疫靶向治療提供了基礎(chǔ)。利妥昔單抗是一種嵌合鼠/人的單克隆抗體，該抗體可與縱貫細(xì)胞膜的CD20抗原特異性結(jié)合。已經(jīng)有報(bào)道用利妥昔治療慢性淋巴細(xì)胞增殖性疾病[1-3]，但是在臨床上 B-CLL病人對(duì)利妥昔治療出現(xiàn)低反應(yīng)現(xiàn)象[4]，除其他因素外，腫瘤細(xì)胞的CD20抗原密度可能是影響治療效果的因素[5-6]。一直以來(lái)，表征細(xì)胞表面抗原密度最常用的是流式細(xì)胞儀，流式表征正常和B-CLL B細(xì)胞表面CD20抗原密度的研究結(jié)果表明[7-12]，CD20在B-CLL B細(xì)胞上的表達(dá)比在正常B細(xì)胞上的表達(dá)還要低，這為解釋B-CLL對(duì)利妥昔治療出現(xiàn)低反應(yīng)提供了一定的依據(jù)。

AFM已經(jīng)被廣泛應(yīng)用于生物領(lǐng)域中，并作為研究解決生物問(wèn)題的有力工具。利用AFM可以在細(xì)胞水平上對(duì)樣品進(jìn)行成像[13-15]。隨著對(duì)AFM功能的深入研究，目前可以利用原子力顯微鏡空間和力譜高分辨的獨(dú)特優(yōu)勢(shì)，通過(guò)細(xì)胞和功能化針尖之間抗原抗體的相互作用或受體配體相互作用，來(lái)探測(cè)蛋白分子間作用力及細(xì)胞表面蛋白分子的分布[16-21]，這方面研究已經(jīng)成為國(guó)內(nèi)外研究的新熱點(diǎn)。對(duì)此，本課題組也有相關(guān)的報(bào)道[22-24]。本文首次嘗試結(jié)合共聚焦顯微鏡和原子力顯微鏡對(duì)人正常和慢性淋巴白血病外周血單個(gè)B淋巴細(xì)胞表面的CD20抗原的分布情況進(jìn)行分析和比較，為臨床上針對(duì) CD20分子治療的藥物選擇提供一定的參考。

1 材料與方法

1.1 試劑和儀器

淋巴細(xì)胞分離液購(gòu)自AXIS SHIELD PoC AS公司；RPMI1640培養(yǎng)液購(gòu)自Gibgo公司；CD20+微珠和MS柱購(gòu)自Mihenyi Biotech公司；鏈霉親和素偶聯(lián)發(fā)射中心波長(zhǎng)655 nm (紅色) 的量子點(diǎn) (QD565)購(gòu)自Sigma公司；其他所用的試劑皆為分析純，實(shí)驗(yàn)用水為三次蒸餾水。Zeiss激光共聚焦掃描熒光顯微鏡 (LS M510，Zeiss，德國(guó))；原子力顯微鏡 (Autoprobe CP Research，Thermomicroscopes 公司，美國(guó))；免疫磁珠分選儀 (Mihenyi Biotech公司，德國(guó))。

1.2 方法

1.2.1 外周血取樣

B慢性淋巴白血病病人外周血取自暨南大學(xué)附屬第一臨床醫(yī)院血液科，正常外周血取自健康志愿者。

1.2.2 單個(gè)核細(xì)胞分離

分別抽取健康人和B-CLL病人新鮮外周血，加肝素鈉抗凝，用密度梯度離心法分離獲得外周血單個(gè)核細(xì)胞。具體如下：以等體積 RPMI1640培養(yǎng)液稀釋肝素抗凝血，小心鋪在細(xì)胞分離液之液面上(稀釋液∶淋巴細(xì)胞分離液為 2∶1)，2 000 r/min離心15 min，收集環(huán)狀乳白色淋巴細(xì)胞層，單個(gè)核細(xì)胞沉淀經(jīng)RPMI1640培養(yǎng)液反復(fù)洗2次即得所需細(xì)胞。

1.2.3 免疫磁珠分選法分離CD20+B細(xì)胞

將得到的單個(gè)核細(xì)胞用 80 μL的緩沖液對(duì) 107個(gè)細(xì)胞進(jìn)行重懸，加入20 μL的CD20微珠，混勻，4 ℃~8 ℃孵育15 min，加入1 mL緩沖液洗滌細(xì)胞，1 500 r/min離心10 min，完全去除上清，用500 μL緩沖液重懸，所得細(xì)胞懸液加入 MS分選柱中，收集先行流出的未標(biāo)記細(xì)胞組分，并用500 μL緩沖液沖洗MS柱，重復(fù)3次，此時(shí)收集到的懸液為CD20陰性細(xì)胞。將分選柱移出磁場(chǎng)，于柱中加1 mL緩沖液，并用活塞快速將分選柱上滯留的細(xì)胞洗脫下來(lái)，這些細(xì)胞是磁性標(biāo)記的 CD20陽(yáng)性細(xì)胞。由于慢淋病人外周血單個(gè)核細(xì)胞中CD20+B淋巴細(xì)胞占90%以上，故無(wú)需進(jìn)一步純化[25]。

1.2.4 激光掃描共聚焦實(shí)驗(yàn)

使用 Zeiss激光共聚焦掃描熒光顯微鏡對(duì)CD20+B淋巴細(xì)胞進(jìn)行成像。將分離出的CD20+B淋巴細(xì)胞滴在經(jīng)多聚賴氨酸處理的蓋玻片上，用 PBS清洗3次，4% (質(zhì)量分?jǐn)?shù)) 多聚甲醛固定15 min，再用PBS清洗3次，加入50 μL 10 mg/L的生物素化的單克隆CD20抗體室溫孵育30 min，PBS清洗除去過(guò)量的抗體，加入 50 μL 1 mg/L鏈霉親和素QD655室溫孵育30 min，PBS清洗除去過(guò)量的鏈霉親和素QD655，封片，對(duì)處理好的載玻片進(jìn)行共聚焦成像。

1.2.5 針尖修飾

AFM 針尖修飾的過(guò)程[26-27]：將 AFM 針尖(UL20B硅探針，力常數(shù)為0.9 N/m) 在乙醇溶液中浸泡5 min，紫外燈下輻照30 min，隨后把針尖浸泡在1 g/L生物素化的牛血清白蛋白溶液中37 ℃孵育過(guò)夜，用PBS洗去過(guò)量的生物素化牛血清白蛋白溶液，再將針尖浸泡在1 g/L鏈霉親和素溶液中室溫孵育30 min，用 PBS洗去過(guò)量的鏈霉親和素，然后將針尖浸泡在10 mg/L生物素化抗人的CD20抗體溶液中4 ℃孵育過(guò)夜，PBS清洗過(guò)量的抗體，最后將針尖浸泡在PBS溶液中4 ℃保存?zhèn)溆茫诳諝庵凶匀涣栏墒褂谩?/p>

1.2.6 AFM樣品制備

分別取正常和B-CLL外周血B淋巴細(xì)胞，滴于玻片上，使其自然鋪展，吸附10 min，然后用 4%多聚甲醛固定15 min，用蒸餾水沖洗3次，室溫自然干燥。立即進(jìn)行AFM掃描。

1.2.7 原子力顯微鏡掃描

將載有細(xì)胞樣品的玻片固定在 AFM 的掃描臺(tái)上，用監(jiān)視器定位所要掃描的樣品區(qū)域，對(duì)樣品進(jìn)行掃描成像，實(shí)驗(yàn)采用100 μm掃描器，空氣中進(jìn)行掃描，以接觸模式成像。所有圖像僅經(jīng)儀器配置軟件 (Thermomicroscopes proscan image processing software version 2.1) 平滑處理，以消除掃描方向上的低頻背景噪音。原子力顯微鏡的力譜用于分析力曲線測(cè)量。所有的力曲線都是在同一加載速率下測(cè)量得到。

2 結(jié)果與分析

2.1 正常和B-CLL外周血CD20+B淋巴細(xì)胞的熒光成像

將量子點(diǎn)標(biāo)記的細(xì)胞樣品放在LSCM下觀察，得到量子點(diǎn)標(biāo)記的外周血 CD20+B淋巴細(xì)胞的激光共聚焦圖像 (圖1)，其中A和D為熒光圖，B和E為微分干涉 (DIC) 圖，C和F為疊加圖。由于CD20+B淋巴細(xì)胞攜帶CD20分子，圖1A和1D中細(xì)胞發(fā)紅光代表 CD20抗原分子表達(dá)于該細(xì)胞上，且分布在細(xì)胞膜表面，核區(qū)未出現(xiàn)，該實(shí)驗(yàn)結(jié)果進(jìn)一步證實(shí)了所分離的細(xì)胞確為 CD20+B淋巴細(xì)胞，并且從圖中可以明顯看出B-CLL外周血CD20+B淋巴細(xì)胞表面 CD20抗原分子表達(dá)量低于正常外周血 CD20+B淋巴細(xì)胞。

2.2 正常和B-CLL外周血CD20+B淋巴細(xì)胞形態(tài)及CD20抗原-抗體特異性相互作用力的AFM分析

對(duì)正常和B-CLL外周血CD20+B淋巴細(xì)胞進(jìn)行了AFM成像 (圖2A和2B)，高分辨的形貌圖像顯示正常外周血CD20+B淋巴細(xì)胞比較光滑，而B-CLL外周血 CD20+B淋巴細(xì)胞較粗糙。為了進(jìn)一步探測(cè)單個(gè)正常和B-CLL外周血CD20+B淋巴細(xì)胞表面受體分子即CD20的分布情況，我們對(duì)這2種淋巴細(xì)胞分別進(jìn)行超微結(jié)構(gòu)成像以達(dá)到定位的目的 (圖2C和 2D)。利用 AFM 力曲線測(cè)量的方法，在 1 μm× 1 μm的膜區(qū)域共測(cè)量了256條力曲線，分析檢測(cè)這2種細(xì)胞的CD20+B淋巴細(xì)胞表面CD20抗原-抗體的特異性相互作用(圖 3A和 3B)，圖中白色的點(diǎn)代表該位置有很強(qiáng)的黏附力，這些可探測(cè)到黏附力的位置就是CD20+B淋巴細(xì)胞表面的 CD20抗原分布，其中代表性的力曲線如圖3C (正常B淋巴細(xì)胞) 和D(B-CLL B淋巴細(xì)胞)。力曲線回收使CD20+B淋巴細(xì)胞表面CD20抗原和CD20抗體修飾的AFM針尖作用斷裂。對(duì)照實(shí)驗(yàn)使用未修飾 CD20抗體的針尖對(duì)正常和 B-CLL CD20+B淋巴細(xì)胞進(jìn)行力曲線測(cè)量，發(fā)現(xiàn)2種細(xì)胞與針尖間均沒有出現(xiàn)明顯的作用力，具有代表性的力曲線如圖3E (正常B淋巴細(xì)胞)和 F (B-CLL B淋巴細(xì)胞)。這個(gè)結(jié)果說(shuō)明了 CD20抗原和對(duì)應(yīng)抗體具有特異性和高親和性，并且抗原-抗體特異性作用力約是非特異性4倍。對(duì)比圖3A和3B，發(fā)現(xiàn)CD20抗原在正常CD20+B淋巴細(xì)胞膜上的分布均勻，小部分有聚集現(xiàn)象，而在 B-CLL CD20+B淋巴細(xì)胞膜上的分布相對(duì)要稀少，這與用LSCM 觀察到的結(jié)果一致。這很可能是因?yàn)?，相?duì)于正常外周血B淋巴細(xì)胞，B-CLL B細(xì)胞是處在B淋巴細(xì)胞成熟的早期階段，并且CD20在B-CLL B細(xì)胞表達(dá)下降是B-CLL的標(biāo)志性特征[4]。

圖1 CD20+B淋巴細(xì)胞CD20分子標(biāo)記QD655激光掃描共聚圖Fig. 1 LSCM images of CD20 molecules of CD20+B lymphocytes labeled with QD565. (A) Fluorescence image of normal CD20+B lymphocyte. (B) DIC image of normal CD20+B lymphocyte. (C) Merged image of normal CD20+B lymphocyte. (D) Fluorescence image of B-CLL CD20+B lymphocyte. (E) DIC image of B-CLL CD20+B lymphocyte. (F) Merged image of B-CLL CD20+B lymphocyte.

圖2 CD20+B淋巴細(xì)胞的原子力圖Fig. 2 AFM images of CD20+B lymphocytes. (A) 3-D image of normal CD20+B lymphocyte (9 μm×9 μm). (B) 3-D image of B-CLL CD20+B lymphocyte (9 μm×9 μm). (C) Ultrastructure image of normal CD20+B lymphocyte (1 μm×1 μm). (D)Ultrastructure image of B-CLL CD20+B lymphocyte (1 μm×1 μm). div: division.

圖3 CD20+B淋巴細(xì)胞的力曲線Fig. 3 Force curves of CD20+B lymphocytes. Map of force curves pots (n=256) recorded using anti-human CD20 functionalized AFM tips and cells (A: normal group, B: B-CLL group, fwhite>fblack, white spots represent the location of CD20 molecules). (C) The force distance curves between the functionalized tip and normal CD20+B lymphocytes. (D) The force distance curves between the functionalized tip and B-CLL CD20+B lymphocytes. (E) The force distance curves between the unfunctionalized tip and normal CD20+B lymphocytes. (F)The force distance curves between the unfunctionalized tip and B-CLL CD20+B lymphocytes.

3 結(jié)論

本實(shí)驗(yàn)通過(guò)結(jié)合LSCM和AFM兩種方法，分析對(duì)比了人正常和外周血單個(gè) CD20+B淋巴細(xì)胞膜表面 CD20抗原的表達(dá)及分布情況。結(jié)果表明，與正常B淋巴細(xì)胞相比，CD20在B-CLL B淋巴細(xì)胞上面的表達(dá)低，分布不均勻。首先，利用LSCM和AFM能進(jìn)一步辨別出B-CLL B淋巴細(xì)胞的異常，結(jié)合形態(tài)學(xué)和其他特征，能夠提高B淋巴細(xì)胞異常診斷的精確性。其次，利妥昔治療能改善患B細(xì)胞白血病人的病情，但是體內(nèi)確切的治療機(jī)制并不清楚，因此B淋巴細(xì)胞表面CD20抗原表達(dá)的多少與臨床上利妥昔治療有一定的關(guān)系[28]。本實(shí)驗(yàn)結(jié)果在一定程度上能解釋臨床上B-CLL病人對(duì)利妥昔反應(yīng)低的原因，并且為針對(duì)抗原 CD20的治療用藥選擇提供一定的參考。

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Distribution and force spectroscopy of CD20 antigen-antibody binding on the B cell surface

Qiulan Wang1, Yuhong Lu2, Shengpu Li1, Mu Wang1, and Jiye Cai1
1 Department of Chemistry, Jinan University, Guangzhou 510632, China
2 Department of Internal Medicine, First Affiliated Hospital of Jinan University, Guangzhou 510632, China