活細(xì)胞提取及應(yīng)用——單個(gè)細(xì)胞級(jí)別的活細(xì)胞提取

 

 

 

 

 

2016年7月14日發(fā)表于Cell雜志。

 

 

 

   由于細(xì)胞異質(zhì)性的存在，單細(xì)胞層面的分析就變得十分重要。目前對(duì)于單細(xì)胞分析的方法主要還是通過化學(xué)、生物學(xué)的方法進(jìn)行裂解后，提取內(nèi)容物進(jìn)行分析，然而這種方法往往會(huì)對(duì)樣本造成一些損傷。直接提取活細(xì)胞具有諸多優(yōu)點(diǎn)，但是操作苦難。如今一種全新使用FluidFM科技的技術(shù)新報(bào)道有望提供一種活細(xì)胞提取新型的簡(jiǎn)易方法。

 

   在本文中，作者對(duì)首先建立了一種使用FluidFM提取細(xì)胞的方法。他們嘗試了各種提取量以確定使用FluidFM能夠?qū)��?xì)胞提取的最大量。在實(shí)驗(yàn)中他們發(fā)現(xiàn)使用FluidFM抽取4 pL的細(xì)胞質(zhì)并觀測(cè)，發(fā)現(xiàn)82%的細(xì)胞依然存活，如圖1B所示。但是如果將抽取量加大到4.5 pL則無細(xì)胞能夠存活。而對(duì)細(xì)胞核而言抽取0.6 pL后，有86%的細(xì)胞依舊存活，如圖1A。而在0.7 pL或以上的體積提取則不可避免的導(dǎo)致細(xì)胞死亡，如圖1B所示。隨后他們對(duì)提取后細(xì)胞的存活狀況進(jìn)行了考察。而后他們繼續(xù)對(duì)提取過的細(xì)胞進(jìn)行觀察，結(jié)果發(fā)現(xiàn)這些細(xì)胞的功能并沒有受到影響。細(xì)胞能夠在提取后約30小時(shí)后正常分裂，如圖1E所示。因此作者認(rèn)為使用FluidFM提取的最大量為細(xì)胞核0.6 pL和細(xì)胞質(zhì)4pL。

圖1. 細(xì)胞存活率檢測(cè) A)原生細(xì)胞的體積分布圖，虛線為平均值，點(diǎn)線為最大值和最小值；B)細(xì)胞提取體積與活細(xì)胞數(shù)之間的關(guān)系。C)活細(xì)胞細(xì)胞核提取后GFP的變化；D)死細(xì)胞細(xì)胞核提取后GFP的變化；E)活細(xì)胞提取后（2.9 pL）后對(duì)細(xì)胞形態(tài)的連續(xù)觀測(cè)。

 

   對(duì)于細(xì)胞亞結(jié)構(gòu)的觀察，往往對(duì)于揭示細(xì)胞病變有著重要的意義。然而細(xì)胞裂解的傳統(tǒng)手段往往會(huì)產(chǎn)生大量的碎片，因此對(duì)細(xì)胞器的觀察造成了諸多困難。在本篇報(bào)道中，作者通過使用FluidFM設(shè)備提取細(xì)胞內(nèi)容物，在低溫環(huán)境下轉(zhuǎn)移到透射電鏡的銅網(wǎng)上，然后進(jìn)行負(fù)染色和揮干，之后將片子放到透射電鏡下觀察，并使用傳統(tǒng)裂解方法得到單細(xì)胞溶液同時(shí)鋪設(shè)在銅網(wǎng)上進(jìn)行對(duì)比。通過觀察他們發(fā)現(xiàn)，使用FluidFM技術(shù)得到的細(xì)胞提取物能夠觀察到大泡狀的結(jié)構(gòu)、小球狀的結(jié)構(gòu)和長(zhǎng)絲類的結(jié)構(gòu)，如圖2C所示。而相比之下細(xì)胞裂解法得到的結(jié)果卻不盡人意，如圖2D所示。

圖2. 細(xì)胞提取物負(fù)染色電鏡圖 A)電鏡樣本制作的示意圖；B)提取液滴在電鏡銅網(wǎng)上的放大圖；C)FluidFM技術(shù)的細(xì)胞提取物電鏡下的圖像；D)普通裂解法的細(xì)胞提取物電鏡下的圖像。

 

   酶活力的檢測(cè)對(duì)于探尋細(xì)胞異質(zhì)性有著十分重要的意義。因此作者也對(duì)FluidFM提取的細(xì)胞提取物進(jìn)行了對(duì)比。首先作者通過β-半乳糖苷酶實(shí)驗(yàn)來測(cè)定提取蛋白的完整性。通過測(cè)定酶解底物產(chǎn)生的熒光素的熒光強(qiáng)度，他們成功觀察到熒光強(qiáng)度隨時(shí)間而增加，說明提取物中的蛋白沒有被破壞，如圖3C、D所示。隨后作者又對(duì)不同細(xì)胞進(jìn)行了不同酶活力的檢驗(yàn)，結(jié)果顯示無論是LacZ轉(zhuǎn)基因HeLa細(xì)胞上還是在測(cè)定HeLa細(xì)胞的Caspase3酶上均取得了成功，如圖3 E、F所示。

圖3. 酶活性分析 A)細(xì)胞提取分析的示意圖；B)將提取的3 pL細(xì)胞提取物放到預(yù)先液封的微孔中；C)酶解底物產(chǎn)生熒光素的熒光強(qiáng)度變化，熒光在1小時(shí)后可見。D)圖形量化熒光強(qiáng)度時(shí)間表；E)LacZ轉(zhuǎn)基因細(xì)胞和非轉(zhuǎn)基因細(xì)胞之間β-半乳糖苷酶活性的差異；F）Caspase3酶活力測(cè)定。

   單細(xì)胞層面的基因表達(dá)通常需要反轉(zhuǎn)錄或者PCR擴(kuò)增，之后使用qPCR測(cè)定。而在此之前往往需要將細(xì)胞裂解，而作者他們采用了與傳統(tǒng)方法不同的策略。他們首先創(chuàng)建了使用FluidFM直接從活細(xì)胞中提取大約0.01 pg RNA，并用普通PCR管合成cDNA并進(jìn)行qPCR檢測(cè)，如圖4A所示。由于如此小的提取量是不能直接放到PCR管里面的，所以他們采取的策略是首先將提取液注入1uL水中，然后再轉(zhuǎn)移到PCR管中，進(jìn)行合成和檢測(cè)如圖6B所示。他們檢測(cè)了兩種管家基因beta-actin (ACTB)beta-2-microglobulin (B2M)以及GFP mRNA的表達(dá)量。在21個(gè)樣本中有90%的樣本成功檢測(cè)到至少1中基因的表達(dá)，其中2/3的樣本可以同時(shí)檢測(cè)到三種基因的表達(dá)。而對(duì)細(xì)胞核的檢測(cè)中，也能夠檢測(cè)到至少一種基因的表達(dá)，如圖4C所示。在對(duì)比同一細(xì)胞同時(shí)提取細(xì)胞質(zhì)（1.7 pL）和細(xì)胞核（1.3 pL）中這三種基因的表達(dá)時(shí)，可以發(fā)現(xiàn)兩者基本相同，如圖4D所示。

圖4 A)單細(xì)胞提取mRNA轉(zhuǎn)錄實(shí)驗(yàn)的示意圖；B)將提取物放入液滴中的方法；C) ERCC spike 為對(duì)照，測(cè)定細(xì)胞質(zhì)中GFP、B2M、ACTB的Ct值；D)對(duì)同一細(xì)胞的細(xì)胞質(zhì)與細(xì)胞核進(jìn)行提取并測(cè)定Ct值。

 

   隨著生物研究越發(fā)趨于微觀化，對(duì)于分析單個(gè)細(xì)胞的需求變得越來越大。但是由于單個(gè)細(xì)胞體積小，所能夠提取出的物質(zhì)相比以前細(xì)胞群落分析來說，難度顯著提高。這不僅對(duì)檢測(cè)儀器的靈敏度有了新的更高的要求，也同時(shí)對(duì)樣本本身的質(zhì)量也提出了更高的指標(biāo)。本篇中使用FluidFM提取活細(xì)胞所取得的樣本質(zhì)量相比于傳統(tǒng)裂解手段有了明顯的提高，從而取得了令人滿意的結(jié)果。另外這種方法控制提取量后，甚至能夠做到不殺死細(xì)胞的情況下完成提取，這使得對(duì)于對(duì)單個(gè)細(xì)胞代謝測(cè)定的追蹤成為了可能。

 

 

   多功能單細(xì)胞顯微操作系統(tǒng)--FluidFM BOT，是將原子力系統(tǒng)、微流控系統(tǒng)、細(xì)胞培養(yǎng)系統(tǒng)為一體的單細(xì)胞操作系統(tǒng)。主要功能包括單細(xì)胞注射、單細(xì)胞提取以及單細(xì)胞分離。

   FluidFM BOT極大的方便了單細(xì)胞水平的研究，尤其適合應(yīng)用于精準(zhǔn)醫(yī)療、單細(xì)胞生物學(xué)、單細(xì)胞質(zhì)譜、單細(xì)胞基因編輯、藥物研發(fā)等領(lǐng)域。

 

 

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