活細胞提取及應(yīng)用——單個細胞級別的活細胞提取
2016年7月14日發(fā)表于Cell雜志。
活細胞提取及應(yīng)用——單個細胞級別的活細胞提取
由于細胞異質(zhì)性的存在，單細胞層面的分析就變得十分重要。目前對于單細胞分析的方法主要還是通過化學、生物學的方法進行裂解后，提取內(nèi)容物進行分析，然而這種方法往往會對樣本造成一些損傷。直接提取活細胞具有諸多優(yōu)點，但是操作苦難。如今一種全新使用FluidFM科技的技術(shù)新報道有望提供一種活細胞提取新型的簡易方法。
1.為什么要進行活體單細胞提取
隨著技術(shù)的發(fā)展，對于細胞的研究開始向單細胞領(lǐng)域的分析靠攏。隨著細胞異質(zhì)性的發(fā)現(xiàn)以來，人們開始更多地認識到即使基因型完全相同，但由于基因表達的不同，也會導致細胞的表型出現(xiàn)差異，從而使得每個細胞的功能、組成等各方面也不完全相同。而這種差異是在生物界廣泛存在的，即使同源細胞群中也是存在的。因此對于單細胞層面的分子分析，成為了揭示病理特征，細胞應(yīng)激等方面的重要手段。
在目前的單細胞分析手段中也面臨著諸多挑戰(zhàn)。目前主要的方法是使用流式細胞儀或者微流控芯片對細胞進行分選并隔離單細胞，然后通過裂解的方式對細胞進行分析。這種高通量分選的方式雖然快速，但是本身需要將細胞從原始的培養(yǎng)環(huán)境中取出，這樣始終會導致細胞內(nèi)部一些生物信息的丟失或損傷。而且由于細胞破壞方式使用的是化學或者生物手段裂解，所使用的裂解液也會對細胞之中的一些組分產(chǎn)生破壞。因此單細胞分析始終面臨著重大的挑戰(zhàn)。
因此，學者開始嘗試從活細胞中直接提取其成分。目前已經(jīng)有諸多不同的活細胞提取技術(shù)得到了建立，并且通過這類的方法所提取分析內(nèi)容的方法所獲得的結(jié)果往往要好于裂解的方式。因此活細胞提取技術(shù)是一種更加無損的獲得細胞內(nèi)組分的方式。
2.使用FluidFM設(shè)備提取細胞并不會影響細胞活力
在本文中，作者對首先建立了一種使用FluidFM提取細胞的方法。他們嘗試了各種提取量以確定使用FluidFM能夠?qū)毎崛〉淖畲罅俊Ｔ趯嶒炛兴麄儼l(fā)現(xiàn)使用FluidFM抽取4 pL的細胞質(zhì)并觀測，發(fā)現(xiàn)82%的細胞依然存活，如圖1B所示。但是如果將抽取量加大到4.5 pL則無細胞能夠存活。而對細胞核而言抽取0.6 pL后，有86%的細胞依舊存活，如圖1A。而在0.7 pL或以上的體積提取則不可避免的導致細胞死亡，如圖1B所示。隨后他們對提取后細胞的存活狀況進行了考察。而后他們繼續(xù)對提取過的細胞進行觀察，結(jié)果發(fā)現(xiàn)這些細胞的功能并沒有受到影響。細胞能夠在提取后約30小時后正常分裂，如圖1E所示。因此作者認為使用FluidFM提取的最大量為細胞核0.6 pL和細胞質(zhì)4pL。
圖1. 細胞存活率檢測 A)原生細胞的體積分布圖，虛線為平均值，點線為最大值和最小值；B)細胞提取體積與活細胞數(shù)之間的關(guān)系。C)活細胞細胞核提取后GFP的變化；D)死細胞細胞核提取后GFP的變化；E)活細胞提取后（2.9 pL）后對細胞形態(tài)的連續(xù)觀測。
3.使用FluidFM單細胞提取物的三種應(yīng)用
3.1透射電鏡負染色觀測
3.1透射電鏡負染色觀測
對于細胞亞結(jié)構(gòu)的觀察，往往對于揭示細胞病變有著重要的意義。然而細胞裂解的傳統(tǒng)手段往往會產(chǎn)生大量的碎片，因此對細胞器的觀察造成了諸多困難。在本篇報道中，作者通過使用FluidFM設(shè)備提取細胞內(nèi)容物，在低溫環(huán)境下轉(zhuǎn)移到透射電鏡的銅網(wǎng)上，然后進行負染色和揮干，之后將片子放到透射電鏡下觀察，并使用傳統(tǒng)裂解方法得到單細胞溶液同時鋪設(shè)在銅網(wǎng)上進行對比。通過觀察他們發(fā)現(xiàn)，使用FluidFM技術(shù)得到的細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)和長絲類的結(jié)構(gòu)，如圖2C所示。而相比之下細胞裂解法得到的結(jié)果卻不盡人意，如圖2D所示。
圖2. 細胞提取物負染色電鏡圖 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)的細胞提取物電鏡下的圖像；D)普通裂解法的細胞提取物電鏡下的圖像。
3.2酶活力的檢測
酶活力的檢測對于探尋細胞異質(zhì)性有著十分重要的意義。因此作者也對FluidFM提取的細胞提取物進行了對比。首先作者通過β-半乳糖苷酶實驗來測定提取蛋白的完整性。通過測定酶解底物產(chǎn)生的熒光素的熒光強度，他們成功觀察到熒光強度隨時間而增加，說明提取物中的蛋白沒有被破壞，如圖3C、D所示。隨后作者又對不同細胞進行了不同酶活力的檢驗，結(jié)果顯示無論是LacZ轉(zhuǎn)基因HeLa細胞上還是在測定HeLa細胞的Caspase3酶上均取得了成功，如圖3 E、F所示。
圖3. 酶活性分析 A)細胞提取分析的示意圖；B)將提取的3 pL細胞提取物放到預先液封的微孔中；C)酶解底物產(chǎn)生熒光素的熒光強度變化，熒光在1小時后可見。D)圖形量化熒光強度時間表；E)LacZ轉(zhuǎn)基因細胞和非轉(zhuǎn)基因細胞之間β-半乳糖苷酶活性的差異；F）Caspase3酶活力測定。
4.3單細胞級轉(zhuǎn)錄檢測
單細胞層面的基因表達通常需要反轉(zhuǎn)錄或者PCR擴增，之后使用qPCR測定。而在此之前往往需要將細胞裂解，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直接從活細胞中提取大約0.01 pg RNA，并用普通PCR管合成cDNA并進行qPCR檢測，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，進行合成和檢測如圖6B所示。他們檢測了兩種管家基因beta-actin (ACTB)beta-2-microglobulin (B2M)以及GFP mRNA的表達量。在21個樣本中有90%的樣本成功檢測到至少1中基因的表達，其中2/3的樣本可以同時檢測到三種基因的表達。而對細胞核的檢測中，也能夠檢測到至少一種基因的表達，如圖4C所示。在對比同一細胞同時提取細胞質(zhì)（1.7 pL）和細胞核（1.3 pL）中這三種基因的表達時，可以發(fā)現(xiàn)兩者基本相同，如圖4D所示。
圖4 A)單細胞提取mRNA轉(zhuǎn)錄實驗的示意圖；B)將提取物放入液滴中的方法；C) ERCC spike 為對照，測定細胞質(zhì)中GFP、B2M、ACTB的Ct值；D)對同一細胞的細胞質(zhì)與細胞核進行提取并測定Ct值。
總結(jié)
隨著生物研究越發(fā)趨于微觀化，對于分析單個細胞的需求變得越來越大。但是由于單個細胞體積小，所能夠提取出的物質(zhì)相比以前細胞群落分析來說，難度顯著提高。這不僅對檢測儀器的靈敏度有了新的更高的要求，也同時對樣本本身的質(zhì)量也提出了更高的指標。本篇中使用FluidFM提取活細胞所取得的樣本質(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胞的情況下完成提取，這使得對于對單個細胞代謝測定的追蹤成為了可能。
多功能單細胞顯微操作系統(tǒng)——瑞士 Cytosurge FluidFM BOT
多功能單細胞顯微操作系統(tǒng)--FluidFM BOT，是將原子力系統(tǒng)、微流控系統(tǒng)、細胞培養(yǎng)系統(tǒng)為一體的單細胞操作系統(tǒng)。主要功能包括單細胞注射、單細胞提取以及單細胞分離。
FluidFM BOT極大的方便了單細胞水平的研究，尤其適合應(yīng)用于精準醫(yī)療、單細胞生物學、單細胞質(zhì)譜、單細胞基因編輯、藥物研發(fā)等領(lǐng)域。
注射、提取、分選一體化的單細胞操縱解決方案
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1. Actis, P., Maalouf, M.M., Kim, H.J., Lohith, A., Vilozny, B., Seger, R.A., and Pourmand, N. (2014). Compartmental genomics in living cells revealed by single- cell nanobiopsy. ACS Nano 8, 546–553.
2. Amara, A., and Mercer, J. (2015). Viral apoptotic mimicry. Nat. Rev. Microbiol. 13, 461–469.
3. Bengtsson, M., Sta° hlberg, A., Rorsman, P., and Kubista, M. (2005). Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Res. 15, 1388–1392.
4. Bertrand, R., Solary, E., O’Connor, P., Kohn, K.W., and Pommier, Y. (1994). Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res. 211, 314–321.
5. Cai, X., Evrony, G.D., Lehmann, H.S., Elhosary, P.C., Mehta, B.K., Poduri, A., and Walsh, C.A. (2014). Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep. 8, 1280–1289.
6. Grindberg, R.V., Yee-Greenbaum, J.L., McConnell, M.J., Novotny, M., O’Shaughnessy, A.L., Lambert, G.M., Arau´ zo-Bravo, M.J., Lee, J., Fishman, M., Robbins, G.E., et al. (2013). RNA-sequencing from single nuclei. Proc. Natl. Acad. Sci. USA 110, 19802–19807.
7. Guillaume-Gentil, O., Potthoff, E., Ossola, D., Do¨ rig, P., Zambelli, T., and Vorholt, J.A. (2013). Force-controlled fluidic injection into single cell nuclei. Small 9, 1904–1907.
8. Guillaume-Gentil, O., Potthoff, E., Ossola, D., Franz, C.M., Zambelli, T., and Vorholt, J.A. (2014). Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388.
9. Hashimshony, T., Wagner, F., Sher, N., and Yanai, I. (2012). CEL-Seq: singlecell RNA-Seq by multiplexed linear amplification. Cell Rep. 2, 666–673.
10. Jiang, L., Schlesinger, F., Davis, C.A., Zhang, Y., Li, R., Salit, M., Gingeras, T.R., and Oliver, B. (2011). Synthetic spike-in standards for RNA-seq experiments. Genome Res. 21, 1543–1551.
11. Kovarik, M.L., and Allbritton, N.L. (2011). Measuring enzyme activity in single cells. Trends Biotechnol. 29, 222–230.
12. Kuipers, M.A., Stasevich, T.J., Sasaki, T., Wilson, K.A., Hazelwood, K.L., McNally, J.G., Davidson, M.W., and Gilbert, D.M. (2011). Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload. J. Cell Biol. 192, 29–41.
13. Liebherr, R.B., Hutterer, A., Mickert, M.J., Vogl, F.C., Beutner, A., Lechner, A., Hummel, H., and Gorris, H.H. (2015). Three-in-one enzyme assay based on single molecule detection in femtoliter arrays. Anal. Bioanal. Chem. 407, 7443–7452.
14. Lo, S.J., and Yao, D.J. (2015). Get to understand more from single-cells: current studies of microfluidic-based techniques for single-cell analysis. Int. J. Mol. Sci. 16, 16763–16777.
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