活細(xì)胞提取及應(yīng)用——單個(gè)細(xì)胞級別的活細(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對樣本造成一些損傷。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)胞提取新型的簡易方法。

 

   在本文中，作者對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，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 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）后對細(xì)胞形態(tài)的連續(xù)觀測。

 

   對于細(xì)胞亞結(jié)構(gòu)的觀察，往往對于揭示細(xì)胞病變有著重要的意義。然而細(xì)胞裂解的傳統(tǒng)手段往往會產(chǎn)生大量的碎片，因此對細(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。

 

   酶活力的檢測對于探尋細(xì)胞異質(zhì)性有著十分重要的意義。因此作者也對FluidFM提取的細(xì)胞提取物進(jìn)行了對比。首先作者通過β-半乳糖苷酶實(shí)驗(yàn)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(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，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(dá)量。在21個(gè)樣本中有90%的樣本成功檢測到至少1中基因的表達(dá)，其中2/3的樣本可以同時(shí)檢測到三種基因的表達(dá)。而對細(xì)胞核的檢測中，也能夠檢測到至少一種基因的表達(dá)，如圖4C所示。在對比同一細(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 為對照，測定細(xì)胞質(zhì)中GFP、B2M、ACTB的Ct值；D)對同一細(xì)胞的細(xì)胞質(zhì)與細(xì)胞核進(jìn)行提取并測定Ct值。

 

   隨著生物研究越發(fā)趨于微觀化，對于分析單個(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。這不僅對檢測儀器的靈敏度有了新的更高的要求，也同時(shí)對樣本本身的質(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，這使得對于對單個(gè)細(xì)胞代謝測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。

 

 

參考文獻(xiàn)：
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.
15. Meister, A., Gabi, M., Behr, P., Studer, P., Vo¨ ro¨ s, J., Niedermann, P., Bitterli, J., Polesel-Maris, J., Liley, M., Heinzelmann, H., and Zambelli, T. (2009). FluidFM: combining atomic force microscopy and nanofluidics in a universal liquid delivery system for single cell applications and beyond. Nano Lett. 9, 2501–2507.
16. Nagaraj, N., Wisniewski, J.R., Geiger, T., Cox, J., Kircher, M., Kelso, J., Pa¨ a¨ bo, S., and Mann, M. (2011). Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548.
17. Nawarathna, D., Turan, T., and Wickramasinghe, H.K. (2009). Selective probing of mRNA expression levels within a living cell. Appl. Phys. Lett. 95, 83117. O’Huallachain, M., Karczewski, K.J., Weissman, S.M., Urban, A.E., and Snyder, M.P. (2012). Extensive genetic variation in somatic human tissues. Proc. Natl. Acad. Sci. USA 109, 18018–18023.
18. Osada, T., Uehara, H., Kim, H., and Ikai, A. (2003). mRNA analysis of single living cells. J. Nanobiotechnology 1, 2.
19. Pfeiffer-Guglielmi, B., Dombert, B., Jablonka, S., Hausherr, V., van Thriel, C., Scho¨ bel, N., and Jansen, R.P. (2014). Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons. BMC Neurosci. 15, 70.
20. Picelli, S., Faridani, O.R., Bjo¨ rklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smartseq2. Nat. Protoc. 9, 171–181.
21. Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y., and Tyagi, S. (2006). Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309.
22. Ramsko¨ ld, D., Luo, S., Wang, Y.C., Li, R., Deng, Q., Faridani, O.R., Daniels, G.A., Khrebtukova, I., Loring, J.F., Laurent, L.C., et al. (2012). Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782.
23. Rissin, D.M., Kan, C.W., Campbell, T.G., Howes, S.C., Fournier, D.R., Song, L., Piech, T., Patel, P.P., Chang, L., Rivnak, A.J., et al. (2010). Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 28, 595–599.
24. Rondelez, Y., Tresset, G., Tabata, K.V., Arata, H., Fujita, H., Takeuchi, S., and Noji, H. (2005). Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nat. Biotechnol. 23, 361–365.
25. Saha-Shah, A., Weber, A.E., Karty, J.A., Ray, S.J., Hieftje, G.M., and Baker, L.A. (2015). Nanopipettes: probes for local sample analysis. Chem. Sci. 6, 3334–3341.
26. Sarkar, A., Kolitz, S., Lauffenburger, D.A., and Han, J. (2014). Microfluidic probe for single-cell analysis in adherent tissue culture. Nat. Commun. 5, 3421.
27. Schmid, A., Kortmann, H., Dittrich, P.S., and Blank, L.M. (2010). Chemical and biological single cell analysis. Curr. Opin. Biotechnol. 21, 12–20.
28. Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V.I., Lao, K., and Surani, M.A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat. Protoc. 5, 516–535.
29. Taniguchi, K., Kajiyama, T., and Kambara, H. (2009). Quantitative analysis of gene expression in a single cell by qPCR. Nat. Methods 6, 503–506.
30. Van Gelder, R.N., von Zastrow, M.E., Yool, A., Dement, W.C., Barchas, J.D., and Eberwine, J.H. (1990). Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663–1667.
31. Veyer, D.L., Maluquer de Motes, C., Sumner, R.P., Ludwig, L., Johnson, B.F., and Smith, G.L. (2014). Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection. J. Gen. Virol. 95, 2757–2768.
32. Wachsmuth, M., Weidemann, T., Mu¨ ller, G., Hoffmann-Rohrer, U.W., Knoch, T.A., Waldeck, W., and Langowski, J. (2003). Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys. J. 84, 3353–3363.
33. Wang, D., and Bodovitz, S. (2010). Single cell analysis: the new frontier in ‘omics’. Trends Biotechnol. 28, 281–290.
34. Wasilenko, S.T., Stewart, T.L., Meyers, A.F., and Barry, M. (2003). Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis. Proc. Natl. Acad. Sci. USA 100, 14345–14350.
35. Weis, K. (2003). Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441–451.
36. Wu, M., and Singh, A.K. (2012). Single-cell protein analysis. Curr. Opin. Biotechnol.23, 83–88.
37. Zhao, L., Kroenke, C.D., Song, J., Piwnica-Worms, D., Ackerman, J.J., and Neil, J.J. (2008). Intracellular water-specific MR of microbead-adherent cells: the HeLa cell intracellular water exchange lifetime. NMR Biomed. 21, 159–164.