單個(gè)細(xì)胞別的粘附力測(cè)定

      單細(xì)粘附力的測(cè)定直以來都缺乏種能夠在不改變細(xì)胞性質(zhì)的同時(shí)測(cè)量細(xì)胞整體粘附力的設(shè)備?，F(xiàn)如今FluidFM 技術(shù)的出現(xiàn)改變了這狀況。高精密的流體力探針能夠在感知壓力的同時(shí)通過內(nèi)壓而非蛋白結(jié)合的方式在不改變細(xì)胞性質(zhì)的同時(shí)牢固的抓取細(xì)胞，為單細(xì)胞粘附力測(cè)定提供新的可能。

      當(dāng)今，機(jī)械生物學(xué)是個(gè)新興、迅速發(fā)展的研究域，并著重研究細(xì)胞力學(xué)在細(xì)胞功能乃至整個(gè)生物體水平上的作用，從而揭示細(xì)胞受力對(duì)組織、器官發(fā)育、生理學(xué)以及疾病的起因和進(jìn)展中所發(fā)揮的作用。其中在細(xì)胞層面上的研究主要集中在研究細(xì)胞之間的粘附力和細(xì)胞與基質(zhì)之間的相互作用。其中細(xì)胞與基質(zhì)的作用往往需要通過細(xì)胞表面的整合素受體介導(dǎo)來完成，也是當(dāng)今的研究重點(diǎn)。目前在細(xì)胞-基質(zhì)相互作用力的研究中已經(jīng)有諸多方法被建立，其中有諸多測(cè)量方法是基于AFM（原子力顯微鏡）的，因?yàn)锳FM 能夠定量測(cè)量細(xì)胞-基質(zhì)之間的粘附力，這也使得AFM 成為了測(cè)量細(xì)胞間作用力的常用設(shè)備。

      該類方法主要是用基質(zhì)蛋白包被探針并與細(xì)胞靠近發(fā)生作用并固定在懸臂上，之后通過將細(xì)胞與基質(zhì)進(jìn)行接觸從而通過測(cè)量作用過程中懸臂的彎曲程度來實(shí)現(xiàn)力學(xué)測(cè)量。然而這種方法也有其局限性。為了讓細(xì)胞能夠與懸臂進(jìn)行粘連，就必須使用凝集素、鏈霉親和素或細(xì)胞外基質(zhì)蛋白進(jìn)行預(yù)處理。然而這將無可避免的改變細(xì)胞表面細(xì)胞的功能狀態(tài)和表面整合素分布。另外這種方法僅能讓細(xì)胞與基質(zhì)接觸短的時(shí)間，這導(dǎo)致了這種方法只能應(yīng)用于早期粘附力的測(cè)量。此外受制于材料學(xué)的限制，適合于固定細(xì)胞在探針上并不影響細(xì)胞的強(qiáng)力材料仍有待開發(fā)。因此使用AFM 測(cè)量細(xì)胞粘附力的方法仍然需要改進(jìn)與完善。

      而如今Cytosurge 推出的全新的FluidFM 技術(shù)給粘附力測(cè)量帶來了新的希望。這種技術(shù)結(jié)合了的原子力顯微鏡探測(cè)技術(shù)與微流體控制系統(tǒng)。該技術(shù)能夠直接通過使用中空的原子力探針將細(xì)胞通過負(fù)壓粘附在探針表面，并不需要激活細(xì)胞的任何通路信號(hào)。這樣為粘附力的測(cè)量帶來了大的勢(shì)。方面，這種方法能夠提供遠(yuǎn)比蛋白結(jié)合牢固多的粘附力，能夠?qū)⒓?xì)胞牢固的固定在探針上面，因此能夠用于直接從基質(zhì)上分離。而另方面，由于沒有生物處理，這種方法不會(huì)改變?nèi)魏渭?xì)胞表面的通路，從而能夠得到接近細(xì)胞原生的數(shù)據(jù)。本文就如何使用FluidFM 測(cè)定細(xì)胞粘附力和近期應(yīng)用案例進(jìn)行總結(jié)。

FluidFM 技術(shù)如何測(cè)定細(xì)胞粘附力？
      為了闡述這個(gè)問題，本文引用Scientific Reports 在2017 年發(fā)表的文獻(xiàn)中的方法進(jìn)行闡述。*，細(xì)胞在基質(zhì)上進(jìn)行單層培養(yǎng)時(shí)，吸附在基質(zhì)表面時(shí)主要會(huì)產(chǎn)生兩種不同類型的力，種是細(xì)胞與基質(zhì)之間的粘附力，另種是細(xì)胞與細(xì)胞之間的粘附力。因此對(duì)于細(xì)胞粘附力來說，單個(gè)細(xì)胞的粘附力就是細(xì)胞與基質(zhì)之間的作用力。而單層細(xì)胞的細(xì)胞粘附力則是細(xì)胞之間相互作用力和細(xì)胞基質(zhì)與細(xì)胞之間作用力之和。如下圖所示：

       因此只要同時(shí)測(cè)定單個(gè)細(xì)胞粘附力即可得到細(xì)胞與基質(zhì)之間的相互作用力，而細(xì)胞間的相互作用力則可以通過同時(shí)測(cè)量單層細(xì)胞的細(xì)胞粘附力和單個(gè)細(xì)胞的粘附力做差即可得到，如下公式所示：

Force _cell-cell ≌ Force _Monolayer – Force _Indiv.cell

      以上即為粘附力的計(jì)算方法，為了能夠測(cè)量粘附力Sancho 等使用FluidFM 技術(shù)，通過將探針靠近細(xì)胞直到探針與細(xì)胞接觸，之后開始對(duì)探針腔內(nèi)增加負(fù)壓從而牢固的吸住細(xì)胞。當(dāng)細(xì)胞固定后收回探針并記錄這之間的力學(xué)變化，如下圖所示：

      從圖中顯示出當(dāng)探針開始靠近細(xì)胞后，探針表面開始出現(xiàn)壓力變化，如上圖中的藍(lán)色區(qū)域所示。當(dāng)出現(xiàn)這種變化后就停止下降探針并開始施加負(fù)壓。這時(shí)候由于腔內(nèi)負(fù)壓，探針和細(xì)胞之間的結(jié)合變得緊密，導(dǎo)致探針被細(xì)胞向下拉動(dòng)，從而產(chǎn)生了上邊右圖白色區(qū)域的力學(xué)變化。隨后隨著探針上升，細(xì)胞給以探針的拉力隨之增高，并逐漸達(dá)到臨界，使得細(xì)胞脫離基質(zhì)。這過程的大值即為細(xì)胞粘附力。

      之后作者考察了兩種性質(zhì)截然不同的細(xì)胞的粘附力。種是L929 無細(xì)胞間作用的細(xì)胞，另種是HUAEC 具有細(xì)胞間的相互作用的細(xì)胞，結(jié)果如下圖所示：

      結(jié)果也證實(shí)了粘附力測(cè)定公式。具有細(xì)胞間作用的HUAEC 在單個(gè)細(xì)胞和單層細(xì)胞之間的粘附力存在差異，而無細(xì)胞間相互作用的L929 細(xì)胞則沒有差異。因此FluidFM 技術(shù)能夠很好地幫助研究者研究單細(xì)胞粘附力的性質(zhì)。

FluidFM 測(cè)定細(xì)胞粘附力的應(yīng)用
      隨著時(shí)間推移，越來越多的學(xué)者開始使用FluidFM 技術(shù)進(jìn)行測(cè)定細(xì)胞粘附力。以下就近五年的具有代表性的應(yīng)用進(jìn)行總結(jié)。
      Cohen 等使用FluidFM 技術(shù)對(duì)MCF7-MCF10A、MCF7-HS5 的細(xì)胞粘附力進(jìn)行了測(cè)定，并與以往的文獻(xiàn)進(jìn)行對(duì)比，發(fā)現(xiàn)其數(shù)據(jù)與Hossein 等測(cè)定的結(jié)果相符。如下圖所示：

      使用FluidFM 技術(shù)對(duì)MCF7-MCF10A、MCF7-HS5 細(xì)胞粘附力進(jìn)行測(cè)定 a. 使用FluidFM 測(cè)定細(xì)胞粘附力全過程；b. MCF7-HS5 的細(xì)胞粘附力測(cè)試結(jié)果；c. MCF7-MCF10A 的細(xì)胞粘附力測(cè)試結(jié)果。

      Jaatinen 等通過使用FluidFM 技術(shù)研究外加電流對(duì)C2C12 小鼠成肌細(xì)胞粘附力的影響中發(fā)現(xiàn)隨著外周電流的增加，細(xì)胞形態(tài)發(fā)生改變，與基質(zhì)接觸面積降低。當(dāng)電流劑量高過11As/m²后細(xì)胞形態(tài)急劇改變，粘附力等參數(shù)發(fā)生明顯變化，甚至死亡。如下圖所示：

      FluidFM 測(cè)定C2C12 細(xì)胞粘附力 a.使用FluidFM 測(cè)定粘附力顯微鏡圖；b.施加12.3As/m²電流和空白對(duì)照組的粘附力譜線；c.粘附力與電流之間的量效關(guān)系圖。

      Sankaran 等使用FluidFM 來研究共價(jià)和非共價(jià)的表面整合素受體對(duì)細(xì)胞粘附力的影響。通過測(cè)定發(fā)現(xiàn)兩者均可有效增加細(xì)胞的粘附能力，并且效果近似。

      使用FluidFM 技術(shù)測(cè)定共價(jià)鍵與非共價(jià)鍵之間的整合素受體RGD 之間的區(qū)別 a. FluidFM 測(cè)定粘附力的示意圖； b. 細(xì)胞粘附力測(cè)定前后顯微鏡示意圖； c.測(cè)定粘附力時(shí)候的力學(xué)曲線圖；d. 大粘附力圖。

      Sancho 等通過FluidFM 技術(shù)使用了種非常有趣的測(cè)量方法來測(cè)量MSX1 過表達(dá)對(duì)細(xì)胞骨架的影響，他們將10μm 的小膠球固定在探針上，之后使用探針去壓細(xì)胞直到探針壓力達(dá)到2 nN，通過壓痕曲線來分析細(xì)胞骨架變化。通過對(duì)比發(fā)現(xiàn)過量表達(dá)MSX1 細(xì)胞的硬度顯著比普通細(xì)胞高。如下圖所示：

      使用FluidFM 技術(shù)測(cè)定HUAEC 中MSX1 過表達(dá)對(duì)細(xì)胞骨架的影響。a. HUAEC 細(xì)胞的免疫熒光染色phalloidin（上）、vimentin（下）（綠色）Hoechst（藍(lán)色）；b. HUAEC 細(xì)胞的免疫熒光染色phalloidin（紅色）、vinculin（綠色）TOPRO-3（藍(lán)色）；c. 每個(gè)克隆中vinculin 陽性面積；d. 使用FluidFM 技術(shù)壓細(xì)胞的示意圖；e. 吸取10μm 珠子；f. 空白細(xì)胞下壓時(shí)的力學(xué)譜線；g. MSX 過表達(dá)細(xì)胞下壓時(shí)的力學(xué)譜線，更深的凹陷和平滑的斜率表示較低的剛度； h.用膠體壓痕法測(cè)定細(xì)胞剛度的測(cè)量結(jié)果。

總結(jié)
      細(xì)胞粘附力測(cè)定在細(xì)胞生命科學(xué)研究中起著至關(guān)重要的作用，然而傳統(tǒng)手段中有著各種各樣的局限性，這主要原因是缺乏種有效能夠抓取細(xì)胞并進(jìn)行力學(xué)測(cè)定的手段?，F(xiàn)如今FluidFM 技術(shù)在細(xì)胞粘附力測(cè)定中的使用，使得研究者們有了種能夠有效、低損的方式抓取細(xì)胞，并配合著原子力顯微鏡的測(cè)量的性，從而能夠真正意義上的做到、無損、快速的測(cè)量單細(xì)胞粘附力，幫助研究者尋找細(xì)胞粘附力與細(xì)胞生命發(fā)展、腫瘤細(xì)胞轉(zhuǎn)移之間的關(guān)系。

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