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單個細胞別的粘附力測定

更新時間:2018-05-15點擊次數:1101

單個細胞別的粘附力測定

 

      單細粘附力的測定直以來都缺乏種能夠在不改變細胞性質的同時測量細胞整體粘附力的設備。現如今FluidFM 技術的出現改變了這狀況。高精密的流體力探針能夠在感知壓力的同時通過內壓而非蛋白結合的方式在不改變細胞性質的同時牢固的抓取細胞,為單細胞粘附力測定提供新的可能。

 

      當今,機械生物學是個新興、迅速發展的研究域,并著重研究細胞力學在細胞功能乃至整個生物體水平上的作用,從而揭示細胞受力對組織、器官發育、生理學以及疾病的起因和進展中所發揮的作用。其中在細胞層面上的研究主要集中在研究細胞之間的粘附力和細胞與基質之間的相互作用。其中細胞與基質的作用往往需要通過細胞表面的整合素受體介導來完成,也是當今的研究重點。目前在細胞-基質相互作用力的研究中已經有諸多方法被建立,其中有諸多測量方法是基于AFM(原子力顯微鏡)的,因為AFM 能夠定量測量細胞-基質之間的粘附力,這也使得AFM 成為了測量細胞間作用力的常用設備。

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

      而如今Cytosurge 推出的全新的FluidFM 技術給粘附力測量帶來了新的希望。這種技術結合了的原子力顯微鏡探測技術與微流體控制系統。該技術能夠直接通過使用中空的原子力探針將細胞通過負壓粘附在探針表面,并不需要激活細胞的任何通路信號。這樣為粘附力的測量帶來了大的勢。方面,這種方法能夠提供遠比蛋白結合牢固多的粘附力,能夠將細胞牢固的固定在探針上面,因此能夠用于直接從基質上分離。而另方面,由于沒有生物處理,這種方法不會改變任何細胞表面的通路,從而能夠得到接近細胞原生的數據。本文就如何使用FluidFM 測定細胞粘附力和近期應用案例進行總結。

 

FluidFM 技術如何測定細胞粘附力?
      為了闡述這個問題,本文引用Scientific Reports 在2017 年發表的文獻中的方法進行闡述。*,細胞在基質上進行單層培養時,吸附在基質表面時主要會產生兩種不同類型的力,種是細胞與基質之間的粘附力,另種是細胞與細胞之間的粘附力。因此對于細胞粘附力來說,單個細胞的粘附力就是細胞與基質之間的作用力。而單層細胞的細胞粘附力則是細胞之間相互作用力和細胞基質與細胞之間作用力之和。如下圖所示:

       因此只要同時測定單個細胞粘附力即可得到細胞與基質之間的相互作用力,而細胞間的相互作用力則可以通過同時測量單層細胞的細胞粘附力和單個細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算方法,為了能夠測量粘附力Sancho 等使用FluidFM 技術,通過將探針靠近細胞直到探針與細胞接觸,之后開始對探針腔內增加負壓從而牢固的吸住細胞。當細胞固定后收回探針并記錄這之間的力學變化,如下圖所示:

 

      從圖中顯示出當探針開始靠近細胞后,探針表面開始出現壓力變化,如上圖中的藍色區域所示。當出現這種變化后就停止下降探針并開始施加負壓。這時候由于腔內負壓,探針和細胞之間的結合變得緊密,導致探針被細胞向下拉動,從而產生了上邊右圖白色區域的力學變化。隨后隨著探針上升,細胞給以探針的拉力隨之增高,并逐漸達到臨界,使得細胞脫離基質。這過程的大值即為細胞粘附力。

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

      結果也證實了粘附力測定公式。具有細胞間作用的HUAEC 在單個細胞和單層細胞之間的粘附力存在差異,而無細胞間相互作用的L929 細胞則沒有差異。因此FluidFM 技術能夠很好地幫助研究者研究單細胞粘附力的性質。

 

FluidFM 測定細胞粘附力的應用
      隨著時間推移,越來越多的學者開始使用FluidFM 技術進行測定細胞粘附力。以下就近五年的具有代表性的應用進行總結。
      Cohen 等使用FluidFM 技術對MCF7-MCF10A、MCF7-HS5 的細胞粘附力進行了測定,并與以往的文獻進行對比,發現其數據與Hossein 等測定的結果相符。如下圖所示:

 

      使用FluidFM 技術對MCF7-MCF10A、MCF7-HS5 細胞粘附力進行測定 a. 使用FluidFM 測定細胞粘附力全過程;b. MCF7-HS5 的細胞粘附力測試結果;c. MCF7-MCF10A 的細胞粘附力測試結果。

 

      Jaatinen 等通過使用FluidFM 技術研究外加電流對C2C12 小鼠成肌細胞粘附力的影響中發現隨著外周電流的增加,細胞形態發生改變,與基質接觸面積降低。當電流劑量高過11As/m2后細胞形態急劇改變,粘附力等參數發生明顯變化,甚至死亡。如下圖所示:

 

      FluidFM 測定C2C12 細胞粘附力 a.使用FluidFM 測定粘附力顯微鏡圖;b.施加12.3As/m2電流和空白對照組的粘附力譜線;c.粘附力與電流之間的量效關系圖。

 

      Sankaran 等使用FluidFM 來研究共價和非共價的表面整合素受體對細胞粘附力的影響。通過測定發現兩者均可有效增加細胞的粘附能力,并且效果近似。

      使用FluidFM 技術測定共價鍵與非共價鍵之間的整合素受體RGD 之間的區別 a. FluidFM 測定粘附力的示意圖; b. 細胞粘附力測定前后顯微鏡示意圖; c.測定粘附力時候的力學曲線圖;d. 大粘附力圖。

 

      Sancho 等通過FluidFM 技術使用了種非常有趣的測量方法來測量MSX1 過表達對細胞骨架的影響,他們將10μm 的小膠球固定在探針上,之后使用探針去壓細胞直到探針壓力達到2 nN,通過壓痕曲線來分析細胞骨架變化。通過對比發現過量表達MSX1 細胞的硬度顯著比普通細胞高。如下圖所示:

      使用FluidFM 技術測定HUAEC 中MSX1 過表達對細胞骨架的影響。a. HUAEC 細胞的免疫熒光染色phalloidin(上)、vimentin(下)(綠色)Hoechst(藍色);b. HUAEC 細胞的免疫熒光染色phalloidin(紅色)、vinculin(綠色)TOPRO-3(藍色);c. 每個克隆中vinculin 陽性面積;d. 使用FluidFM 技術壓細胞的示意圖;e. 吸取10μm 珠子;f. 空白細胞下壓時的力學譜線;g. MSX 過表達細胞下壓時的力學譜線,更深的凹陷和平滑的斜率表示較低的剛度; h.用膠體壓痕法測定細胞剛度的測量結果。

 

總結
      細胞粘附力測定在細胞生命科學研究中起著至關重要的作用,然而傳統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胞并進行力學測定的手段。現如今FluidFM 技術在細胞粘附力測定中的使用,使得研究者們有了種能夠有效、低損的方式抓取細胞,并配合著原子力顯微鏡的測量的性,從而能夠真正意義上的做到、無損、快速的測量單細胞粘附力,幫助研究者尋找細胞粘附力與細胞生命發展、腫瘤細胞轉移之間的關系。

 

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