北京生物醫(yī)學(xué)工程

基于原子力顯微鏡力曲線陣列模式獲得兔角膜基質(zhì)細(xì)胞彈性模量的方法研究

Study on methods of obtain elastic modulus of rabbit keratocytes by force volume model of AFM

作者：陳昕妍曾正張海霞李林

單位：首都醫(yī)科大學(xué)生物醫(yī)學(xué)工程學(xué)院，臨床生物力學(xué)應(yīng)用基礎(chǔ)研究北京市重點(diǎn)實(shí)驗(yàn)室(北京 100069)

關(guān)鍵詞：細(xì)胞彈性模量; 原子力顯微鏡; 力曲線陣列; Snedden 模型; 角膜基質(zhì)細(xì)胞

分類號(hào)：R318.01

出版年·卷·期（頁(yè)碼）：2020·39·6（574-581）

摘要：

目的細(xì)胞的力學(xué)特性與自身的生物學(xué)特性及某些疾病息息相關(guān)，原子力顯微鏡（atomic force microscope, AFM）可提供多種掃描模式及測(cè)試方法，而關(guān)于細(xì)胞彈性模量的獲得方法目前尚未統(tǒng)一，本研究擬探討通過(guò)原子力顯微鏡力曲線陣列模式（force volume）獲得細(xì)胞彈性模量的方法。方法首先通過(guò)酶消化法提取正常兔眼角膜基質(zhì)細(xì)胞，進(jìn)行培養(yǎng)并通過(guò)波形蛋白（vimentin）免疫熒光染色進(jìn)行鑒定。通過(guò)原子力顯微鏡力曲線陣列測(cè)試，選定9個(gè)100×100 μm2區(qū)域，獲得每個(gè)區(qū)域均布的1 024點(diǎn)的力-壓痕深度曲線，利用Snedden模型擬合曲線獲得相應(yīng)測(cè)試點(diǎn)彈性模量。結(jié)果波形蛋白（vimentin）免疫熒光染色鑒定結(jié)果證實(shí)提取細(xì)胞免疫熒光波形蛋白鑒定陽(yáng)性，確定細(xì)胞為角膜基質(zhì)細(xì)胞。通過(guò)原子力顯微鏡測(cè)試得到每個(gè)區(qū)域約有3~5個(gè)完整細(xì)胞，擬合得到細(xì)胞的彈性模量中央與邊緣處略有差別。細(xì)胞中央處擬合得彈性模量約為53.1 kPa±6.35 kPa。結(jié)論原子力顯微鏡力曲線陣列模式可以較為簡(jiǎn)易方便地獲得眾多細(xì)胞數(shù)據(jù)，更為細(xì)致的獲得細(xì)胞整體力學(xué)特性，有效地避免了實(shí)驗(yàn)操作與壓入點(diǎn)不同帶來(lái)的誤差。

Objective The mechanical properties of cells are closely related to their biological characteristics and some diseases. AFM（atomic force microscope) provide several scan and test models, while there is no uniform method to obtain the elastic modulus of cells so far. In this study, the elastic modulus of cells was obtained by force volume model of AFM. Methods Normal rabbit keratocytes were extracted by enzyme digestion, then identified by Vimentin immunofluorescence staining. Through the force volume model of AFM, nine 100×100 μm2 regions were tested and the force-indent depth curves of 1024 points of each area were obtained. The elastic modulus was obtained through the fitting of force-indent depth curves via Snedden model. Results The immunofluorescence staining was positive, confirmed that the cell is keratocytes. There were about 3-5 complete cells in each tested region, and the elastic modulus of the cells was slightly different between the center and the edge area. The elastic modulus at the center of the cell was approximately 53.1 kPa±6.35 kPa. Conclusions The force volume model of AFM can obtain cell data in a simple and convenient way. And the overall mechanical properties of keratocytes are obtained in detail, which effectively avoids the errors caused by different experimental operations and pressing points.

參考文獻(xiàn)：

1. Li YS, Chen J, Wang LL, et al. Experimental verification of the elastic formula for the aspirated length of a single cell considering the size and compressibility of cell during micropipette aspiration[J]. Annals of Biomedical Engineering, 2018, 46(7):1026–1037 .
2. Hassell JR, Birk DE. The molecular basis of corneal transparency[J]. Experimental Eye Research, 2010, 91(3):326-335.
3. Petroll WM, Miron-Mendoza M. Mechanical interactions and crosstalk between corneal keratocytes and the extracellular matrix[J]. Experimental Eye Research, 2015, 133:49-57.
4. 張海霞, 張迪, 秦曉, 等. 角膜生物力學(xué)特性研究進(jìn)展[J]. 科技導(dǎo)報(bào),2018, 36(13):23-29.
Zhang HX, ZhanG D, Qin X, et al. Progress of research on corneal biomechanical properties[J]. Science & Technology Review, 2018, 36(13): 23-29.
5. Zhang HX, Khan MA, Zhang D, et al. Corneal biomechanical properties after FS-LASIK with residual bed thickness less than 50% of the original corneal thickness[J]. Journal of Ophthalmology, 2018: 2752945.
6.于夢(mèng)瑤，秦曉，張海霞，等，角膜膠原交聯(lián)后應(yīng)力重分布的有限元研究[J].北京生物醫(yī)學(xué)工程，2019, 38(6):598-604.
Yu MY, Qin X, Zhang HX, et al. Finite element analysis of stress redistribution after corneal collagen cross-linking[J]. Beijing Biomedical Engineering, 2019, 38(6):598-604.
7. Kieβling TR, Herrera M, Nnetu KD, et al. Analysis of multiple physical parameters for mechanical phenotyping of living cells [J]. European Biophysics Journal, 2013, 42(5):383-394.
8. Iyer S, Woodworth CD, Gaikwad RM, et al. Towards nonspecific detection of malignant cervical cells with fluorescent silica beads[J]. Small, 2009, 5(20):2277-2284.
9. 王浩. 基于納米鑷子的細(xì)胞粘彈性測(cè)試方法研究[D]. 哈爾濱：哈爾濱工業(yè)大學(xué), 2014.
 Wang H. Characterizing viscoelastic properties of the living cell with a probe nanotweezer [D]. Harbin: Harbin Institute of Technology, 2014.
10. Lim CT, Zhou EH, Li A, et al. Experimental techniques for single cell and single molecule biomechanics[J]. Materials Science and Engineering: C, 2006, 26(8):1278-1288
11. Bucchianico SD, Poma AM, Giardi MF, et al. Atomic force microscope nanolithography on chromosomes to generate single-cell genetic probes[J]. Journal of Nanobiotechnology, 2011, 9: 27.
12. Guilak F, Tedrow JR, Burgkart R. Viscoelastic properties of the cell nucleus[J]. Biochemical and Biophysical Research Communications, 2000, 269(3): 781-786.
13.Hochmuth RM. Micropipette aspiration of living cells[J]. Journal of Biomechanics, 2000, 33(1):15-22.
14.Dao M, Lim CT, Suresh S. Mechanics of the human red blood cell deformed by optical tweezers[J]. Journal of the Mechanics and Physics of Solids, 2003, 51(11-12): 2259-2280.
15.宋建民. 基于AFM的細(xì)胞核力學(xué)特性原位測(cè)試的研究[D]. 哈爾濱：哈爾濱工業(yè)大學(xué), 2015.
 Song JM. In situ quantification of living cell nucleus mechanical propertity with atomic force microscope[D]. Harbin: Harbin Institute of Technology, 2015.
16. Binnig G, Garcia N, Rohrer H. Conductivity sensitivity of inelastic scanning tunneling microscopy[J]. Physical Review B Condensed Matter, 1985, 32(2):1336-1338.
17. Puntheeranurak T, Wildling L, Gruber HJ, et al. Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells[J]. Journal of Cell Science, 2006, 119(14):2960-2967.
18. Yu J, Wang Q, Shi X, et al. Single-molecule force spectroscopy study of interaction between transforming growth factor β1 and its receptor in living cells[J]. The Journal of Physical Chemistry B, 2007, 111(48):13619-13625.
19. Chaudhuri O, Parekh SH, Lam WA, et al. Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells[J]. Nature Methods, 2009, 6(5):383-392.
20. Liang X, Shi X, Ostrovidov S, et al. Probing stem cell differentiation using atomic force microscopy[J]. Applied Surface Science, 2016, 366: 254-259.
21. Lekka G. Discrimination between normal and cancerous cells using AFM[J]. Bionanoscience, 2016, 6(1):65-80.
22. Reich A, Meurer M, Eckes B, et al. Surface morphology and mechanical properties of fibroblasts from scleroderma patients[J]. Journal of Cellular and Molecular Medicine, 2009, 13(8B):1644-1652.
23.于洋, 李林, 李?yuàn)檴? 等. 大鼠小梁細(xì)胞的體外培養(yǎng)及彈性模量的測(cè)量[J]. 北京生物醫(yī)學(xué)工程,2019,38(2):145-150.
Yu Y, Li L, Li SS, et al. Culture in vitro and stiffness measurement of rat trabecular meshwork cells[J]. Beijing Biomedical Engineering, 2019, 38(2):145-150.
24.Wang C, Li L, Liu Z. Experimental research on the relationship between the stiffness and the expressions of fibronectin proteins and adaptor proteins of rat trabecular meshwork cells[J]. BMC Ophthalmology, 2017, 17: 268.
25. Liu H, Wen J, Xiao Y, et al. In situ mechanical characterization of the cell nucleus by atomic force microscopy[J].ACS Nano, 2014, 8(4): 3821-3828.
26. Eghiaian F, Rigato A, Scheuring S. Structural, mechanical, and dynamical variability of the actin cortex in living cells[J]. Biophysical Journal, 2015, 108: 1330-1340.
27.李杰, 李霞, 譚少健, 等. 牛角膜基質(zhì)細(xì)胞的兩步酶消化法高效分離及體外培養(yǎng)觀察[J]. 中華實(shí)驗(yàn)眼科雜志, 2011, 29(5): 398-401.
Li J, Li X, Tan SJ, et al. Efficient isolation of bovine keratocytes utilizing two step enzymatic digestion[J].Chinese Journal of Experimental Ophthalmology, 2011, 29(5): 398-401.
28.張璐, 李妍, 胡竹林. 角膜基質(zhì)細(xì)胞的表型轉(zhuǎn)化及其預(yù)防瘢痕形成的研究[J]. 國(guó)際眼科縱覽, 2018, 42(3): 194-198.
Zhang L，Li Y, Hu ZL. Henotypic transformation of corneal stromal cells and prevention of scar formation[J]. International Review of Ophthalmology, 2018, 42(3): 194-198.
29.劉玉喬, 王亞宇, 史亮. 生理狀態(tài)下人牙周膜成纖維細(xì)胞的原子力顯微鏡下的觀察[J]. 牙體牙髓牙周病學(xué)雜志, 2018, 28(9):511-514, 524.
Liu YQ, Wang YY, Shi L. Observation of the morphology of living human periodontal ligament cells with atomic force microscope[J]. Chinese Journal of Conservative Dentistry, 2018, 28(9): 511-514, 524.
30.Radmacher M. Measuring the elastic properties of living cells by the atomic force microscope[J]. Methods in Cell Biology, 2002, 68: 67-90.
31.Dokukin ME, Sokolov I. On the measurements of rigidity modulus of soft materials in nanoindentation experiments at small depth[J]. Macromolecules, 2012, 45(10): 4277-4288.
32.Guo Q, Xia Y, Sandig M, et al. Characterization of cell elasticity correlated with cell morphology by atomic force microscope[J]. Journal of Biomechanics, 2012, 45(2): 304-309
33.嚴(yán)拓, 孫蓉, 鄧華, 等. 角膜基質(zhì)細(xì)胞在p(HEMA-MMA)改性前后的粘附率、形貌與力學(xué)性能變化[J]. 分析測(cè)試學(xué)報(bào), 2010, 29(3): 232-236.
Yan T, Sun R, Deng H, et al. Attachment, morphology and biomechanical changes of keratocytes cultured on unmodified and modified p(HEMA-MMA) hydrogel[J]. Journal of Instrumental Analysis, 2010, 29(3): 232-236.
34.Raghunathan VK, Thomasy SM, Strфm MP, et al. Tissue and cellular biomechanics during corneal wound injury and repair[J]. Acta Biomaterialia, 2017, 58: 291-301.
35.栗亞. 基于納米探針技術(shù)的NSCLC細(xì)胞表面形貌和力學(xué)特性研究[D]. 哈爾濱: 哈爾濱工業(yè)大學(xué), 2013.
Li Y. Investigation of morphology and mechanical properties of NSCLC cells based on nano-indentation [D]. Harbin: Harbin Institute of Technology, 2013.
36.李淑萍. 基于AFM的細(xì)胞力學(xué)特性實(shí)驗(yàn)研究[D]. 哈爾濱: 哈爾濱工業(yè)大學(xué), 2014.
Li SP. The research on mechanics properties of cells based on AFM[D]. Harbin: Harbin Institute of Technology, 2014.
37. Ursell TS, Nguyen J, Monds RD, et al. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization[J]. Proceeding of the National Academy of Sciences of the United States of America, 2014, 111(11): E1025–E1034.

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