杨氏模量及细胞骨架重塑对肝癌细胞侵袭的影响

Effect of Young＇ s modulus and cytoskeleton remodeling on invasion of hepatocarcinoma cell

目的利用原子力显微镜（AFM）研究肝癌细胞侵袭能力与细胞骨架力学重塑的关系，探明肝癌发生、发展及侵袭过程中的力学特性。方法以4种不同侵袭能力肝癌细胞系（MHCC-97H细胞、MHCC-97L细胞、SMMC-7721细胞、Huh-7细胞）为研究对象，通过AFM测定细胞力学参数（杨氏模量），采用免疫荧光染色研究不同侵袭能力肝癌细胞的细胞骨架重塑特性，并利用细胞划痕实验检测细胞迁移能力的差异对力学数据进行验证。采用Kruskal—WallisH检验比较组间差异。结果AFM结果显示，按细胞侵袭力由低至高，Huh-7细胞胞质区杨氏模量为1602．43（845．48，3317．25）Pa，SMMC-7721细胞为1055．28（367．48，2280．77）Pa，MHCC-97L细胞为1026．78（369．20，2019．96）Pa，MHCC-97H细胞为503．12（366．11，700．31）Pa；Huh-7细胞胞核区杨氏模量为2823．98（1262．78，4440．07）Pa，SMMC-7721细胞为1313．43（590．71，2678．62）Pa，MHCC-97L细胞为1285．17（583．29，1961．19）Pa，MHCC-97H细胞为655．57（441．29，943．39）Pa；随着细胞侵袭能力增强，胞质区和胞核区杨氏模量均逐渐降低（χ^2=472．78、622．43，P均〈0．01）。免疫荧光结果显示，侵袭能力越强，细胞骨架完整性越差，细胞微丝分布越紊乱。划痕实验中MHCC-97H细胞24h迁移率为46．67％，48h迁移率为86．47％；MHCC-97L细胞24h迁移率为45．70％，48h迁移率为82．86％；SMMC-7721细胞24h迁移率为39．41％，48h迁移率为79．85％；Huh-7细胞24h迁移率为34．60％，48h迁移率为72．09％，呈现出随细胞侵袭能力增强而迁移率逐渐增加的趋势。结论侵袭能力越强的肝癌细胞其杨氏模量越低，细胞越软，形变能力越强，细胞骨架完整性越差，结构越趋于紊乱，反之亦然。

Objective To investigate the correlation between invasion ability and cytoskeleton remodeling of hepatocarcinoma cell by atomic force microscopy （AFM） and to explore mechanical properties during genesis, development and invasion of hepatocellular carcinoma （HCC）. Methods Four HCC cell lines （MHCC-97H, MHCC-97L, SMMC-7721, Huh-7） with different invasive ability were studied. Mechanical parameter （Young＇s modulus） was measured by AFM. The pattern of cytoskeleton remodeling of HCC cell lines with different invasive ability was detected by immunofluorescent staining. The difference of cell invasive ability was tested by cell scratch experiment in other to verify mechanical data. Kruskal-Wallis H test was used to compare the differences between groups. Results The results of AFM indicated that YoungPs modulus of cytoplasma area and nucleus area decreased gradually as cell invasion ability increased （χ^2 = 472.78, 622.43, both P〈0.01）. According to invasive ability from low to high, Young＇s modulus of cell cytoplasm area of Huh-7, SMMC-7721, MHCC-97L and MHCC-97H were 1 602.43 （845.48, 3 317.25） Pa, 1 055.28 （367.48, 2 280.77） Pa, 1 026.78 （369.20, 2 019.96） Pa and 503.12 （366.11, 700.31） Pa, respectively. Young＇s modulus of cell nucleus area of Huh-7, SMMC- 7721, MHCC-97L and MHCC-97H were 2 823. 98 （1 262.78, 4 440. 07） Pa, 1 313. 43 （590. 71, 2 678.62） Pa, 1 285. 17 （583.29, 1 961. 19） Pa and 655.57 （441.29, 943.39） Pa, respectively. The results of irnmunofluorescent staining showed that the stronger the cell invasive ability, the worse cytoskeletal integrity and more irregular cell microfilament distribution. In cell scratch assay, the migration rate of MHCC-97H was 46.67% in 24 h and 86.47% in 48 h, that of MHCC-97L was 45.70% in 24 h and 82.86% in 48 h, that of SMMC-7721 was 39.41% in 24 h and 79.85% in 48 h and that of Huh-7 was 34.60% in 24 h and 72.09% in 48 h, which showed that the cell migration orderly in creased as the cell invasion ability increased. Conclusions It seemed that HCC with higher invasive ability had lower Young＇s modulus, softer cell, stronger deformability, worse cytoskeleton integrity and more irregular cell structure, and vice versa.