肝癌细胞力学特性及其与细胞周期的关系研究
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摘要
肿瘤作为一种恶性疾病，严重地危害人类健康，已经成为人类生命的第二号杀手。其恶性表现之一就是它的侵袭转移性，这是引起肿瘤晚期恶化，导致死亡的直接原因。无论对临床医学或是基础理论来说，研究并阐明肿瘤细胞侵袭和转移的机制都是头等重大的课题。恶性肿瘤常见的转移途径主要有淋巴道转移、血道转移和种植性转移，通常肿瘤经血道转移需经历浸润性生长→侵蚀基底膜→穿透内皮细胞→血道运输→靶器官微血管附着→穿出内皮及在靶组织形成转移灶等，这是一个十分复杂的多阶段连续过程。近年来尽管肿瘤的诊断、治疗取得了较大进展，但关于肿瘤细胞的侵袭转移机制至今尚未完全阐明，肿瘤手术切除后的复发转移是影响疗效的主要原因之一，因此有关肿瘤细胞侵袭转移机制的实验研究不仅有重要的理论意义，而且有重要的应用价值。
原发性肝癌是世界上十大常见癌症之一，自20世纪90年代以来其发病率已上升为恶性肿瘤的第二位，本文在分析研究国内外有关肝癌细胞力学特性研究方法及其研究状况的基础上，把细胞力学和细胞生物学有机结合起来，建立了肝癌细胞力学特性研究的实验方法和分析模型，应用微管吸吮技术从细胞周期的角度研究了肝癌细胞的粘弹性特征以及肝癌细胞与IV型胶原裱衬表面、人脐静脉内皮细胞的粘附力学特征。其主要研究工作和结果、结论如下：
1．肝癌细胞力学特性研究的方法学
建立了微管吸吮技术研究肝癌细胞力学特性的实验方法和分析方法，采用标准线性固体模型描述肝癌细胞的粘弹性特征。表明微管吸吮技术是一种较好的在单个细胞水平上研究肝癌细胞粘弹特性、肝癌细胞与IV型胶原和内皮细胞粘附力学特性的先进手段。
2．肝癌细胞的同步化
利用流式细胞技术分析未经同步处理的大鼠肝癌细胞（HTC）的细胞周期，其各周期时相细胞的百分比为G0/G1期44.97%、G2/M期12.67%、S期42.36%；采取胸腺嘧啶脱氧核苷和秋水仙碱顺序阻断后释放培养的方法可获得同步化于G1期的HTC细胞，G1期平均同步率为69.79%；采取胸腺嘧啶脱氧核苷双阻断后释放培养的方法可获得S期HTC细胞，S期平均同步率为98.32%。
用同样的方法对人肝癌细胞（SMMC-7721）进行同步，结果是：G1期平均同步率为66.11%，S期平均同步率为95.82%，而未经同步处理的SMMC-7721细胞各周期时相百分比为G0/G1期53.51%、G2/M期11.01%、S期35.48%。两种肝癌细胞G1期和S期的同步率在4hr内均稳定维持于这一水平，能满足后续微管吸吮实验的要求。
3．肝癌细胞的粘弹性特征
利用微管吸吮技术从细胞周期角度研究了HTC细胞的粘弹特征，结果发现HTC细胞具有高弹性系数和低粘性系数的粘弹特征；G1期HTC细胞与S期相比，其刚性增大，蠕变时间短，初始变形和最大变形程度均小于S期，提示G1期HTC细胞经血道转移时可能更容易在血流中存活。本文还结合细胞骨架对HTC细胞的粘弹性特征进行了分析。
4．肝癌细胞与IV型胶原的粘附力学特性
HTC细胞与IV型胶原的粘附力在研究剂量范围内随胶原浓度的增加而增大，呈现出明显的浓度依赖关系。G1期HTC细胞与S期比较，呈现出粘附和去粘附的活跃状态。在相同的条件下，G1期HTC细胞与IV型胶原的粘附力比S期相应值大，粘附时间更快，提示在HTC细胞侵蚀基底膜阶段，G1期细胞可能起更大的作用。本文还从粘附分子的角度对HTC细胞与IV型胶原的粘附力学特征进行了分析。
5．肝癌细胞与内皮细胞的粘附力学特性
本文首先建立了肝癌细胞与内皮细胞粘附力分析的实验模型，接着应用流式细胞技术检测肝癌细胞上integrin β1的表达，最后研究了不同细胞周期肝癌细胞与内皮细胞的粘附力学特性。结果表明：integrin β1粘附分子在SMMC-7721细胞上高表达，但其表达量呈现周期差异，这种粘附分子在SMMC-7721细胞与人脐静脉内皮细胞（HUVECs）的粘附过程中起着重要作用；SMMC-7721细胞与HUVECs的粘附力随粘附时间的变化而变化，也表现出粘附和去粘附的行为特征；S期SMMC-7721细胞上integrin β1的表达比G1期相应值高，与HUVECs的粘附力比G1期细胞相应值大，提示在肝癌细胞与内皮细胞的粘附过程中，S期细胞可能起主要作用。
本文的研究结果可进一步揭示肝癌细胞生长、侵袭、转移过程与肝癌细胞力学特性的相关关系，为全面认识肝癌的转移机制及临床上肝癌的预防和治疗提供定量的参考依据
肝癌细胞,细胞周期,粘弹性,粘附,微管吸吮技术
原发性肝癌是世界上十大常见癌症之一，自20世纪90年代以来其发病率已上升为恶性肿瘤的第二位，本文在分析研究国内外有关肝癌细胞力学特性研究方法及其研究状况的基础上，把细胞力学和细胞生物学有机结合起来，建立了肝癌细胞力学特性研究的实验方法和分析模型，应用微管吸吮技术从细胞周期的角度研究了肝癌细胞的粘弹性特征以及肝癌细胞与IV型胶原裱衬表面、人脐静脉内皮细胞的粘附力学特征。其主要研究工作和结果、结论如下：
1．肝癌细胞力学特性研究的方法学
建立了微管吸吮技术研究肝癌细胞力学特性的实验方法和分析方法，采用标准线性固体模型描述肝癌细胞的粘弹性特征。表明微管吸吮技术是一种较好的在单个细胞水平上研究肝癌细胞粘弹特性、肝癌细胞与IV型胶原和内皮细胞粘附力学特性的先进手段。
2．肝癌细胞的同步化
利用流式细胞技术分析未经同步处理的大鼠肝癌细胞（HTC）的细胞周期，其各周期时相细胞的百分比为G0/G1期44.97%、G2/M期12.67%、S期42.36%；采取胸腺嘧啶脱氧核苷和秋水仙碱顺序阻断后释放培养的方法可获得同步化于G1期的HTC细胞，G1期平均同步率为69.79%；采取胸腺嘧啶脱氧核苷双阻断后释放培养的方法可获得S期HTC细胞，S期平均同步率为98.32%。
用同样的方法对人肝癌细胞（SMMC-7721）进行同步，结果是：G1期平均同步率为66.11%，S期平均同步率为95.82%，而未经同步处理的SMMC-7721细胞各周期时相百分比为G0/G1期53.51%、G2/M期11.01%、S期35.48%。两种肝癌细胞G1期和S期的同步率在4hr内均稳定维持于这一水平，能满足后续微管吸吮实验的要求。
3．肝癌细胞的粘弹性特征
利用微管吸吮技术从细胞周期角度研究了HTC细胞的粘弹特征，结果发现HTC细胞具有高弹性系数和低粘性系数的粘弹特征；G1期HTC细胞与S期相比，其刚性增大，蠕变时间短，初始变形和最大变形程度均小于S期，提示G1期HTC细胞经血道转移时可能更容易在血流中存活。本文还结合细胞骨架对HTC细胞的粘弹性特征进行了分析。
4．肝癌细胞与IV型胶原的粘附力学特性
HTC细胞与IV型胶原的粘附力在研究剂量范围内随胶原浓度的增加而增大，呈现出明显的浓度依赖关系。G1期HTC细胞与S期比较，呈现出粘附和去粘附的活跃状态。在相同的条件下，G1期HTC细胞与IV型胶原的粘附力比S期相应值大，粘附时间更快，提示在HTC细胞侵蚀基底膜阶段，G1期细胞可能起更大的作用。本文还从粘附分子的角度对HTC细胞与IV型胶原的粘附力学特征进行了分析。
5．肝癌细胞与内皮细胞的粘附力学特性
本文首先建立了肝癌细胞与内皮细胞粘附力分析的实验模型，接着应用流式细胞技术检测肝癌细胞上integrin β1的表达，最后研究了不同细胞周期肝癌细胞与内皮细胞的粘附力学特性。结果表明：integrin β1粘附分子在SMMC-7721细胞上高表达，但其表达量呈现周期差异，这种粘附分子在SMMC-7721细胞与人脐静脉内皮细胞（HUVECs）的粘附过程中起着重要作用；SMMC-7721细胞与HUVECs的粘附力随粘附时间的变化而变化，也表现出粘附和去粘附的行为特征；S期SMMC-7721细胞上integrin β1的表达比G1期相应值高，与HUVECs的粘附力比G1期细胞相应值大，提示在肝癌细胞与内皮细胞的粘附过程中，S期细胞可能起主要作用。
本文的研究结果可进一步揭示肝癌细胞生长、侵袭、转移过程与肝癌细胞力学特性的相关关系，为全面认识肝癌的转移机制及临床上肝癌的预防和治疗提供定量的参考依据
肝癌细胞,细胞周期,粘弹性,粘附,微管吸吮技术
引文
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