在细胞生物学领域电场最初主要应用于形成细胞膜电穿孔以利于大分子能够通过细胞膜[1, 2]。细胞膜电穿孔模型中,膜孔的形成主要由于短时间内细胞膜两端电荷不平衡,引起带电荷的磷脂层移动形成孔隙。该技术目前已经广泛应用于细胞的基因研究、药物转化[3, 4]以及肿瘤的电化学疗法[5, 6, 7, 8]。传统电穿孔技术所利用的脉冲电场脉冲宽度往往在微秒至毫秒范围内,电场强度则位于1 kV/cm范围内;不同于之前的技术,纳秒脉冲电场采用的脉冲宽度更短(10~300 ns)、强度也更大(10 kV/cm)[9, 10, 11, 12, 13, 14]。在短时间内,这些电场脉冲能够释放出极大的能量,但是由于脉冲持续时间短,该能量并不会引起明显的温度变化。正是利用纳秒脉冲电场治疗的非产热性,其在肿瘤治疗方面有独特的优势:不仅可以保护神经、血管等重要的热敏感组织免受损伤[15, 16],还因纤维结缔组织的坏死减少而使疤痕最小化[17]。随着第一例人体内基底细胞癌治疗的顺利开展[18],纳秒脉冲电场技术将会逐渐应用于临床工作中,但是其治疗的细胞学机制至今仍未完全阐明。本文结合最新的研究进展,围绕纳秒脉冲电场对细胞的分子生物学作用机制进行简要的回顾。
1 纳秒脉冲电场致细胞膜通透性的改变理论上来讲,纳秒脉冲电场因其持续时间短,虽能够使细胞外膜带电,因未达到细胞膜穿孔阈值,可避免由此产生的细胞外膜效应,这短暂作用能够对细胞内膜产生一定的影响。Jurkat细胞在接受纳秒脉冲电场处理后,实验组荧光示踪的钙离子可从细胞内膜系统转移到细胞质中,引起细胞质钙离子浓度的增加,提示细胞内膜亦存在穿孔现象[13]。已有的实验结果显示,细胞在脉冲宽度600 ns、强度1~2 kV/cm的纳秒脉冲电场作用下,大分子染料如碘化丙啶(PI)、YO-PRO-1并不能顺利通过细胞膜,而铊离子、钙离子能够自由通过[19, 20, 21]。PI、YO-PRO-1、钙离子、铊离子分子直径分别为1.500 nm、1.000 nm、0.462 nm、0.392 nm,提示纳米孔的大小应在0.462~1.000 nm范围内[19, 20, 21]。进一步的实验证实纳米孔的大小可随着电场强度的增强而增大,电场达到一定强度时,纳米孔足以使PI染料分子通过[22]。当外加电场强度在一定阈值以下时,纳米孔的形成是可逆的。纳米孔的形成分为生成期和修复期,修复期的持续时间要远远长于生成期。纳米孔的形成速度与电场强度成正比,但是其修复时间往往与电场强度不相关。细胞膜上形成的纳米孔在高强度电场下往往要比低强度电场下更为无序、大小不均[23, 24]。值得一提的是,纳米孔的形成要求电场强度在一定阈值以上。如果电场强度在纳米孔形成阈值以下,则可能产生其他的生物学效应。如有实验对牛的嗜铬细胞进行单次低剂量的纳秒脉冲电刺激,在不形成纳米孔的情况下,电压门控钙离子通道激活,钙离子发生细胞质内流[25],提示纳米孔并不是钙离子内流的唯一通道。
2 纳秒脉冲电场致磷脂酰丝氨酸转位纳秒脉冲电场对于细胞膜的另一个重要作用是磷脂酰丝氨酸的转位——从细胞膜内侧面外翻至细胞膜外侧面[26, 27, 28]。正常情况下,磷脂酰丝氨酸主要分布于细胞膜内侧面,主要通过钙离子依赖的酶维持该分布的不均衡性[29]。目前一般认为磷脂酰丝氨酸外翻的主要成因是电刺激致纳米孔形成后,排布于纳米孔内周的磷脂酰丝氨酸侧向位移,即磷脂双分子层的内外侧翻转。同时,脉冲时间越长、强度越大、电击次数越多,磷脂酰丝氨酸外翻的可能性越大。低剂量的纳秒脉冲电场可只形成纳米孔,而不形成磷脂酰丝氨酸的外翻[30]。
3 纳秒脉冲电场致细胞内源性凋亡 3.1 细胞活性氧产生Pakhomova课题组[31]首先报道了纳秒脉冲电场作用后细胞可因发生氧化应激反应而产生大量活性氧。活性氧作为凋亡因子,至少可从三条通路诱导细胞凋亡:DNA双链的断裂[32, 33]、Bid介导的线粒体膜通透性改变[34]、Fas参与的凋亡小体的形成[28]。活性氧具有钙离子浓度依赖性,即减少活性氧产生可通过降低细胞内钙离子浓度来实现[35]。活性氧并不是细胞凋亡的决定因子,可能作为协同因子诱导细胞的凋亡[36]。
3.2 细胞内钙离子浓度的增加接受纳秒脉冲电场处理后细胞内钙离子浓度的上调是目前研究的热点之一。通过纳米孔的形成、钙离子通道的开启,细胞外、内膜系统的钙离子可瞬间涌入细胞质,从而使得短时间内细胞质钙离子浓度急剧上升[37, 38, 39, 40, 41]。钙离子作为第二信使,是细胞内许多生化反应的调节因子。钙离子与胞内活性氧的生成相关[36],参与调节胞内囊泡对细胞膜的融合修复[42]、溶酶体对损伤细胞器的吞噬清除[43]以及钙蛋白酶介导的线粒体诱导的细胞凋亡[44]。
4 纳秒脉冲电场致细胞外源性凋亡外源性凋亡途径是新近发现的另一种可能解释纳秒脉冲电场治疗作用、诱导凋亡的通路。不同于内源性凋亡途径,该途径通过激活细胞膜上Fas/CD95受体,信号转导引起Caspase3的激活,最终诱导细胞的凋亡。纳秒脉冲电场处理可以刺激细胞内源性FasL表达量的增加,增强Fas/CD95-Caspase3通路信号,诱导细胞转向凋亡。SiRNA干扰FasL基因表达后细胞的存活率显著提高,反证了Fas/CD95-Caspase3信号通路在细胞凋亡途径中所发挥的作用[45]。不同细胞系经处理后Fas/CD95信号强弱有差异[46, 47],侧面解释了不同细胞对于纳秒脉冲电场治疗效果的敏感度不一致。
5 纳秒脉冲电场致细胞坏死一些研究发现纳秒脉冲电场处理后细胞发生肿胀[14, 48, 49],且细胞的死亡时间远远短于细胞凋亡过程所需的时间[50]。高强度的纳秒脉冲电场处理后,细胞膜上纳米孔大量形成,导致短时间内溶液中的大量水分渗透进入细胞质而致细胞肿胀,最终引起细胞破裂。因此纳秒脉冲电场处理后并不是所有的细胞都会发生凋亡,其中一部分细胞可能会转向坏死。
6 纳秒脉冲电场激发细胞自噬细胞自噬是细胞在受到细胞内外不良刺激后激活的细胞自我保护机制。自噬程序的激活意味着细胞将损伤细胞膜或者细胞器转移到溶酶体降解,并且将原料再利用,尽可能地将损伤效应最小化。尽管细胞自噬是细胞的自我修复和生存机制,但是其同样可以诱导细胞的程序性死亡。Ullery等[51]观察到纳秒脉冲电场处理后细胞质中出现了细胞自噬的早、中、晚期分子标记物,提示其中有自噬过程参与其中。纳秒脉冲电场处理后,细胞膜不可避免地会受到一定程度的损伤,这时细胞内的溶酶体会自动启动清除损伤成分,以维持细胞内的正常代谢平衡。此外,纳秒脉冲电场还会损伤溶酶体。如果电场强度足够大,能够正常行使功能的溶酶体的修复能力不足,那么细胞命运可能由自噬修复转归为死亡[52]。
7 纳秒脉冲电场破坏细胞骨架之前对纳秒脉冲电场治疗作用的研究往往着眼于其对细胞内、外膜以及细胞器的生物学效应。对于亚细胞结构的研究鲜有涉及。Stacey团队[53, 54, 55]的一系列研究发现,高强度纳秒脉冲电场还对细胞骨架形态产生影响。一般认为,细胞骨架支撑着细胞膜,起着稳定细胞膜结构、保持细胞正常形态的作用[56]。高强度的纳秒脉冲电场刺激还可能通过破坏细胞微丝骨架,影响细胞膜的稳定性,进一步导致细胞形态异常而导致细胞死亡。
8 结 语纳秒脉冲电场对细胞的作用机制是多方面、多层次的。它不仅可以穿透细胞膜形成纳米孔,引起钙离子内流;同时还可作用于细胞内膜,引起内质网损伤,线粒体膜电位差的改变;再者还可以损伤细胞微丝骨架,导致细胞形态的变化。因此,细胞最终的命运转归,是细胞内外一系列的物理、生化综合因素相互作用的结果。
纳秒脉冲电场在肿瘤治疗方面已经显示出多种优势,具有极其广阔的应用前景。然而,与之不相对应的是目前纳秒脉冲电场对细胞的作用机制的研究仍较为初步,相关知识了解还十分有限,尚未有一个系统的模型或理论可用于解释、预测其作用机制。因此进一步阐明电场处理后细胞不同转归结局的原因,摸索出电场的最佳处理条件,以及体内外实验可能引起的治疗效果的差异,都将是我们接下来亟待解决的关键问题。
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