细胞膜骨架蛋白4.1R对光动力效应的影响

Effects of membrane skeleton protein 4.1R on the efficiency of photodynamic therapy

目的 探讨细胞膜骨架蛋白4.1R对光动力效应的影响.方法 将4.1R基因敲除型（4.1R-/-）和野生型（4.1R＋/＋）小鼠胚胎成纤维细胞（MEF）分别以0.25、0.50、1.00、1.50、2.00mmol/L浓度的5-氨基乙酰丙酸（5-ALA）孵育,72、96、120、180、240mJ/cm2剂量的450 nm蓝光照射进行光动力处理,细胞增殖检测试剂盒（CCK-8）检测细胞存活率;经不同浓度5-ALA孵育的细胞,以荧光分光光度法检测胞内光敏剂原卟啉的荧光强度;以1.00 mmol/L的5-ALA孵育细胞后激光共聚焦显微镜观察胞内产生的原卟啉在细胞中的定位;Western印迹法检测原卟啉合成限速酶亚铁螯合酶（FECH）与胆色素原脱氨基酶（HMBS）蛋白表达水平.结果 5-ALA光动力处理均能杀伤两种细胞,其杀伤作用与5-ALA的浓度、孵育时间及光照射剂量相关,但两者PDT杀伤效果不同.当1.00mmol/L的5-ALA孵育4.0h且光照射剂量为120mJ/cm2时,4.1R-/-MEF存活率显著高于4.1R＋/＋MEF[（46.9±7.1）％比（12.5±2.1）％,P＜0.001].5-ALA孵育细胞后产生的原卟啉分布于整个胞质中,但两种细胞中原卟啉的浓度不同,在1.00 mmol/L的5-ALA孵育4.0h时,4.1R＋/＋MEF内原卟啉荧光值显著高于4.1R-/-MEF（124.2±3.5比34.6 ±3.8,P＜0.001）.Western印迹结果表明FECH与HMBS在两种细胞中的表达水平差异无统计学意义.结论 蛋白4.1R的缺失造成细胞内原卟啉合成量降低,PDT效果下降,但并未影响ALA代谢过程,推测蛋白4.1R影响了5-ALA的跨膜转运,进而影响细胞内原卟啉的合成,最终影响了PDT杀伤效应.

Objective To explore the effects of membrane skeleton protein 4.1R on the efficiency of photodynamic therapy （PDT）.Methods 4.1R gene knockout and wild-type mouse embryonic fibroblasts （MEFs） were incubated with various concentrations of 5-aminolevulinic acid （5-ALA） （0.25,0.50,1.00,1.50 and 2.00 mmoL/L）,followed by exposure to 450 nm light at a dose of 72,96,120,180,240 mJ/ cm2.Cell counting kit 8 （CCK-8） assay was used to assess the survival rate after PDT treatment.Laser confocal microscopy was used to observe the location of photo-sensitizer protoporphyrin and fluorescence spectrophotometer for detecting the fluorescent intensity of intracellular protoporphyrin.The protein levels of rate-limiting enzyme of protoporphyrin synthesis,ferrochelatase （FECH） and hydroxymethylbilane synthase （HMBS） were determined by Western blot.Results Both cell lines were killed after 5-aminolevulinic acid （5-ALA）-PDT and its efficacy was dependent on 5-ALA concentration,incubation duration and light dose.The cell survival rates of 4.1R-/-MEF were significantly higher than those of 4.1R＋/＋ MEF （46.9％ ± 7.1％ vs 12.5％ ±2.1％,P ＜0.001） after PDT treatment with a light dose of 120 mJ/cm2 mediated by 5-ALA 1.00 mmol/L.After incubation with 1.00 mmol/L 5-ALA,protoporphyrin was distributed throughout cytoplasm in both cell lines while the fluorescent intensity of 4.1R ＋/＋ MEF was higher than that of 4.1R-/-MEF （124.2±3.5 vs 34.6 ±3.8,P ＜0.001）.Western blot showed that no difference of FECH and HMBS protein level was found in two cell lines.Conclusions A lack of protein 4.1R may attenuate the intracellular protoporphyrin level and the photo-cytotoxicity of PDT.No cellular change of ALA metabolic activity is found.Protein 4.1R may be involved in the ALA uptake in MEF cells so that the cellular level of protoporphyrin ultimately affects the PDT efficiency.