CAJ | 학술논문

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.
목적 탐토세포막골가단백4.1R대광동력효응적영향.방법 장4.1R기인고제형(4.1R-/-)화야생형(4.1R+/+)소서배태성섬유세포(MEF)분별이0.25、0.50、1.00、1.50、2.00mmol/L농도적5-안기을선병산(5-ALA)부육,72、96、120、180、240mJ/cm2제량적450 nm람광조사진행광동력처리,세포증식검측시제합(CCK-8)검측세포존활솔;경불동농도5-ALA부육적세포,이형광분광광도법검측포내광민제원계람적형광강도;이1.00 mmol/L적5-ALA부육세포후격광공취초현미경관찰포내산생적원계람재세포중적정위;Western인적법검측원계람합성한속매아철오합매(FECH)여담색소원탈안기매(HMBS)단백표체수평.결과 5-ALA광동력처리균능살상량충세포,기살상작용여5-ALA적농도、부육시간급광조사제량상관,단량자PDT살상효과불동.당1.00mmol/L적5-ALA부육4.0h차광조사제량위120mJ/cm2시,4.1R-/-MEF존활솔현저고우4.1R+/+MEF[(46.9±7.1)％비(12.5±2.1)％,P＜0.001].5-ALA부육세포후산생적원계람분포우정개포질중,단량충세포중원계람적농도불동,재1.00 mmol/L적5-ALA부육4.0h시,4.1R+/+MEF내원계람형광치현저고우4.1R-/-MEF(124.2±3.5비34.6 ±3.8,P＜0.001).Western인적결과표명FECH여HMBS재량충세포중적표체수평차이무통계학의의.결론 단백4.1R적결실조성세포내원계람합성량강저,PDT효과하강,단병미영향ALA대사과정,추측단백4.1R영향료5-ALA적과막전운,진이영향세포내원계람적합성,최종영향료PDT살상효응.
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.