ALA-PDT通过内质网应激途径诱导瘢痕疙瘩成纤维细胞凋亡的机制

The Mechanism of Apoptosis of Keloid Fibroblasts Induced by ALA-PDT Through Endoplasmic Reticulum Stress Pathway

目的通过内质网应激(endoplasmic reticulum stress, ERS)途径研究5-氨基酮戊酸(aminolevulnilic acid, ALA)联合光动力疗法(photodynamic therapy, PDT)诱导人瘢痕疙瘩成纤维细胞(keloid fibroblasts, KFB)凋亡的机制。方法采用组织块培养法人工培养KFB,经免疫组织化学染色鉴定后,取第3～10代KFB进行干预研究。KFB分为5组,A组:空白对照组(无光敏剂无光照),B组:光敏剂组,C组:光照组,D组:死亡受体和线粒体途径抑制剂+光敏剂+光照组,E组:光敏剂+光照组。ALA 4 mmol/L,PDT 80 J/cm^2干预后,CCK-8检测KFB存活率,流式细胞仪检测KFB凋亡率,Western blot检测Caspase-3及ESR途径的标志性蛋白GRP78、PERK和CHOP的表达丰度。结果原代KFB呈长梭形或三角形贴壁成长,波形蛋白免疫鉴定为阳性;CCK8检测及流式细胞仪显示:D、E组KFB活性抑制、凋亡增加;Western blot检测显示:D、E组中Caspase-3以及ESR途径标志性蛋白GRP78、PERK和CHOP较A组的表达均明显上调(P<0.05);D组与E组相比,D组的Caspase-3表达降低(P<0.05),而其中GRP78、PERK、CHOP的表达差异无统计学意义(P>0.05)。结论 ALA-PDT能够诱导KFB凋亡,且ERS途径可能参与了ALA-PDT诱导KFB凋亡的过程。

Objective To investigate the mechanism of keloid fibroblast(KFB) apoptosis induced by aminolevulinic acid photodynamic therapy(ALA-PDT)in vitro through the endoplasmic reticulum stress(ESR) pathway. Methods KFB were cultivated by tissue block adherent method. The 3rd ~10th generation of the logarit-hmic growth phase fibroblasts after immunocytochemistry were used for further research. KFB were respectively treated with PBS,ALA,PDT,combination of Caspase-8, 9 inhibitor, ALA and PDT, combination of ALA and PDT, which were defined as A, B, C, D, E group.Flow cytometry instrument(Annexin V/PI staining) was performed to measure the apoptosis rates of KFB. Western blot was used to detect the expression of Caspase-3, ESR markers such as GRP78,PERK,CHOP. Results The original generation of KFB showed long fusiform or triangle adherent growth, and were identified as positive vimentin with immunocytochemistry staining. The proliferation activity of KFB was restrained and apoptosis was induced with ALA-PDT intervention, which were detected by CCK-8 and flow cytometry instrument. The expression of Caspase-3, GRP78, PERK and CHOP increased in D and E groups, compared with negative control group(P < 0.05). The expression of Caspase 3 were lower in D than E group(P < 0.05). There was no statistical difference of GRP78, PERK and CHOP between D and E group(P > 0.05). Conclusion KFB apoptosis can be induced by ALA-PDT through the endoplasmic reticulum stress pathway.