半胱氨酸天冬氨酸蛋白酶3在血卟啉单甲醚光动力学疗法中对瘢痕成纤维细胞的作用

Effects of caspase 3 on hypertrophic scar fibroblasts in hematoporphyrin monomerthyl ether- photodynamic therapy （HMME-PDT）

目的 探讨半胱氨酸天冬氨酸蛋白酶3（caspase 3）在增生性瘢痕成纤维细胞（HSF）经光敏剂血卟啉单甲醚（HMME）诱导的凋亡效应中的作用。方法 将原代培养的HSF分为对照组、HMME-光动力疗法（PDT）组和caspase 3抑制剂（Z-DEVD-FMK）组，经caspase 3-异硫氰酸荧光素（FITC）-碘化丙啶（PI）染色后在荧光显微镜下观察各组细胞内caspase 3的荧光强度。收集各组细胞，在活性caspase 3-FITC单染后，经流式细胞术检测活性caspase 3阳性细胞百分率；在PI单染后，经流式细胞术检测细胞凋亡率。结果 对照组和caspase 3抑制剂组HSF胞质中caspase 3-FITC荧光微弱，PDT组荧光显著增强；对照组活性caspase 3阳性细胞百分率低，HMME-PDT组（30.86% ± 1.21%）上升，caspase 3抑制剂组（2.46% ± 0.18%）降低，后两组间比较，t = 21.76，P 〈 0.05。PI染色分析表明，对照组凋亡率（2.45% ± 0.22%）低，PDT组（30.54% ± 3.78%）显著升高，两组比较，t = 35.90，P 〈 0.05；caspase 3抑制剂组凋亡率（10.46% ± 2.15%）低于PDT组，但显著高于对照组（t = 27.97，P 〈 0.05）。结论 HMME-PDT诱导HSF发生的凋亡效应与caspase 3的激活密切相关，但该凋亡效应并不依赖于caspase 3。

Objective To investigate the role of caspase 3 in HMME-induced apoptosis in hypertrophic scar fibroblasts（HSFs）. Methods Fibroblasts were obtained from 10 patients with untreated hypertrophic scar, and subjected to a primary culture. After 4 to 6 passages of culture, the HSFs were divided into 3 groups to remain untreated （control group）, be treated with HMME followed by photodynamic therapy （HMME-PDT group）, or the combination of HMME and Z-DEVD-FMK followed by photodynamic therapy （caspase 3 inhibitor group）. At 12 hours after the therapy, HSFs were collected and immunofluorescence microscopy was used to observe the fluorescence intensity of caspase 3 after staining with fluorescein isocyanate （FITC） and popodium iodide （PI）, flow cytometry was performed to determine the percentage of caspase 3-positive HSFs and apoptosis rate in HSFs after single staining with FITC and PI respectively. Results The fluorescence intensity of caspase 3 was weak in the control group and caspase 3 inhibitor group, but was strong in the HMME-PDT group. An increased percentage of caspase 3-positive HSFs was noted in the HMME-PDT group compared with the control group and caspase 3 inhibitor group （30.86% ± 1.21% vs. 3.12% ± 0.28% and 2.46% ± 0.18%, t = 19.92, 21.76, both P 〈 0.05）. The apoptosis rate in HSFs was significantly higher in the HMME-PDT group and caspase 3 inhibitor group than in the control group（30.54% ± 3.78% and 10.46% ± 2.15% vs. 2.45% ± 0.22%, t = 35.90, 27.97, both P 〈 0.05）, and higher in the HMME-PDT group than in the caspase 3 inhibitor group. Conclusions The apoptosis in HSFs induced by HMME-PDT is closely related to the activation of caspase 3, while caspase 3 seems to be dispensable for the apoptosis.