成纤维细胞的自噬性溶解死亡在增生性瘢痕消退过程中的作用

doi:10.3969/j.issn.1674-8115.2022.01.007

摘要/Abstract

摘要：

目的·探索增生性瘢痕消退成熟过程中，成纤维细胞是否发生自噬性溶解死亡。方法·收集上海交通大学医学院附属瑞金医院烧伤科2018年6月—2019年6月烧伤患者16例的瘢痕组织，分为增生组（对照组，8例）和消退组（试验组，8例）2组。采用透射电镜观察细胞组织内的自噬情况。体外分别培养2组患者的成纤维细胞。建立缺氧模型，分别于12、24、48 h时点收集细胞，采用透射电镜观察成纤维细胞的自噬情况。Calcein/PI荧光染料试剂盒检测活/死细胞的数量。免疫荧光显微镜下观察细胞的自噬和凋亡。流式细胞仪检测细胞凋亡和自噬性溶解死亡的数量。Western blotting观察低氧诱导因子-1（hypoxia inducible factor 1，HIF-1）、自噬蛋白区域衍生肽beclin-1、微管相关蛋白轻链-3（microtuble-associated protein light chain 3，LC3）、半胱氨酸蛋白酶-3（caspase-3）、半胱氨酸蛋白酶-9的表达变化。2组间定量资料采用Student's t检验，多组间定量资料的分析使用One-way ANOVA检验。结果·电镜显示增生性瘢痕中存在自噬小体，退行性瘢痕中存在自噬性溶解死亡。体外培养细胞电镜观察结果与体内细胞的结果一致。死亡细胞在24 h显著增加，在48 h进一步增加。其中，细胞死亡类型主要为自噬性溶解死亡，少量凋亡。这与LC3的高表达有关。结论·增生性瘢痕演变过程中，除了细胞凋亡以外，自噬性溶解死亡可能是引起瘢痕消退成熟的主要方式，其中LC3作用明显。

关键词: 增生性瘢痕, 成纤维细胞, 自噬, 细胞凋亡, 自噬性溶解死亡, 微管相关蛋白轻链-3（LC3）

Abstract:

Objective·To investigate whether autosis occurred in fibroblasts during hypertrophic scar regression.

Methods·The scar tissues of 16 burn patients were collected from June 2018 to June 2019 in the Burn Department of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. They were divided into two groups: hyperplasia group (control group, 8 cases) and regression group (experimental group, 8 cases). Autophagy was observed by transmission electron microscopy. Fibroblasts of the two groups were cultured in vitro to establish a hypoxia model. The fibroblasts were collected at 12, 24 and 48 h respectively, and autophagy was observed by transmission electron microscopy. Live/dead cells were detected by using Calcein /PI fluorescent dye kit. The autophagy and apoptosis were observed by immunofluorescence. The numbers of apoptosis and autophagic death were detected by flow cytometry. Then the expression of hypoxia inducible factor1 (HIF-1), beclin-1, microtuble-associated protein light chain 3 (LC3), caspase-3 and caspase-9 were assayed at the protein level. Student's t test was used for quantitative data between the two groups, and One-way ANOVA test was used for quantitative data among multiple groups.

Results·The electron microscopy showed that autophagosome existed in hyperplasia scar, and autosis occured in the regressive scar. In vitro study by electron microscopy was consistent with in vivo tissue observation. The dead cells had a marked increase at 24 hours, and further increased at 48 hours. Among them, the cell death type was mainly autosis, and a small amount of apoptosis. High expression of LC3 was responsible for this.

Conclusion·In addition to apoptosis, autosis may be the major cell death during hypertrophic scar regression, and LC3 plays an important role.

Key words: hypertrophic scar, fibroblast, autophagy, apoptosis, autosis, microtuble-associated protein light chain 3 (LC3)

中图分类号:

R644

张剑, 宋菲, 王西樵. 成纤维细胞的自噬性溶解死亡在增生性瘢痕消退过程中的作用[J]. 上海交通大学学报(医学版), 2022, 42(1): 44-50.

Jian ZHANG, Fei SONG, Xiqiao WANG. Role of autosis of fibroblasts in hypertrophic scar regression[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(1): 44-50.

图/表 6

图1 增生期和消退期瘢痕组织自噬的透射电镜观察Note：A. Autophagy of scar fibroblasts during hyperplasia. B—D. Autophagy and autosis of scar fibroblasts during regression. Red arrows—Autophagosome and autosis.

Fig 1 Transmission electron microscope observation of autophagy in proliferative and regressive scar

图2 不同时间点成纤维细胞的自噬观察Note：A. Autophagy of fibroblasts at 0 h. B. Autosis of fibroblasts at 12 h. C. Autosis of fibroblasts at 24 h. D. Autosis of fibroblasts at 48 h. Red arrow—Autophagosome and autosis.

Fig 2 Investigation of autophagy in fibroblasts at different time points

图3 成纤维细胞在缺氧和缺营养条件下的细胞死亡情况Note：White arrow—dead cells.

Fig 3 Cell death of fibroblasts under hypoxia and nutritional deficiency

图4 成纤维细胞缺氧和缺营养条件下自噬和凋亡共染色结果Note：White arrow—apoptotic cells.

Fig 4 Co-staining of autophagy and apoptosis in fibroblasts under hypoxia and nutritional deficiency

图5 流式细胞仪检测成纤维细胞自噬性死亡和细胞凋亡Note：①P=0.030, ②P=0.039, ③P=0.020, ④P=0.041, ⑤P=0.038, ⑥P=0.031, ⑦P=0.038, ⑧P=0.042, ⑨P=0.021, ⑩P=0.036, compared with 0 h.

Fig 5 Assay of cell autosis and apoptosis by flow cytometry

图6 Western blotting检测细胞自噬和凋亡Note：①P=0.035, ②P=0.031, ③P=0.025, ④P=0.021, ⑤P=0.024, ⑥P=0.019, ⑦P=0.015, ⑧P=0.011, ⑨P=0.036, ⑩P=0.031, ?P=0.033, ?P=0.042,?P=0.037, ?P=0.011, compared with 0 h.

Fig 6 Assay of apoptosis- and autophagy-related proteins by Western blotting

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