暨南大学生命科学技术学院洪岸/陈小佳团队：聚乙二醇原子化灰硒构建纳米硒促进皮肤组织创面修复|BAM

近期，暨南大学生命科学技术学院洪岸/陈小佳团队和暨南大学化学与材料学院郑文杰教授在科爱出版创办的期刊Bioactive Materials上联合发表原创性文章：聚乙二醇原子化灰硒构建纳米硒促进皮肤组织创面修复。通过SD大鼠、斑马鱼多模式生物结合转录组分析发现聚乙二醇原子化的纳米硒在安全剂量下通过Wnt，FGFR，VEGFR通路促进皮肤组织创面修复。

01

研究内容简介

组织再生与许多疾病密切相关。再生速度缓慢、炎症调控异常或者再生过程中各种细胞修复速度不协调均可引起不完全再生，以致于原本具有一定再生能力的皮肤组织在损伤后修复仍旧存在障碍，尤其是代谢异常的机体内。因此，为了潜在地解决这一临床需求，设计制备具有促进组织再生的药物分子非常关键。

研究团队发现以灰硒和聚乙二醇为原料合成零氧化态的纳米硒。通过转录组分析，发现纳米硒在安全剂量下促进与组织再生相关的信号通路。同时，作者在SD大鼠和斑马鱼两种模式生物中验证了纳米硒在安全剂量下不仅促进皮肤的再生，而且促进了高血糖引起的再生障碍的修复 。

一、 聚乙二醇原子化灰硒的制备

在这篇文章中，作者采用聚乙二醇高温原子化灰硒制备得到纳米硒（图1A）。通过马尔文粒度仪（图1B）、透射电镜（图1C）、原子力显微镜（图1D）等多种实验证明制备的纳米硒是粒径在100-200 nm的球形结构。接着作者采用RNA-seq转录组分析，发现经纳米硒处理后的斑马鱼尾鳍组织，与组织再生相关的Wnt，FGFR，VEGFR信号通路及其下游的分子PI3K-Akt被激活（图1G,H），这提示我们聚乙二醇原子化灰硒制备的纳米硒可能具有促进组织再生的功效。

Fig. 1. (A) Schematic illustration synthesis of Nano-Se. (B) Particle size distribution of the Nano-Se. (C) TEM image of Nano-Se. Scale bars: 200 nm. (D) Atomic Force Microscope (AFM) image and particle size data analysis of Nano-Se. (E) Principal component analysis (PCA) was performed based on differentially expressed genes from regenerated tail fin the of two groups. Each data point corresponds to the PCA analysis of each sample. (F) Heat maps of significantly upregulated and downregulated genes (fold change ≥2 and P < 0.05). (G) KEGG pathway enrichment analysis of the identified differentially expressed genes. The 30 most significantly enriched pathways are shown. (H) Heat maps of significantly upregulated genes related to FGF, VEGF, Wnt pathway and selenoprotein.

二、 纳米硒促进皮肤组织创面的修复

为了验证聚乙二醇原子化灰硒制备的纳米硒具有促进皮肤组织修复的作用。作者采用成年斑马鱼切尾构建组织再生模型（图2A），发现随着时间的推移纳米硒在安全剂量下（100 nM）显著的促进斑马鱼尾鳍的组织再生（图2B,C），同时作者以纳米金、纳米硫作为对照，发现这种促进斑马鱼尾鳍再生的作用是纳米硒特有的（图2D）。除此之外，作者采用SD大鼠通过背部全皮层损伤模型评估纳米硒的促进皮肤组织修复的作用，发现纳米硒在100 nM的浓度下加速大鼠背部皮肤组织的创面愈合（图2E,F）。此部分，作者采用斑马鱼、SD大鼠两种模式生物验证了聚乙二醇原子化灰硒制备的纳米硒在安全剂量下显著的促进皮肤组织的创面愈合，且这种作用效果在不同的物种间具有保守性。

Fig. 2. (A) Schematic illustration of calculating the regeneration rate of zebrafish caudal fin. (B) Representative images of the Nano-Se accelerates caudal fin regeneration. dpa: days past amputation. (C) Regeneration rate of zebrafish caudal fin treated with Nano-Se. (3 independent biological repeats with a total n = 29). (D) Regeneration rate of adult zebrafish caudal fin treated with Nano-S, Nano-Au, Nano-Se (3 independent biological repeats with a total n = 15). (E) Representative images of Nano-Se in a dorsal cortex injury SD rats model. (F) Wound healing rate of Nano-Se in a dorsal cortex injury SD rats model (3 independent biological repeats with a total n = 15). (Mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

三、纳米硒促进血管新生

在皮肤创面修复过程中，新生血管扮演着重要的角色。为了验证纳米硒在组织再生过程中是否具有促进血管新生的作用，作者采用双转基因斑马鱼Tg: (fli1:EGFP/gata1:mCherry) 即血管和血液分别标记绿色和红色荧光，验证纳米硒促进新生血管的作用，结果显示纳米硒在100 nM 的浓度下显著促进血管的新生且新生的血管中有血液的流动（图3A,B）。同时作者采用鸡胚尿囊膜实验（图3C）和人血管内皮细胞迁移实验（图3D,E），综合在体内、体外水平证明了聚乙二醇原子化灰硒制备的纳米硒具有促进血管新生的作用。

Fig. 3. (A) Representative fluorescence microscopy images for angiogenesis in fin regeneration by transgenosis zebrafish (fli1:EGFP/gata1:mCherry) which blood vessels were labeled with green fluorescence and blood with red fluorescence, the adult zebrafish were treated with different concentrations of Nano-Se (0, 50, 100 and 200 nM). (B) Regeneration rate of fin angiogenesis treated with Nano-Se (3 independent biological repeats with a total n = 13). (C) Nano-Se promotes angiogenesis of allantoic membrane of chicken embryo. (D) Wound healing assay to evaluate the migration of HUVEC cells after being treated with Nano-Se (0, 25, 50 and 100 nM) and DMEM with 10% FBS. Cells were wounded and monitored using a microscope 12 h. The red areas represent migrating cells. (E) Migration rate of HUVEC cells induced by Nano-Se (3 independent biological repeats with a total n = 9). (Mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

四、 纳米硒通过VEGFR信号通路促进血管新生

为了验证纳米硒促进皮肤组织修复的作用机制，根据图1的转录组测序结果。作者采用VEGFR2的抑制剂（PTK787）作用于转基因斑马鱼Tg: (fli1:EGFP) 即血管标记绿色。实验结果发现当VEGFR2信号被抑制后，纳米硒促进皮肤组织的修复的作用被抑制，但是当VEGFR2信号抑制后，纳米硒仍能促进斑马鱼尾鳍的组织基质生长（图4），这提示我们纳米硒促进皮肤组织再生的作用不仅仅是通过VEGFR2促进血管新生发挥作用的，还会通过调控别的信号通路促进组织的基质生长。

Fig. 4. (A) Representative images for caudal fin regeneration in adult zebrafish, the adult zebrafish were treated with Nano-Se (100 nM) and PTK787 (100 nM) (an inhibitor of VEGFR2/KDR). (B) Representative fluorescence microscopy images for angiogenesis in caudal fin by transgenosis zebrafish (fli1:EGFP), the adult zebrafish were treated with Nano-Se (100 nM) and PTK787 (100 nM) (an inhibitor of VEGFR2/KDR) (C) Regeneration rate of caudal fin regeneration treated with Nano-Se (3 independent biological repeats with a total n = 13). (D) Angiogenesis rate of caudal fin treated with Nano-Se (3 independent biological repeats with a total n = 13). (Mean values ± SD, *：Significant difference compared with control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #: Significant difference between PTK787 and PTK787+ Nano-Se group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, n.s: not significant difference between PTK787 and PTK787+ Nano-Se group, N.S: Not significant difference compared with control group).

五、 纳米硒通过FGFR/Wnt信号通路促进皮肤组织的基质生长

为了验证纳米硒促进皮肤组织基质生长的作用机制，结合图1的转录组测序结果，作者采用FGFR的抑制剂（AZD4547）和Wnt的抑制剂（LGK974）作用于转基因斑马鱼Tg: (fli1:EGFP) 即血管标记绿色。实验结果发现当FGFR信号（图5）和Wnt信号（图6）通路被抑制后，纳米硒促进皮肤组织基质生长的作用被抑制。以上实验结果说明聚乙二醇原子化灰硒制备的纳米硒在安全剂量下，一方面通过VEGFR信号通路促进血管新生，另一方面通过FGFR/Wnt信号通路促进组织的基质生长，最终达到促进皮肤组织创面的修复作用。

Fig. 5. (A) Representative images for caudal fin regeneration in adult zebrafish, the adult zebrafish were treated with Nano-Se (100 nM) and AZD4547 (50 nM) (an inhibitor of FGFR). (B) Representative fluorescence microscopy images for angiogenesis in caudal fin by transgenosis zebrafish (fli1:EGFP), the adult zebrafish were treated with Nano-Se (100 nM) and AZD4547 (50 nM) (an inhibitor of FGFR) (C) Regeneration rate of caudal fin regeneration treated with Nano-Se (4 independent biological repeats with a total n = 15). (D) Angiogenesis rate of caudal fin treated with Nano-Se (4 independent biological repeats with a total n = 15). (E) Wound healing assay to evaluate the migration of HFF cells after being treated with Nano-Se (0, 25, 50 and 100 nM) and DMEM with 10% FBS. Cells were wounded and monitored using a microscope 12 h. The red areas represent migrating cells. (F) Wound healing assay to evaluate the migration of Balb/c 3T3 cells after being treated with Nano-Se (0, 25, 50 and 100 nM) and DMEM with 10% FBS. Cells were wounded and monitored using a microscope 12 h. The red areas represent migrating cells. (G) Migration rate of HFF cells induced by Nano-Se (3 independent biological repeats with a total n = 9). (H) Migration rate of Balb/c 3T3 cells induced by Nano-Se (3 independent biological repeats with a total n = 9). (Mean values ± SD, *：Significant difference compared with control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #: Significant difference between AZD4547 and AZD4547+ Nano-Se group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, n.s: not significant difference between AZD4547 and AZD4547+ Nano-Se group, N.S: Not significant difference compared with control group).

Fig. 6. (A) Representative images for caudal fin regeneration in adult zebrafish, the adult zebrafish were treated with Nano-Se (100 nM) and LGK974 (10 nM) (an inhibitor of Wnt pathway). (B) Representative fluorescence microscopy images for angiogenesis in caudal fin by transgenosis zebrafish (fli1:EGFP), the adult zebrafish were treated with different concentrations of Nano-Se (100 nM) and LGK974 (10 nM) (an inhibitor of Wnt pathway) (C) Regeneration rate of fin regeneration treated with Nano-Se (3 independent biological repeats with a total n = 12). (D) Angiogenesis rate of transgenosis fin treated with Nano-Se (3 independent biological repeats with a total n = 12). (Mean values ± SD, *：Significant difference compared with control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #: Significant difference between LGK974 and LGK974+ Nano-Se group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, n.s: not significant difference between LGK974 and LGK974+Nano-Se group, N.S: Not significant difference compared with control group).

六、纳米硒促进糖尿病难愈性皮肤创面的修复

在皮肤修复过程中，糖尿病引起皮肤组织溃烂的修复是临床上的治疗瓶颈。在此作者探究纳米硒是否对高血糖引起的皮肤修复障碍具有一定的作用，作者通过过度饮食和腹腔注射STZ破坏β-胰岛细胞构建高血糖斑马鱼和糖尿病大鼠模型，并验证了高血糖斑马鱼的皮肤再生具有障碍性（图7A）。当纳米硒处理高血糖斑马鱼和糖尿病大鼠后，发现显著促进高血糖状态下皮肤组织的修复作用。

Fig. 7. (A) Representative images for zebrafish caudal fin regeneration by overdiet (6 times a day) and intraperitoneal injection of streptozocin (STZ); (B) Representative images for caudal fin regeneration by overdiet and intraperitoneal injection STZ with transgenosis zebrafish (coroa1: EGFP) which inflammatory cells are labeled with green fluorescence, the adult zebrafish were treated with Nano-Se (100 nM); (C) Regeneration rate of fin regeneration treated with overdiet and STZ (3 independent biological repeats with a total n = 24). (D) Regeneration rate of fin regeneration treated with Nano-Se (3 independent biological repeats with a total n = 12). (E) Representative images of Nano-Se in a dorsal cortex injury SD diabetic rats model. (F) Wound healing rate of Nano-Se in a dorsal cortex injury SD diabetic rats model (3 independent biological repeats with a total n = 15). (Mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

综上所述，作者通过聚乙二醇原子化灰硒制备纳米硒，发现该纳米硒下安全剂量范围，一方面通过VEGFR信号通路促进血管新生，另一方面通过FGFR/Wnt信号通路促进组织基质的生长，共同促进皮肤组织再生。同时对由于高血糖引起的皮肤再生障碍仍具有一定的修复作用。本研究拓宽了硒元素在生物医药领域的应用，同时也为临床上糖尿病难愈性创面修复提供的更多的解决方案。

02

论文第一/通讯作者简介

第一作者：曹洁琼

暨南大学临床医学博士后。主要从事活性成分（蛋白、多肽和化合物等）在组织再生修复的作用机制研究及其相关纳米生物材料的研究。

第一作者：张逸波

暨南大学临床医学博士后。主要从事活性成分（蛋白、多肽和化合物等）在组织再生修复的作用机制研究及其相关纳米生物材料的研究。

通讯作者：洪岸

暨南大学教授，博士生导师。长期从事基因工程药物及其关键技术的研究，主要围绕FGFs、FGFRs受体及其功能开展研究。主持承担国家自然科学基金重大研究计划培育项目、面上项目、国家科技部新药创制重大专项、863项目、973项目课题等各项目。已发表研究论文100多篇，授权发明专利26项，研发两个基因工程一类新药，获得省部级科技奖多项。

通讯作者：郑文杰

暨南大学教授，博士生导师。主要从事无机化学、配位化学、生物无机化学的教学科研工作。在硒杂环化合物、硒的生物有机化等领域主持并完成了多项省部级科研项目，包括国家自然科学基金和省、教育部多项基金；获厅局级奖项两项（广东省高教厅科技进步奖，广东省教育厅自然科学奖）。在国内外各类学术期刊上发表重要研究论文90余篇。在纳米硒及其制备和抗肿瘤药物先导化合物方面申请专利5项。

通讯作者：陈小佳

暨南大学研究员，博士生导师。主要从事：（1）以酪氨酸激酶受体为靶点的蛋白、多肽和小分子化合物靶向药物的研究开发；（2）酪氨酸激酶受体FGFR2及其配体FGF2在不同组织和细胞的功能与作用，以及相关的上下游分子间调控关系及其机制研究。近年来主持承担国家自然科学基金面上项目、广东省重点研发计划项目课题、广东省科技计划项目、广州市科技计划项目等多个项目。已发表研究论文40多篇，中国授权发明专利近20项，美国授权发明专利1项，PCT国际专利申请2项。

03

资助信息

该研究获国家自然科学基金(No. 81902801)、国家自然科学基金(No.8217329)、广东省生物工程医学重点实验室基金(No. 2014B030301050)、广东省“脑疾病治疗关键技术”项目基金(No.2018b030332001)、中国博士后基金(No. 2019m663375)，广州科技项目(No.20212210007)的支持，并得到了西安交通大学何旺骁教授的支持和帮助。

04

原文信息

Jieqiong Cao^#, Yibo Zhang^#, Peiguang Zhang, Zilei Zhang, Bihui Zhang, Yanxian Feng, Zhixin Li, Yiqi Yang, Qilin Meng, Liu He, Yulin Cai, Zhenyu Wang, Jie Li, Xue Chen, Hongwei Liu, An Hong*, Wenjie Zheng*, Xiaojia Chen*. 

Turning gray selenium into a nanoaccelerator of tissue regeneration by PEG modification. 

Bioactive Materials, 15, (2022) 131-144.

点击阅读原文，查看文章详情

Bioactive Materials是一本高质量英文期刊，目前已经被SCIE、PubMed Central、Scopus、Embase收录。同时本刊还入选了2019年中国科技期刊卓越行动计划--“高起点新刊”项目。

2022年Bioactive Materials 获得影响因子16.874 ，在Materials Science，Biomaterials领域排名第一。

位于《2021年中国科学院文献情报中心期刊分区表》1区，TOP期刊。

CiteScore 2021: 14.3。