摘要
目的探讨构建自交联重组胶原蛋白水凝胶的可行性及其对紫外线诱导的光老化模型修复作用。方法2023年3—12月,于西北大学陕西省可降解生物医学材料重点实验室,使用不同浓度的重组胶原蛋白通过自交联制备重组胶原蛋白水凝胶,表征了水凝胶材料的物理机械性能,用MTT法、溶血试验检测水凝胶生物相容性;建立人皮肤成纤维细胞光老化模型,通过RT-qPCR检测炎症因子及ECM生成、降解相关基因表达量探究水凝胶分子水平改善光老化效果。建立紫外照射小鼠光老化模型,通过大体观察以评估水凝胶改善光老化效果,通过HE染色观察水凝胶的组织兼容性和降解性,通过RT-qPCR及免疫荧光染色探讨水凝胶作用机制。结果4 mg/ml、20 mg/ml和40 mg/ml的GEL-Ⅰ水凝胶均表现出良好的物理机械性能。生物相容性研究显示不同浓度的水凝胶细胞存活率>100%,具有促细胞增殖作用,且与重组Ⅰ型胶原蛋白的浓度呈正相关;3组水凝胶溶血率均<5%。qPCR显示HSF光老化模型中,与模型组相比,3个浓度的水凝胶组细胞中Bcl-2和TGF-β1显著升高(P<0.05),Caspase-3和Caspase-9显著下调(P<0.05),细胞凋亡程度显著降低。MMP-9基因水平下调(P<0.05),Col-Ⅰ、Col-Ⅲ和Vimentin基因显著上调(P<0.05)。光老化小鼠模型中,与模型组相比,水凝胶组的小鼠皮肤形成的皱纹更少。HE染色显示胶原蛋白纤维表达量丰富且紧密、角质层更加光滑;与模型组相比,GEL-Ⅰ能更好地降低活性氧含量,提高SOD活性,降低MDA表达(P<0.05),且呈浓度依赖性趋势,40 mg/ml组与对照组相比活性氧、SOD水平差异有统计学意义(P<0.05);天狼猩红染色显示水凝胶组组织中胶原蛋白含量显著增加(P<0.05),40 mg/ml组与对照组相比胶原含量显著增加(P<0.05);免疫荧光染色显示肿瘤坏死因子α、白细胞介素6、基质金属蛋白酶-9含量呈显著下降(P<0.05),且呈浓度依赖性。结论重组Ⅰ型胶原水凝胶可改善氧化应激和炎症微环境,降低皮肤组织活性氧,下调促HSF凋亡细胞因子和基质金属肽酶9,促进皮肤光老化细胞的功能恢复,提升细胞外基质成分表达,可有效改善光损伤皮肤。
Objective To investigate the feasibility of constructing self-crosslinked recombinant type Ⅰ collagen hydrogels and their role in repairing UV-induced photoaging models.Methods Recombinant collagen hydrogels were prepared by self-cross-linking using different concentrations of recombinant collagen at the Key Laboratory of Degradable Biomedical Materials of Shaanxi Province,Northwestern University,from July 2021 to December 2022,and the pore size distribution,swelling properties,rheological properties and pushing force of the hydrogels were examined by scanning electron microscopy(SEM),swelling test,rheometer,and universal material testing machine.The hydrogel biocompatibility was analyzed by MTT method and haemolysis test.A photoaging model of human skin fibroblast cells was established,and and the expression levels of inflammatory factors and genes related to ECM generation and degradation were detected by RT-qPCR.A UV-irradiated mice photoaging model was established to evaluate the effect of hydrogel on photoaging by gross observation,and the histocompatibility and degradation of hydrogel were observed by HE staining,and the mechanism of hydrogel action was investigated by RT-qPCR and immunofluorescence staining.Results The GEL-Ⅰhydrogels at 4 g/ml,20 mg/ml and 40 mg/ml showed good physico-mechanical properties.Biocompatibility studies showed that the cell survival rate of different concentrations of hydrogels was greater than 100%,with pro-cell proliferative effects,and positively correlated with the concentration of recombinant collagen type Ⅰ.The hemolysis rate of the three groups of hydrogels was less than 5%.qPCR showed that in the HSF photo-aging model,compared with the model group,the three concentrations of hydrogel group cells in the Bcl-2 and TGF-β1 were significantly elevated(P<0.005),Caspase-3 and Caspase-9(P<0.05),and Caspase-3 and Caspase-9(P<0.05),respectively.Caspase-3 and Caspase-9 were significantly down-regulated(P<0.05),and the degree of apoptosis was significantly reduced;the level of MMP-9 gene was down-regulated(P<0.05),and Col-Ⅰ,Col-Ⅲ,and vimentin genes were significantly up-regulated(P<0.05).In the photoaging mouse model,less wrinkles were formed in the skin of mice in the hydrogel group compared with the model group;HE staining showed abundant and tight expression of collagen fibers and smoother stratum corneum;compared with the model group,GEL-Ⅰ could better reduce the ROS content,increase the SOD activity,and reduce the expression of MDA(P<0.05)with a concentration-dependent manner,and the ROS,SOD,and MDA levels were significantly up-regulated in the 40 mg/ml group compared with the control group(P<0.05),while the ROS and SOD levels of the 40 mg/ml group compared with the control group were significantly up-regulated.ROS and SOD levels were significantly different compared with the control group(P<0.05);Wolf scarlet staining showed that the collagen content in the tissue of the hydrogel group was significantly increased(P<0.05),and the collagen content in the 40 mg/ml group was significantly increased compared with the control group(P<0.05);immunofluorescent staining showed that the contents of TNF-α,IL-6 and MMP-9 decreased significantly(P<0.05)in a concentration-dependent manner.Conclusions The recombinant type Ⅰ collagen hydrogel could improve the oxidative stress and inflammatory microenvironment,reduce ROS in skin tissue,down-regulate HSF necrosis cell factors and matrix metallopeptidase 9,promote the functional recovery of skin photoaging cells and enhance the expression of ECM components,which can effectively improve photodamaged skin.
作者
黄长瑾
雷桓
唐晓军
Huang Changjin;Lei Huan;Tang Xiaojun(Department of Craniomaxillofacial Surgery,Plastic Surgery Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100144,China;Shaanxi Key Laboratory of Degradable Biomedical Materials,School of Chemical Engineering,Northwest University/Shanxi R&D Center of Biomaterials and Fermentation Engineering,School of Chemical Engineering,Northwest University/Biotech and Biomed Research Institute,Northwest University,Xi′an 710069,China)
出处
《中华医学美学美容杂志》
2024年第2期163-172,共10页
Chinese Journal of Medical Aesthetics and Cosmetology
基金
国家重点研发计划(2019YFA0905200)
中国医学科学院整形外科医院重大项目基金资助(Z2018006)。
关键词
光
皮肤衰老
皮肤光老化修复
重组Ⅰ型胶原
氧化应激
细胞外基质
Light
Skin aging
Recombinant type Ⅰ collagen
Reduce inflammation
Skin photoaging repair
Matrix metallopeptidase 9
Extracellular matrix
