摘要
目的探讨小檗碱对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制。方法采用实验研究方法。配制含小檗碱终质量浓度分别为0(不含小檗碱)、1.25、2.50、5.00、10.00、20.00、40.00、80.00、160.00μg/mL的小鼠真皮成纤维细胞(MDF)正常糖完全培养基(以下简称常规培养基)。取原代MDF,分别用常规培养基和MDF高糖(30 mmol/L葡萄糖)完全培养基(以下简称高糖培养基)培养,收集第3~6代细胞进行以下实验。取采用常规培养基培养的细胞,饥饿处理12 h后更换为含不同浓度小檗碱的常规培养基培养48 h,采用细胞计数试剂盒8(CCK-8)筛选小檗碱最适作用浓度(样本数为6),用于后续细胞实验。取2种培养基培养的细胞,将采用常规培养基培养的细胞纳入正常对照组,将采用高糖培养基培养的细胞按照随机数字表法(分组方法下同)分为单纯高糖组和高糖+小檗碱组。分别用相应的培养基培养48 h后,采用CCK-8检测细胞活力,采用划痕试验和Transwell实验检测细胞24 h迁移能力,采用实时荧光定量反转录PCR法和蛋白质印迹法分别检测细胞中血小板源性生长因子(PDGF)、血管内皮生长因子(VEGF)、转化生长因子β1(TGF-β1)、基质金属蛋白酶9(MMP-9)和胱天蛋白酶3(caspase-3)的mRNA和蛋白表达水平,前述实验的样本数分别为6、3、9、3、6。取15只8周龄雄性BALB/c小鼠,构建糖尿病小鼠模型后在其背部制作全层皮肤缺损创面,分为单纯糖尿病组、糖尿病+低浓度(25μg/mL)小檗碱组和糖尿病+高浓度(75μg/mL)小檗碱组,分别作相应处理,每组5只鼠。伤后0(即刻)、3、7、14、21 d,采用ImageJ软件测量创面面积。伤后21 d,采用苏木精-伊红染色和Masson染色分别检测创面组织病理学变化及胶原生成情况,采用免疫组织化学法和实时荧光定量反转录PCR法分别检测创面组织中MMP-9、PDGF、TGF-β1、VEGF、CD31和caspase-3的蛋白和mRNA表达水平。动物实�
Objective To investigate the influences and mechanism of berberine on wound healing of full-thickness skin defects in diabetic mice.Methods The experimental research method was adopted.Mouse dermal fibroblasts(MDF)conventional-glucose complete medium(hereinafter referred to as conventional medium)were prepared with final mass concentrations of berberine of 0(no berberine),1.25,2.50,5.00,10.00,20.00,40.00,80.00,and 160.00μg/mL,respectively.Primary MDF were cultured using conventional medium and MDF high-glucose(30 mmol/L glucose)complete medium(hereinafter referred to as high-glucose medium),and the 3rd to 6th passage cells were collected for the following experiments.Cells cultured in conventional medium were taken and subjected to starvation treatment for 12 hours,and then cultured in conventional media containing different concentrations of berberine for 48 hours to screen out the optimal working concentration of berberine using the cell counting kit 8(CCK-8),the sample number was 6,and the selected optimal berberine concentration was used for subsequent cell culture experiments.Cells cultured in 2 media were taken,of which the cells cultured in conventional medium were included to the normal control group;cells cultured in high-glucose medium were divided into high-glucose alone group and high-glucose+berberine group according to the random number table(the same grouping method below).After 48 h of cultivation,cell viability was detected by CCK-8,cell migration capacity was evaluated by scratch test and Transwell assays,and mRNA and protein expression levels of platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),transforming growth factor-β1(TGF-β1),matrix metalloproteinase 9(MMP-9),and cysteine aspartic acid specific protease(caspase-3)in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting,respectively,and the sample numbers of the aforementioned experiments were 6,3,9,3,and 6,respectively.Fifteen 8-we
作者
郑力铭
刘钟元
颜鸿宇
李恒飞
张志文
Zheng Liming;Liu Zhongyuan;Yan Hongyu;Li Hengfei;Zhang Zhiwen(College of Acupuncture and Orthopedics,Hubei University of Chinese Medicine,Wuhan 430061,China;Department of Infection,Hubei Provincial Hospital of Traditional Chinese Medicine Affiliated to Hubei University of Chinese Medicine,Wuhan 430061,China;Department of Orthopedics and Traumatology,Hubei Provincial Hospital of Traditional Chinese Medicine,Affiliated to Hubei University of Chinese Medicine,Wuhan 430061,China)
出处
《中华烧伤与创面修复杂志》
CAS
CSCD
北大核心
2023年第11期1072-1082,共11页
Chinese Journal of Burns And Wounds
基金
湖北省自然科学基金项目(2022CFB406)
武汉市知识创新专项项目(2023020201010173)。
