补骨脂素联合转化生长因子β1诱导骨髓间充质干细胞向软骨细胞的分化  

Psoralen combined with transforming growth factor beta 1 induces the differentiation of bone marrow mesenchymal stem cells into chondrocytes

作　　者：高子茏 李婷 吕政 沈骅睿 Gao Zilong;Li Ting;Lyu Zheng;Shen Huarui(Clinical Medicine College of Chengdu University of TCM,Chengdu 610075,Sichuan Province,China;School of Pharmacy,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Affiliated Hospital of TCM,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]成都中医药大学临床医学院,四川省成都市610075 [2]西南医科大学药学院,四川省泸州市646000 [3]西南医科大学附属中医医院,四川省泸州市646000

出　　处：《中国组织工程研究》2022年第30期4884-4888,共5页Chinese Journal of Tissue Engineering Research

基　　金：四川省中医药管理局项目(2021MS463),项目负责人:沈骅睿;四川省科技厅-泸州市人民政府-泸州医学院联合项目(14JC0162),项目负责人:沈骅睿;西南医科大学--西南医科大学附属中医医院联合项目(2020XYLH-013),项目负责人:沈骅睿。

摘　　要：背景:软骨细胞受损后再生能力较差,以干细胞为基础的治疗方案已成为关节软骨修复的新型治疗方法。目的:观察补骨脂素联合转化生长因子β1诱导大鼠骨髓间充质干细胞向软骨细胞分化的作用。方法:全骨髓贴壁法分离培养SD大鼠骨髓间充质干细胞,CCK-8实验筛选出补骨脂素促进骨髓间充质干细胞增殖的有效浓度;将第3代骨髓间充质干细胞分为正常对照组(含体积分数为10%胎牛血清、100 U/mL双抗的低糖DMEM培养液)、补骨脂素组(在正常对照组基础上添加10^(-7)mol/L地塞米松、50 mg/L维生素C、10μmol/L补骨脂素)、补骨脂素联合转化生长因子β1组(在正常对照组基础上添加10μg/L转化生长因子β1、10^(-7)mol/L地塞米松、50 mg/L维生素C、10μmol/L补骨脂素)、阳性对照组(在正常对照组基础上添加10μg/L转化生长因子β1、10^(-7)mol/L地塞米松、50 mg/L维生素C)。诱导21 d后,Ⅱ型胶原免疫细胞化学染色鉴定Ⅱ型胶原表达;RT-PCR和Western blot分别检测软骨分化标记基因Ⅱ型胶原、SOX-9的mRNA和蛋白表达。结果与结论:①免疫细胞化学染色显示,正常对照组未见阳性细胞,其余3组均可见Ⅱ型胶原阳性细胞;②与正常对照组比较,补骨脂素组、联合组及阳性对照组SOX-9、Ⅱ型胶原mRNA和蛋白表达明显增加(P<0.05),其中联合组效果最佳(P<0.01),且明显优于阳性对照组及补骨脂素组(P<0.05);③结果表明,在一定的浓度范围内,补骨脂素可以促进骨髓间充质干细胞增殖;补骨脂素可以诱导骨髓间充质干细胞成软骨分化,其与转化生长因子β1联合使用具有协同促进作用。BACKGROUND:Because of the poor regeneration ability of chondrocytes after damage,the use of stem cells as a basic treatment for repairing cartilage damage has become a popular treatment method.OBJECTIVE:To observe the effect of psoralen combined with transforming growth factorβ1 on the differentiation of rat bone marrow mesenchymal stem cells into chondrocytes.METHODS:Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and cultured by marrow adherent method.The effective concentration to promote bone marrow mesenchymal stem cells proliferation was selected by CCK-8 assay.Bone marrow mesenchymal stem cells were assigned to four groups.In the normal control group,cells were treated with low-sugar DMEM with 10%fetal bovine serum and 100 U/mL double antibodies.In the psoralen group,cells were treated with low-sugar DMEM with 10%fetal bovine serum,100 U/mL double antibodies,10^(-7) mol/L dexamethasone,50 mg/L vitamin C,and 10μmol/L psoralen.In the combination group,cells were treated with low-sugar DMEM with 10%fetal bovine serum,100 U/mL double antibodies,10μg/L transforming growth factorβ1,10^(-7) mol/L dexamethasone,50 mg/L vitamin C,and 10μmol/L psoralen.In the positive control group,cells were treated with low-sugar DMEM with 10%fetal bovine serum,100 U/mL double antibodies,10μg/L transforming growth factorβ1,10^(-7) mol/L dexamethasone,and 50 mg/L vitamin C.After 21 days,the expression of type Ⅱ collagen was detected by immunocytochemistry.RT-PCR and western blot assay were utilized to detect the expression levels of differentiation marker SOX-9 and type Ⅱ collagen mRNA and protein.RESULTS AND CONCLUSION:(1)Immunocytochemical staining showed that no positive cells were found in the normal control group,while type Ⅱ collagen positive cells were found in the other three groups.(2)Compared with the normal control group,the expression of SOX9 and type Ⅱ collagen mRNA and protein increased significantly in the psoralen group,combination group and positive control group(P<0.05),and

关 键 词：补骨脂素 骨髓间充质干细胞 软骨细胞 成软骨分化 转化生长因子Β Ⅱ型胶原 SOX-9蛋白 诱导分化 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]