中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (30): 4884-4888.doi: 10.12307/2022.770

• 干细胞因子及调控因子 stem cell factors and regulatory factors • 上一篇    下一篇

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

高子茏1，李  婷2，吕  政1，沈骅睿3   

  1. 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
  • 收稿日期:2021-05-21 接受日期:2021-07-23 出版日期:2022-10-28 发布日期:2022-03-29
  • 通讯作者: 沈骅睿，副教授，西南医科大学附属中医医院，四川省泸州市 646000
  • 作者简介:高子茏，男，1991年生，四川省成都市人，汉族，2020年成都中医药大学毕业，硕士，主要从事中药诱导干细胞分化的研究。
  • 基金资助:
    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

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

Gao Zilong1, Li Ting2, Lyu Zheng1, Shen Huarui3   

  1. 1Clinical Medicine College of Chengdu University of TCM, Chengdu 610075, Sichuan Province, China; 2School of Pharmacy, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 3Affiliated Hospital of TCM, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2021-05-21 Accepted:2021-07-23 Online:2022-10-28 Published:2022-03-29
  • Contact: Shen Huarui, Associate professor, Affiliated Hospital of TCM, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Gao Zilong, Master, Clinical Medicine College of Chengdu University of TCM, Chengdu 610075, Sichuan Province, China
  • Supported by:
    Project of Sichuan Administration of Traditional Chinese Medicine, No. 2021MS463 (to SHR); Joint Project of Sichuan Science and Technology Department-Luzhou Municipal People's Government-Luzhou Medical College, No. 14JC0162 (to SHR); Joint Project of Southwest Medical University—Affiliated Hospital of TCM, Southwest Medical University, No. 2020XYLH-013 (to SHR)

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摘要：

文题释义：
转移生长因子β：是软骨发育过程中重要的生长因子，它能诱导间充质干细胞向多种细胞分化，如心肌细胞、软骨细胞及神经细胞等。
Ⅱ型胶原：是细胞外基质的主要组成部分，是软骨细胞的特征性标志。经过Ⅱ型胶原蛋白免疫组化染色后细胞胞浆呈棕红色或棕黄色，胞浆异染深浅不一，染色越深，代表Ⅱ型胶原蛋白的表达越高。

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、10 μmol/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10 μg/L转化生长因子β1、10-7 mol/L地塞米松、50 mg/L维生素C、10 μmol/L补骨脂素)、阳性对照组(在正常对照组基础上添加10 μg/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。

https://orcid.org/0000-0001-5202-167X (高子茏) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

Abstract: 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 factor β1 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 10 μmol/L psoralen. In the combination group, cells were treated with low-sugar DMEM with 10% fetal bovine serum, 100 U/mL double antibodies, 10 μg/L transforming growth factor β1, 10-7 mol/L dexamethasone, 50 mg/L vitamin C, and 10 μmol/L psoralen. In the positive control group, cells were treated with low-sugar DMEM with 10% fetal bovine serum, 100 U/mL double antibodies, 10 μg/L transforming growth factor β1, 10-7 mol/L dexamethasone, and 50 mg/L vitamin C. After 21 days, the expression of type ll 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 ll collagen mRNA and protein.  
RESULTS AND CONCLUSION: (1) Immunocytochemical staining showed that no positive cells were found in the normal control group, while type ll 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 the combination group had the best effect (P < 0.01), and it was significantly better than that in the positive control group and the psoralen group (P < 0.05). (3) The results show that within a certain concentration range, psoralen can promote the proliferation of bone marrow mesenchymal stem cells. Psoralen can induce bone marrow mesenchymal stem cells to differentiate into cartilage, and its combined use with transforming growth factor β1 has a synergistic promoting effect.

Key words: psoralen, bone marrow mesenchymal stem cells, chondrocytes, chondrogenic differentiation, transforming growth factor β, type ll collagen, SOX9 protein, induced differentiation

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