中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (19): 2978-2984.doi: 10.12307/2022.374

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

基于CRISPR/Cas9技术编辑RAB39B基因对骨髓间充质干细胞软骨分化的影响

马笃军1，2，彭力平1，周紫琼1，赵  静2，朱厚均2，蒋顺琬1，钟  静2，佘锐豪2   

  1. 1广州中医药大学第四临床医学院，广东省深圳市   518033；2广州中医药大学，广东省广州市   510006
  • 收稿日期:2021-06-28 修回日期:2021-08-04 接受日期:2021-08-31 出版日期:2022-07-08 发布日期:2021-12-28
  • 通讯作者: 马笃军，硕士，副主任医师，硕士研究生导师，广州中医药大学第四临床医学院，广东省深圳市 518033；广州中医药大学，广东省广州市 510006
  • 作者简介:马笃军，男，1985年生，2013年湖南中医药大学毕业，硕士，副主任医师，硕士研究生导师，主要从事中医药联合干细胞对关节软骨修复的研究。
  • 基金资助:
    国家自然科学基金项目(81804124)，项目负责人：马笃军；广东省自然科学基金(2018A0303130138)，项目负责人：马笃军

Effect of RAB39B gene based on CRISPR/Cas9 technology on cartilage differentiation of bone marrow mesenchymal stem cells

Ma Dujun1, 2, Peng Liping1, Zhou Ziqiong1, Zhao Jing2, Zhu Houjun2, Jiang Shunwan1, Zhong Jing2, She Ruihao2   

  1. 1Fourth Clinical Medical College of Guangzhou University of Chinese medicine, Shenzhen 518033, Guangdong Province, China; 2Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China
  • Received:2021-06-28 Revised:2021-08-04 Accepted:2021-08-31 Online:2022-07-08 Published:2021-12-28
  • Contact: Ma Dujun, Master, Associate chief physician, Master’s supervisor, Fourth Clinical Medical College of Guangzhou University of Chinese medicine, Shenzhen 518033, Guangdong Province, China; Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China
  • About author:Ma Dujun, Master, Associate chief physician, Master’s supervisor, Fourth Clinical Medical College of Guangzhou University of Chinese medicine, Shenzhen 518033, Guangdong Province, China; Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81804124 (to MDJ); the Natural Science Foundation of Guangdong Province, China, No. 2018A0303130138 (to MDJ)

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摘要：

文题释义：
骨髓间充质干细胞：也称为骨髓基质成纤维细胞，是一类起源于中胚层的成体干细胞，具有自我更新及多向分化潜能，可根据所处的微环境不同，进行细胞迁移、定植、增殖与分化，其中分化潜能属于目前的研究热点，可分化为多种间质组织，如脂肪、骨骼、软骨、骨髓造血组织等。
CRISPR/Cas9基因编辑技术：是一种基因治疗方法，是继TALEN基因编辑技术之后又一重大突破，该技术通过RNA指导Cas9核酸酶对靶向基因进行特定DNA剪接编辑，相较其他基因编辑效率更高，Cas9系统的载体构建与使用也更加便捷，并已应用在各物种及疾病模型中，是目前广泛使用的基因编辑技术。

背景：基于前期研究，课题组通过蛋白组学和高通量测序筛选出RAB39B基因与骨髓间充质干细胞的增殖、软骨分化可能存在相关性。
目的：探讨利用CRISPR/Cas9技术编辑敲除RAB39B基因对骨髓间充质干细胞软骨分化的影响。
方法：利用 CRISPR/Cas9 慢病毒感染方法，构建敲低RAB39B基因稳转细胞系，将兔骨髓间充质干细胞分为正常对照组、空载对照组、RAB39B基因敲除组，通过Western blot鉴定CRISPR/Cas9系统对RAB39B的敲除效果，CCK-8法检测骨髓间充质干细胞增殖活性，流式细胞仪检测骨髓间充质干细胞凋亡率，qRT-PCR检测骨髓间充质干细胞成软骨分化COLⅡ、COLⅩ基因mRNA表达，Western blot检测骨髓间充质干细胞成软骨分化相关通路RHOA、LIMK、Sox9蛋白表达。
结果与结论：①敲除RAB39B基因质粒的慢病毒感染兔骨髓间充质干细胞48-96 h后荧光显著表达；Western blot检测验证RAB39B基因敲除后骨髓间充质干细胞中RAB39B蛋白表达显著降低(P < 0.01)；②与正常对照组、空载对照组比较，RAB39B基因敲除组细胞增殖活性显著降低(P < 0.01)；细胞凋亡率增加(P < 0.01)；COLⅡ、COLⅩ mRNA表达显著降低(P < 0.01)；RHOA、LIMK、Sox9蛋白表达显著降低(P < 0.01)；③结果表明，CRISPR/Cas9系统编辑敲低RAB39B基因后能够抑制骨髓间充质干细胞增殖，同时促进细胞凋亡，降低其软骨细胞分化能力，说明RAB39B基因可能对骨髓间充质干细胞增殖、软骨分化能力产生影响，其具体作用机制有待进一步研究。

https://orcid.org/0000-0002-8050-2849 (马笃军) 

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: 基因编辑, RAB39B基因, 骨髓间充质干细胞, 软骨细胞, 软骨分化

Abstract: BACKGROUND: Based on previous research, the research team used proteomics and high-throughput sequencing to screen the correlation between the RAB39B gene and the proliferation and cartilage differentiation of bone marrow mesenchymal stem cells.  
OBJECTIVE: To investigate the effect of RAB39B gene knockout by CRISPR/Cas9 gene editing technology on cartilage differentiation of bone marrow mesenchymal stem cells.
METHODS:  CRISPR/Cas9 lentivirus infection method was used to construct stable transfection cell line of RAB39B gene knockdown. Rabbit bone marrow mesenchymal stem cells were divided into normal control group, no-load control group, and RAB39B knockout group. The knockout effect of CRISPR/Cas9 system on RAB39B was evaluated by western blot assay. The proliferation activity of bone marrow mesenchymal stem cells was detected by CCK-8 assay. The apoptosis rate of bone marrow mesenchymal stem cells was detected by flow cytometry. The mRNA expression of COLII and COLX was detected by qRT-PCR. The expression levels of RHOA, LIMK, and Sox9 proteins in chondrogenic differentiation related pathways of bone marrow mesenchymal stem cells were detected by western blot assay.  
RESULTS AND CONCLUSION: (1) Lentivirus carrying knockout RAB39B gene plasmid was found to be highly expressed in rabbit bone marrow mesenchymal stem cells 48-96 hours after infection. Western blot assay showed that the expression of RAB39B protein in bone marrow mesenchymal stem cells was significantly decreased after RAB39B knockout (P < 0.01). (2) Compared with the normal control group and the empty control group, the cell proliferation activity of the RAB39B gene knockout group was significantly reduced (P < 0.01); the apoptosis rate was increased (P < 0.01); the expression of COLII and COLX mRNA was significantly reduced (P < 0.01); RHOA, LIMK, and Sox9 protein expression was significantly decreased (P < 0.01). (3) These findings indicate that RAB39B gene knockout by CRISPR/Cas9 system can inhibit the proliferation of bone marrow mesenchymal stem cells, promote cell apoptosis, and reduce the chondrocyte differentiation ability, which indicates that RAB39B gene may affect the proliferation and chondrocyte differentiation ability of bone marrow mesenchymal stem cells, and its specific mechanism needs further study.

Key words: gene editing, RAB39B gene, bone marrow mesenchymal stem cells, chondrocytes, cartilage differentiation

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