利用CRISPR/Cas9靶向敲除Piezo1基因对小鼠间充质干细胞成骨分化的影响研究

doi:10.3969/j.issn.1674-8115.2023.09.002

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (9): 1080-1088.doi: 10.3969/j.issn.1674-8115.2023.09.002

• 论著 · 基础研究 • 上一篇

利用CRISPR/Cas9靶向敲除Piezo1基因对小鼠间充质干细胞成骨分化的影响研究

高昕¹(), 杨屹羚¹(), 黄湘如¹, 代庆刚², 江凌勇¹()

^1.上海交通大学医学院附属第九人民医院口腔颅颌面科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^2.上海交通大学医学院附属第九人民医院口腔第二门诊部，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011

收稿日期:2023-03-21 接受日期:2023-09-08 出版日期:2023-09-28 发布日期:2023-09-28
通讯作者: 江凌勇 E-mail:gxgaoxin@sjtu.edu.cn;yangyiling2017@foxmail.com;jianglingyong@sjtu.edu.cn
作者简介:高昕（1996—），女，硕士生；电子信箱：gxgaoxin@sjtu.edu.cn
杨屹羚（1994—），女，硕士生；电子信箱：yangyiling2017@foxmail.com。*为并列第一作者。
基金资助:
国家自然科学基金(82071083);上海市自然科学基金(21ZR1436900);上海市优秀学术/技术带头人计划(20XD1422300);上海交通大学医学院附属第九人民医院交叉研究基金(JYJC202116);上海申康医院发展中心临床创新三年行动计划(SHDC2020CR4084);上海交通大学医学院附属第九人民医院生物样本库项目(YBKB201919);上海交通大学医学院附属第九人民医院原创探索项目(JYYC003);上海交通大学医学院生物材料与再生医学研究院联合攻关项目(2022LHB02);上海交通大学医学院“双百人”项目(20221809)

Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9

GAO Xin¹(), YANG Yiling¹(), HUANG Xiangru¹, DAI Qinggang², JIANG Lingyong¹()

^1.Department of Oral and Maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
^2.The 2nd Dental Centre, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200011, China

Received:2023-03-21 Accepted:2023-09-08 Online:2023-09-28 Published:2023-09-28
Contact: JIANG Lingyong E-mail:gxgaoxin@sjtu.edu.cn;yangyiling2017@foxmail.com;jianglingyong@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(82071083);Natural Science Foundation of Shanghai(21ZR1436900);Program of Shanghai Academic/Technology Research Leader(20XD1422300);Cross-disciplinary Research Fund of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202116);Clinical Research Plan of Shanghai Hospital Development Center(SHDC2020CR4084);Project of Biobank of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(YBKB201919);Original Exploration Project of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYYC003);Biomaterials and Regenerative Medicine Institute Cooperative Research Project of Shanghai Jiao Tong University School of Medicine(2022LHB02);"Two-hundred Talents" Program of Shanghai Jiao Tong University School of Medicine(20221809)

摘要/Abstract

摘要：

目的·应用CRISPR/Cas9系统对小鼠间充质干细胞C3H10T1/2进行靶向Piezo1基因稳定敲除，探究Piezo1对间充质干细胞成骨分化能力的影响。方法·根据CRISPR/Cas9原理，设计2条靶向Piezo1基因的单链指导RNA（single guide RNA，sgRNA），利用Lenti-Cas9-GFP和Lenti-U6-sgRNA-mCherry载体分别构建表达Cas9的慢病毒和表达sgRNA的慢病毒。将2种慢病毒感染C3H10T1/2细胞，利用流式细胞分选技术对GFP和mCherry阳性的细胞进行筛选；挑取单克隆细胞，经PCR扩增及琼脂糖凝胶电泳验证，最终得到Piezo1基因片段敲除的单克隆细胞系。序列测定、实时荧光定量PCR（quantitative realtime PCR，qPCR）及细胞免疫荧光技术对构建的Piezo1基因敲除细胞（Piezol knockout C3H10T1/2，CPK）进行敲除效率验证。CCK-8实验检测敲除Piezo1对细胞增殖的影响；对构建成功的Piezo1基因敲除细胞进行体外成骨诱导分化，并进行碱性磷酸酶（alkaline phosphatase，ALP）染色及茜素红染色，利用qPCR探究敲除Piezo1对细胞的成骨相关基因mRNA水平的影响。结果·琼脂糖凝胶电泳结果显示，敲除Piezo1的单克隆细胞菌液通过PCR扩增后产物出现阳性克隆。单克隆细胞的测序结果显示，敲除Piezo1的单克隆细胞的Piezo1基因在第4外显子中提前形成终止密码子TGA，无法正确翻译蛋白；qPCR验证了CPK中Piezo1基因在mRNA水平被抑制；免疫荧光显示CPK中Piezo1基因的敲除效率较高，基本阻碍Piezo1在细胞中的表达。CCK-8实验显示敲除Piezo1后细胞增殖能力下降（P<0.05）；ALP染色及茜素红染色结果显示敲除Piezo1后细胞成骨能力降低（P<0.05），且CPK中成骨相关基因如Ⅰ型胶原蛋白A1（α 1 type Ⅰ collagen，Col1a1）、Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、成骨细胞特异性转录因子（osterix，Osx）以及碱性磷酸酶（alkaline phosphatase，Alp）的mRNA表达水平降低（均P<0.05）。结论·利用CRISPR/Cs9基因编辑系统成功构建靶向敲除Piezo1基因的C3H10T1/2细胞系。敲除Piezo1能抑制小鼠间充质干细胞C3H10T1/2的成骨分化。

关键词: Piezo1, CRISPR/Cas9, 间充质干细胞, 成骨分化

Abstract:

Objective ·To investigate the effect of Piezo1 on osteogenic differentiation of mouse mesenchymal stem cells C3H10T1/2 cell line based on CRISPR/Cas9 system that can achieve stable gene knockout. Methods ·According to the principle of CRISPR/Cas9 target design principle, two single guide RNAs (sgRNAs) were designed to construct lentivirus expressing Cas9 and lentivirus expressing sgRNA by using Lenti-Cas9-GFP and Lenti-U6-sgRNA-mCherry vectors. After the C3H101/2 cells were transfected with two types of lentiviruses, flow cytometry was used to screen mCherry- and GFP-positive cells. The monoclonal cells were selected, and amplified by PCR and agarose gel electrophoresis, and finally the monoclonal cell line with Piezo1 gene fragment knocked out was obtained. Sequencing, quantitative realtime PCR (qPCR) and immunofluorescence were performed to verify the the knockout efficiency of the constructed Piezol knockout C3H10T1/2 cells (CPK). CCK-8 assay was used to detect the effect of knocking out Piezo1 on cell proliferation; in vitro osteogenic induction differentiation was performed on successfully constructed Piezo1 gene knockout cells, and alkaline phosphatase (ALP) staining and alizarin red staining were used to investigate the effect of Piezo1 on osteogenic ability. Results ·Positive clone was obtained in bacterial fluid of monoclonal cell lines with Piezol knocked out after PCR amplification and agarose gel electrophoresis. Sequencing analysis showed that a stop condon TGA was produced in exon 4 of Piezo1 gene in advance, so that the protein could not be translated correctly. qPCR verified that Piezo1 gene in CPK was inhibited at mRNA level; Immunofluorescence showed that the knockout efficiency of Piezo1 gene in CPK was high, which basically hindered the expression of Piezo1 in cells. CCK-8 assay showed that the cell proliferation ability decreased after knocking out Piezo1 (P<0.05); The results of ALP staining and alizarin red staining showed that the osteogenic ability of cells decreased after knocking out Piezo1(P<0.05). The mRNA expression levels of osteogenetic-related genes such as α 1 type Ⅰ collagen (Col1a1),Runt-related transcription factor 2 (Runx-2),osterix (Osx) and alkaline phosphatase (Alp)in CPK decreased significantly (all P<0.05). Conclusion ·Piezo1 knockout C3H10T1/2 cells based on CRISPR/Cas9 system is constructed successfully and the osteogenic activity of stable Piezo1 knockout cell line is hindered significantly.

Key words: Piezo1, CRISPR/Cas9, mesenchymal stem cell, osteogenesis

中图分类号:

Q291

高昕, 杨屹羚, 黄湘如, 代庆刚, 江凌勇. 利用CRISPR/Cas9靶向敲除Piezo1基因对小鼠间充质干细胞成骨分化的影响研究[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1080-1088.

GAO Xin, YANG Yiling, HUANG Xiangru, DAI Qinggang, JIANG Lingyong. Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1080-1088.

图/表 6

表1 sgRNA寡核苷酸序列及引物序列

Tab 1 sgRNA oligonucleotide sequences and primers

Name	Sequences (5′→3′)
sgRNA-1	GTTGAGACACCTTCACCCAA
sgRNA-5	TCTGCCGGGCTTTCCTCGTG
Piezo1 forward primer	ATCAGCATTCCTGAATCACGGATG
Piezo1 reverse primer	CTAGGGCTCTGTTACGCAGAG
Sequencing primer	GGAAAGAATAGTAGACATAATAGC

表2 qPCR引物序列信息

Tab 2 Primer sequences for qPCR

Gene	Forward sequence (5′→3′)	Reverse sequence (5′→3′)
HPRT	GTTAAGCAGTACAGCCCCAAA	AGGGCATATCCAACAACAAACTT
Runx2	CCTCCAGCATCCCTTTCTT	CCTCCAGCATCCCTTTCTT
Osx	CCTTCCCTCACTCATTTCCTGG	TGTTGCCTGGACCTGGTGAGAT
Alp	CGGGACTGGTACTCGGATAA	ATTCCACGTCGGTTCTGTTC
Col1a1	GCTCCTCTTAGGGGCCACT	CCACGTCTCACCATTGGGG

图1 Lenti-U6-sgRNA1-U6-sgRNA5-mCherry重组质粒测序鉴定结果

Fig 1 Sequencing identification of Lenti-U6-sgRNA1-U6-sgRNA5-mCherry recombinant plasmid

图2 Piezo1 基因敲除细胞系的成功构建Note： A. PCR identification of CPK. M—Marker; Lane 1—negative control; Lane 2—wild cell; Lane 3—CPK. B. Base sequence of exon 4 of CON group cells and CPK group cells. C. qPCR results of Piezo1 expression. D. Immunofluorescence staining in CON group and CPK group.

Fig 2 Successful construction of Piezo1-knockout cell line

图3 CCK-8检测 Piezo1 敲除细胞系的增殖能力

Fig 3 Cell proliferation of Piezo1 knockout cell line detected by CCK-8

图4 敲除 Piezo1 基因对C3H10T1/2细胞成骨分化的影响Note： A. ALP staining of CON group and CPK group after osteogenic differentiation induction in vitro. B. Alizarin red staining of CON group and CPK group after osteogenic differentiation induction in vitro. C. qPCR results of osteogenetic-related genes in CON group and CPK group.

Fig 4 Effect of Piezo1 knockout on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2

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利用CRISPR/Cas9靶向敲除Piezo1基因对小鼠间充质干细胞成骨分化的影响研究

Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9

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