التفاصيل البيبلوغرافية
| العنوان: |
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. (Chinese) |
| Alternate Title: |
Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9. (English) |
| المؤلفون: |
高昕, 杨屹羚, 黄湘如, 代庆刚, 江凌勇 |
| المصدر: |
Journal of Shanghai Jiaotong University (Medical Science); Sep2023, Vol. 43 Issue 9, p1080-1088, 9p |
| Abstract (English): |
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. [ABSTRACT FROM AUTHOR] |
| Abstract (Chinese): |
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。. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
Complementary Index |