利用CRISPR/Cas9慢病毒载体构建敲除大鼠肝星状细胞COX-2基因的细胞模型

彭敏; 曹婷; 阳学风; 易世杰; 傅念; 周克兵; 龙建武

doi:10.3969/j.issn.1001-5256.2021.02.018

利用CRISPR/Cas9慢病毒载体构建敲除大鼠肝星状细胞COX-2基因的细胞模型

DOI: 10.3969/j.issn.1001-5256.2021.02.018

南华大学附属南华医院消化内科，湖南衡阳 421002

基金项目:

国家自然科学基金 (81373465);

湖南省自然科学省市联合基金 (2016JJ5010);

湖南省卫生计生委科研课题 (A2017015);

湖南省卫生健康委科研立项课题 (20201938);

衡阳市科技局课题 (2015KJ63)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：彭敏、曹婷、阳学风负责课题设计，资料分析，撰写论文；易世杰、周克兵、龙建武参与收集数据，修改论文；傅念、阳学风负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
彭敏(1990—)，女，主要从事脂肪肝及肝硬化基础与临床方面的研究

通信作者:
曹婷，396406846@qq.com

中图分类号: R575.2
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出版历程
- 收稿日期: 2020-07-29
- 录用日期: 2020-10-12
- 出版日期: 2021-02-20

Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout

Department of Gastroenterology, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan 421002, China

摘要

摘要: 目的通过构建CRISPR/Cas9慢病毒载体，转染HSC-T6细胞，获得能稳定表达Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2^-/-细胞，为后期的功能研究提供良好的工具和手段，为临床上治疗肝纤维化提供新策略。方法设计合成COX-2基因特异性的sgRNA(COX-2-sgRNA-1、COX-2-sgRNA-2、COX-2-sgRNA-3)，并将其连接至GV371载体上，提取重组后的质粒，将其与包装质粒共同转染到293T细胞，形成慢病毒颗粒，荧光法检测病毒滴度。按照MOI值计算病毒最适用量，先将Lenti-Cas9-puro转染至HSC-T6细胞，嘌呤霉素筛选得到HSC-T6-Cas9细胞，再用Lenti-COX-2-sgRNA-EGFP转染至HSC-T6-Cas9细胞获得HSC-T6-COX-2^-/-细胞，通过Cruiser酶切检测及Western Blot等方法在基因和蛋白水平上进行敲除验证。计量资料多组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果通过测序验证COX-2-sgRNA表达载体构建成功。重组表达质粒和包装质粒共同转染到293T细胞，形成慢病毒颗粒，荧光法检测病毒滴度均在1×10⁸以上。成功构建能稳定传代的Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2^-/-细胞模型。HSC-T6-Cas9细胞中LV-Cas9-Puro mRNA相对表达量(541.93±105.76)高于CON组细胞(1.00±0.02)，差异具有统计学意义(t=12.995，P＜0.01)。通过Cruiser酶切检测及Western Blot试验，结果提示CRISPR/Cas9慢病毒表达载体能在靶点起作用，其中COX-2-sgRNA-2敲除作用最明显，并且COX-2蛋白表达水平较CON组和NC组相比显著下降(P值均＜0.05)，提示COX-2-sgRNA有活性。结论成功构建了针对COX-2靶基因的CRISPR/Cas9慢病毒载体，获得稳定的COX-2基因敲除的HSC-T6-COX-2^-/-细胞。
- 肝硬化 /
- 规律成簇间隔短回文重复序列 /
- 慢病毒感染 /
- 基因敲除技术
Abstract: Objective To obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2^-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. Methods The COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2^-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Sequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of > 1×10⁸. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2^-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P < 0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P < 0.05), suggesting that COX-2-sgRNA was active. Conclusion A CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2^-/- cells with stable COX-2 gene knockout are obtained.
- Liver Cirrhosis /
- Clustered Regularly Interspaced Short Palindromic Repeats /
- Lentivirus Infections /
- Gene Knockout Techniques

HTML全文

图 1 sgRNA表达载体阳性克隆转化子测序峰图

注：a，COX-2-sgRNA-1; b, COX-2-sgRNA-2；c，COX-2-sgRNA-3。

下载: 全尺寸图片幻灯片

图 2 不同稀释倍数COX-2-sgRNA慢病毒感染293T细胞的荧光表达情况(×100)

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图 3 puromycin筛选前后HSC-T6-Cas9细胞状态(×100)

注：左右分别是同一视野下光学显微镜图片和荧光显微镜图片。a，CON组；b，puromycin筛选前HSC-T6-Cas9细胞；c，puromycin筛选后HSC-T6-Cas9细胞。

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图 4 扩增曲线

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图 5 熔解曲线

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图 6 HSC-T6-Cas9细胞冻存后复检图片(×100)

注：左右分别是同一视野下光学显微镜图片和荧光显微镜图片。

下载: 全尺寸图片幻灯片

图 7 72 h慢病毒转染HSC-T6细胞图片(×200)

注：左右分别是同一视野下光学显微镜图和荧光显微镜图。a，CON组; b，KO1组; c，KO2组; d，KO3组; e，NC组。

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图 8 PCR产物检测图

注：a，酶切前；b，Cruiser酶切后。

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图 9 注：1, CON组；2, NC组；3，KO组。

转染72 h各组细胞COX-2蛋白表达水平

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图 10 转染96 h各组细胞中COX-2蛋白表达水平

注：1，CON组; 2，NC组; 3，KO组。

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表 1 LV-Cas9-Puro基因的引物序列

基因	引物序列(5′-3′)	扩增片段大小(bp)
LV-Cas9-Puro	上游GCTGAAAACCTATGCCCACC	133
	下游GATTGTCTTGCCGGACTGCT
GAPDH	上游TTCAACGGCACAGTCAAGG	114
	下游CTCAGCACCAGCATCACC

下载: 导出CSV

表 2 PCR引物序列

引物名称	引物序列(5′-3′)
COX-2-E1-SVF	AGTGACTGACAGGCTGTCTTGTC
COX-2-E1-SVR	CACAATGTTCCAGACTCCCTTG
COX-2-E2-SVF	GTGAAAACTGTACTACGCGTAAG
COX-2-E2-SVR	CTTCAAGGCGTTTTGAAAAGGTC
COX-2-E3-SVF	AGTGACTGACAGGCTGTCTTGTC
COX-2-E3-SVR	CACAATGTTCCAGACTCCCTTG

下载: 导出CSV

表 3 转染72 h、96 h各组COX-2蛋白质相对表达情况

组别	72 h COX-2	96 h COX-2
CON组	0.57±0.02	1.14±0.21
KO组	0.11±0.02¹⁾	0.09±0.03¹⁾
NC组	0.55±0.02²⁾	0.49±0.15²⁾
F值	586.926	143.643
P值	＜0.01	＜0.01
注:与CON组比较，1)P＜0.01；与KO组比较，2)P＜0.01。

下载: 导出CSV

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