RCAS-TVA介导的少突胶质谱系细胞在体基因靶向调控研究

doi:10.3969/j.issn.1674-8115.2026.06.011

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (6): 786-794.doi: 10.3969/j.issn.1674-8115.2026.06.011

• 论著 · 技术与方法 • 上一篇

RCAS-TVA介导的少突胶质谱系细胞在体基因靶向调控研究

张潇月¹^,², 沈希¹^,², 李南南¹^,², 洪小琦¹^,²(), 童小萍¹^,²()

^1.上海交通大学医学院附属松江医院妇产科，上海市情绪与情感障碍重点实验室，松江研究院，上海 201600
^2.上海交通大学基础医学院解剖学与生理学系，上海 201318

收稿日期:2026-02-12 接受日期:2026-03-12 出版日期:2026-06-28 发布日期:2026-06-29
通讯作者: 童小萍，研究员，博士；电子信箱：xtong@shsmu.edu.cn
洪小琦，助理研究员，博士；电子信箱：xhong@shsmu.edu.cn。
基金资助:
科技创新2030重大项目(2022ZD0204700);国家自然科学基金(82271466;32471063)

RCAS-TVA-mediated genetic targeting of oligodendrocyte lineage cells in vivo

Zhang Xiaoyue¹^,², Shen Xi¹^,², Li Nannan¹^,², Hong Xiaoqi¹^,²(), Tong Xiaoping¹^,²()

^1.Department of Obstetrics and Gynecology, Songjiang Research Institute, Shanghai Key Laboratory of Emotions and Affective Disorders, Songjiang Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201600, China
^2.Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai 201318, China

Received:2026-02-12 Accepted:2026-03-12 Online:2026-06-28 Published:2026-06-29
Contact: Tong Xiaoping, E-mail: xtong@shsmu.edu.cn
Hong Xiaoqi, E-mail: xhong@shsmu.edu.cn.
Supported by:
Major Project of Science and Technology Innovation 2030(2022ZD0204700);National Natural Science Foundation of China(82271466;32471063)

摘要/Abstract

摘要：

目的·研发一种基于有复制能力的禽肉瘤/白血病病毒（replication-competent avian sarcoma/leukosis virus，RCAS）‒禽肉瘤/白血病病毒A亚群受体（tumor virus receptor A，TVA）基因递送系统的少突胶质谱系细胞体内特异性基因操作技术，以实现对该类细胞的高效标记与靶向调控。方法·以RCAS-EGFP（增强型绿色荧光蛋白，enhanced green fluorescent protein）为载体，分别插入靶向G蛋白偶联受体17（G protein-coupled receptor 17，GPR17）的shRNA序列及Scramble对照序列。病毒包装完成后，使用荧光聚焦单位法测量病毒滴度。利用Olig2-TVA-IRES-Cre转基因小鼠模型，将包装好的RCAS-shRNA（Scramble）-EGFP（对照组）与RCAS-shRNA（GPR17）-EGFP（敲低组）病毒颗粒，通过立体定位注射技术，分别注射到小鼠的海马或者下丘脑弓状核脑区。利用免疫荧光技术，对感染细胞进行少突胶质细胞转录因子2（oligodendrocyte transcription factor 2，Olig2）、神经元标记物神经元核抗原（neuronal nuclei，NeuN）和星形胶质细胞标记物胶质细胞原纤维酸性蛋白（glial fibrillary acidic protein，GFAP）染色，实现对病毒转染的特异性验证。在对照组和敲低组中对目标蛋白GPR17进行免疫荧光染色，验证病毒的干扰效率。通过Western blotting检测敲低前后下丘脑弓状核脑区GPR17蛋白的表达水平。对少突胶质前体细胞标记物血小板衍生生长因子受体α（platelet-derived growth factor receptor α，PDGFRα）和少突胶质细胞标记物CC1进行免疫荧光染色，检测GPR17敲低后对细胞分化的影响。结果·病毒的滴度为5×10⁷ PFU/mL。免疫荧光结果显示，在海马与弓状核脑区，被GFP标记的细胞中与Olig2共标的比例分别高达77.99%和75.02%，并且GFP与NeuN以及GFP与GFAP几乎未见共定位。在海马脑区，对照组病毒感染的细胞中GPR17阳性率为71.85%，而敲低组则显著降至34.34%（P<0.001），GPR17的表达明显减少。在弓状核脑区中，对照组与敲低组GPR17⁺/GFP⁺双阳性细胞占比分别为78.12%和31.72%。Western blotting结果显示，病毒敲减后弓状核脑区GPR17蛋白表达水平显著降低。在海马脑区，GPR17敲低后CC1⁺细胞数量呈增多趋势，但差异无统计学意义，PDGFRα⁺细胞数量显著减少（P<0.001）。在弓状核脑区，GPR17敲低后CC1⁺细胞数量显著增多，PDGFRα⁺细胞数量显著减少（均P<0.001）。结论·利用RCAS‒TVA基因递送系统，能够高效实现在体少突胶质谱系细胞的标记及目的基因的靶向操控。

关键词: 少突胶质谱系细胞, 有复制能力的禽肉瘤/白血病病毒, 禽肉瘤/白血病病毒A亚群受体, G蛋白偶联受体17

Abstract:

Objective ·To develop an in vivo genetic manipulation strategy based on the replication-competent avian sarcoma/leukosis virus (RCAS)‒tumor virus receptor A (TVA) gene delivery system to specifically target oligodendrocyte lineage cells (OLCs), enabling efficient labeling and targeted modulation of these cells. Methods ·Using RCAS-EGFP (enhanced green fluorescent protein) as the backbone vector, shRNA sequences targeting G protein-coupled receptor 17 (GPR17) or a non-targeting Scramble control sequence were inserted. Following viral packaging, viral titers were determined using the fluorescent focus units (FFU). In Olig2-TVA-IRES-Cre transgenic mice, which allow specific RCAS infection of OLCs, stereotactic injections of either the RCAS-shRNA(Scramble)-EGFP (control group) or the RCAS-shRNA(GPR17)-EGFP (knockdown group) were performed into the hippocampus (HPC) or the arcuate nucleus (ARC), respectively. Immunofluorescence staining was conducted for the oligodendrocyte lineage transcription factor 2 (Olig2), the neuronal marker neuronal nuclei (NeuN), and the astrocytic marker glial fibrillary acidic protein (GFAP) to validate viral transduction specificity. To validate knockdown efficiency of the virus, immunofluorescence staining for the target protein GPR17 was performed in both the control group and the knockdown group. The expression levels of GPR17 protein in the ARC were measured by Western blotting before and after knockdown. To evaluate the impact of GPR17 knockdown on cellular differentiation, immunofluorescence staining for the oligodendrocyte precursor cell marker platelet-derived growth factor receptor α (PDGFRα) and the mature oligodendrocyte marker CC1 was performed in both groups. Results ·The packaged viral titer was 5×10⁷ PFU/mL. Immunofluorescence analysis revealed high specificity of infection, with 77.99% and 75.02% of GFP-positive cells co-localizing with Olig2 in the HPC and ARC, respectively. Additionally, co-localization of GFP with NeuN or GFAP was scarcely detected. The knockdown efficiency was confirmed by a significant reduction in GPR17-positive cells among GFP-positive populations. In the HPC, the percentage of GPR17⁺/GFP⁺cells decreased from 71.85% in the control group to 34.34% in the knockdown group (P<0.001), indicating a marked reduction in GPR17 expression. In the ARC, the proportions of GPR17⁺/GFP⁺ double-positive cells were 78.12% in the control group and 31.72% in the knockdown group. Western blotting results showed that the expression level of GPR17 protein in the ARC was significantly reduced following viral knockdown. In the HPC, CC1⁺ mature oligodendrocytes exhibited an increasing trend after GPR17 knockdown, though the difference was not statistically significant. However, PDGFRα⁺ oligodendrocyte precursor cells decreased significantly (P<0.001). In the ARC, knockdown of GPR17 significantly increased CC1⁺ mature oligodendrocytes and reduced PDGFRα⁺ oligodendrocyte precursor cells (both P<0.001). Conclusion ·The RCAS‒TVA gene delivery system enables efficient in vivo labeling and targeted knockdown of genes in oligodendrocyte lineage cells.

Key words: oligodendrocyte lineage cell, replication-competent avian sarcoma/leukosis virus (RCAS), tumor virus receptor A (TVA), G protein-coupled receptor 17 (GPR17)

中图分类号:

张潇月, 沈希, 李南南, 洪小琦, 童小萍. RCAS-TVA介导的少突胶质谱系细胞在体基因靶向调控研究[J]. 上海交通大学学报（医学版）, 2026, 46(6): 786-794.

Zhang Xiaoyue, Shen Xi, Li Nannan, Hong Xiaoqi, Tong Xiaoping. RCAS-TVA-mediated genetic targeting of oligodendrocyte lineage cells in vivo[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(6): 786-794.

图/表 5

图1 质粒构建及病毒包装示意图Note： Schematic of RCAS transfer plasmids encoding either a control shRNA(Scramble) or a GPR17-targeting shRNA(GPR17). The plasmids were transfected into DF-1 cells, and viruses were harvested 72 h post-transfection. The viruses only infect cells expressing TVA. CMV—cytomegalovirus; EGFP—enhanced green fluorescent protein.

Fig 1 Schematic diagram of plasmid construction and viral particles packaging

图2 FFU法测定RCAS-U6-shRNA(Scramble/GPR17)-EGFP病毒滴度的代表性荧光显微镜图像Note： Representative GFP expression pattern at five critical 10-fold serial dilutions of the virus stock: 1.00×102, 1.00×103, 1.00×104, 1.00×105, and 1.00×106.

Fig 2 Representative fluorescence images of RCAS-U6-shRNA(Scramble/GPR17)-EGFP virus titration using the FFU assay

表1 RCAS-U6-shRNA（Scramble/GPR17 ）-EGFP病毒荧光个数

Tab 1 Quantification of RCAS-U6-shRNA（Scramble/GPR17）- EGFP viral fluorescence counts

Group	Dilution
Group	1.00×10²	1.00×10³	1.00×10⁴	1.00×10⁵	1.00×10⁶
RCAS-U6-shRNA(Scramble)-EGFP	N/A	N/A	N/A	45	6
RCAS-U6-shRNA(GPR17)-EGFP	N/A	N/A	N/A	53	5

图3 RCAS-EGFP病毒感染少突胶质谱系细胞的效率Note： A. Schematic illustrating the experimental setup, where RCAS-EGFP was delivered to the hippocampus (HPC) or arcuate nucleus (ARC) of Olig2-TVA-IRES-Cre mice. B/C. Immunofluorescent staining (B) of GFP and Olig2 in cryostat sections of the HPC and ARC, with corresponding statistical analysis (C). Scale bars= 20 μm. n=3 mice. D. Representative immunofluorescence images of GFP (green), NeuN (neuronal marker), and GFAP (astrocytic marker) in the HPC and ARC.

Fig 3 Infection efficiency of RCAS-EGFP virus in oligodendrocyte lineage cells

图4 少突胶质谱系细胞中实现GPR17蛋白的敲低Note： A/B. Representative immunofluorescence staining images and quantification of GFP and GPR17 co-localization following stereotaxic injection of RCAS-U6-shRNA(Scramble/GPR17)-EGFP into the HPC (A) or ARC (B). C. Immunofluorescence staining of CC1 and PDGFRα in cryostat sections of the HPC and ARC, with corresponding statistical analysis. Scale bars=20 μm. n=3 mice. D. Representative immunoblots and densitometric quantification normalized to GAPDH demonstrating efficient GPR17 knockdown in the ARC.

Fig 4 Knockdown of GPR17 protein in oligodendrocyte lineage cells

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RCAS-TVA介导的少突胶质谱系细胞在体基因靶向调控研究

RCAS-TVA-mediated genetic targeting of oligodendrocyte lineage cells in vivo

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