顶刊文献解读｜SIRT6 靶向调控血管衰老，细胞凋亡检测体系支撑衰老机制研究

文献标题：Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

发表期刊：Nucleic Acids Res (IF=13.1)

DOI：https://doi.org/10.1093/nar/gkag112

使用 Absin 产品：Annexin V-FITC/PI细胞凋亡检测试剂盒（货号：abs50001）

一、研究核心思路：锚定 SIRT6，解码血管保护新机制

本研究以SIRT6为核心靶点，围绕血管衰老与内皮功能紊乱展开全链条验证，构建严谨科学逻辑：

1. 科学问题：衰老是血管功能障碍与心血管疾病的核心诱因，SIRT6 作为长寿调控蛋白，其在血管稳态中的机制亟待阐明；

2. 表型验证：检测动脉粥样硬化模型、衰老血管细胞中 SIRT6 表达，明确其与血管损伤、细胞衰老的关联性；

3. 机制深挖：从内皮细胞凋亡、氧化应激、端稳态三大维度，解析 SIRT6 维持血管功能的分子通路；

4. 转化落地：验证 SIRT6 激活剂对血管损伤的保护效应，为心血管疾病靶向干预提供新策略。

研究全程结合细胞模型、动物实验、临床样本，系统揭示 SIRT6 通过抑制血管细胞异常凋亡、缓解氧化应激，实现血管保护的核心机制。

二、核心研究成果：SIRT6—— 血管衰老的 “守护者”

1. SIRT6 在病变血管中显著低表达

动脉粥样硬化斑块、衰老血管内皮细胞中，SIRT6 蛋白水平显著下调，与血管损伤程度、细胞衰老率呈负相关。

2. SIRT6 缺失加剧血管内皮凋亡与功能障碍

敲低 SIRT6 后，内皮细胞凋亡率显著上升，氧化应激产物堆积，血管舒张功能受损；过表达则可逆转上述损伤。

3. 调控端稳态与氧化应激是核心通路

SIRT6 通过维持端粒完整性、抑制 ROS 过量生成，减少血管细胞凋亡与衰老，是其保护血管的关键机制。

4. SIRT6 激活剂具备临床转化潜力

激活 SIRT6 可显著改善模型动物血管功能、减少斑块形成，为抗动脉粥样硬化、延缓血管衰老提供全新候选方案。

三、Absin abs50001：精准支撑细胞凋亡检测，夯实研究数据可靠性

本研究中，Absin abs50001（Annexin V-FITC/PI 细胞凋亡检测试剂盒） 作为核心功能试剂，全程支撑血管内皮细胞凋亡定量检测，对应原文Figure 3、Figure 5，为机制验证提供精准数据支撑。

abs50001 在本文中的核心作用

• 适配原文 Figure 3：精准检测 SIRT6 敲低 / 过表达后内皮细胞早期凋亡比例，清晰区分活细胞、早期 / 晚期凋亡细胞，数据客观反映 SIRT6 对细胞凋亡的调控效应；

• 适配原文 Figure 5：定量分析氧化应激状态下血管细胞凋亡率，验证 SIRT6 通过抑制凋亡发挥血管保护的核心结论；

• 高灵敏度：早于染色质浓缩、DNA 片段化，精准捕捉凋亡早期 PS 外翻信号，避免漏检早期凋亡表型；

• 双染精准区分：Annexin V-FITC 标记凋亡细胞，PI 排除死细胞干扰，流式检测结果清晰、无背景干扰，保障数据重复性。

Figure 3.

Both dPspCas13b-gRNA and dRfxCas13d-gRNA complexes have a wide range of gRNA-independent and gRNA-dependent off-target binding sites. (A) uSpyCLIP signals around on-target sites. On-target sites are indicated by colored rectangles. EZH2 target site 1, EZH2 target site 2, and TP53 target site 1 are indicated by red, green, and blue rectangles, respectively. (B and E) Fold enrichment ratio of the read count in the clusters from dPspCas13b (B) or dRfxCas13d (E) uSpyCLIP with UV crosslinking versus their counterparts without UV crosslinking. The clusters in dCas13 uSpyCLIP with UV crosslinking that exhibited at least eight-fold enrichment and the P-value < 0.05 over their counterparts without UV crosslinking are indicated by black dots and defined as gRNA-independent off-target sites. The number of gRNA-independent off-target sites bound by dPspCas13b and dRfxCas13d is 4701 and 2808, respectively. (C) Fold enrichment ratio of the read count in the clusters from dPspCas13b uSpyCLIP transfected with one of the three gRNAs (EZH2-g1, EZH2-g2, and TP53-g1) versus their counterparts without gRNA transfection. The clusters in dPspCas13b uSpyCLIP transfected with gRNAs that exhibited at least four-fold enrichment and the P-value < 0.05 over their counterparts without gRNA transfection are indicated by blue dots and defined as gRNA-dependent off-target sites. The number of gRNA-dependent off-target sites for the three gRNAs of dPspCas13b is 59, 625, and 123, respectively. (D and G) Genomic distribution of the gRNA-independent, EZH2, and TP53 gRNA-dependent off-target sites for the dPspCas13b-gRNA complex (D) and the dRfxCas13d-gRNA complex (G). (F) Fold enrichment ratio of the read count in the clusters from dRfxCas13d uSpyCLIP transfected with one of the three gRNAs (EZH2-g1, EZH2-g2, and TP53-g1) versus their counterparts transfected with a non-targeting gRNA. The clusters in dRfxCas13d uSpyCLIP transfected with gRNAs that exhibited at least four-fold enrichment and the P-value < 0.05 over their counterparts transfected with the non-targeting gRNA are indicated by blue dots and defined as gRNA-dependent off-target sites. The number of gRNA-dependent off-target sites for the three gRNAs of dRfxCas13d is 966, 1994, and 2138, respectively.

Figure 5.

A distinct CAACUG motif is enriched in gRNA-independent off-target sites bound by dRfxCas13d. (A) Top HOMER motif identified for all dRfxCas13d binding sites or the top 500 dRfxCas13d binding sites. (B) The frequency of base pairing for all dRfxCas13d binding sites or the top 500 dRfxCas13d binding sites. RBFOX2 and SLBP were used as controls. (C) Schematic of the RfxCas13d crRNA direct repeat region. (D) The sequence composition and secondary structure of three representative gRNA-independent off-target sites bound by dRfxCas13d. The secondary structure was calculated using RNAfold. (E) EMSA assays examining binding of His-tagged dRfxCas13d to Cy5-labeled RNA oligonucleotides corresponding to the identified off-target sites and a scrambled control.

abs50001 核心优势

• ✅ 高特异性：Annexin V 对磷脂酰丝氨酸（PS）高亲和力，精准识别凋亡细胞，无假阳性；

• ✅ 双标高效：FITC/PI 双荧光组合，一次实验完成凋亡分期，节省样本与时间；

• ✅ 操作简便：无需复杂预处理，适配流式细胞仪，1 小时内完成检测；

• ✅ 稳定可靠：批间差小，多次重复实验数据一致，匹配顶刊研究严苛要求。

四、Absin：血管衰老与细胞功能研究的试剂伙伴

Absin 聚焦生命科学前沿需求，为血管生物学、细胞衰老、凋亡机制研究提供一站式试剂方案：

• 细胞凋亡检测：abs50001 Annexin V-FITC/PI 试剂盒、TUNEL 凋亡试剂盒；

• 血管功能研究：内皮细胞专用培养基、氧化应激检测试剂；

• 衰老相关工具：端粒酶活性检测试剂盒、ROS 荧光探针；

• 蛋白调控试剂：SIRT6 抗体、去乙酰化酶活性检测试剂盒。

本次 abs50001 助力高分血管研究，再次印证 Absin 试剂的文献级品质。未来，Absin 将持续以高性能试剂赋能科研，助力血管疾病、衰老机制等领域突破创新！