血小板RNA干扰技术的建立与优化

doi:10.19586/j.2095-2341.2026.0036

生物技术进展 ›› 2026, Vol. 16 ›› Issue (3): 665-676.DOI: 10.19586/j.2095-2341.2026.0036

血小板RNA干扰技术的建立与优化

伍宇婷¹^,²(), 梁炜¹^,², 王洪涛¹^,², 刘翠翠¹^,²(), 周家喜¹^,²()

^1.中国医学科学院血液病医院（中国医学科学院血液学研究所），北京协和医学院，血液与健康全国重点实验室，国家血液系统疾病临床医学研究中心，细胞生态海河实验室，天津 300020
^2.天津医学健康研究院，天津 301600

收稿日期:2026-02-10 接受日期:2026-03-31 出版日期:2026-05-25 发布日期:2026-07-14
通讯作者: 刘翠翠,周家喜
作者简介:伍宇婷 E-mail: wuyuting@ihcams.ac.cn
基金资助:
国家杰出青年科学基金项目(82125003);国家自然科学基金重点项目(82430009);天津市杰青项目(24JCJQJC00110);国家自然科学基金面上项目(32571301)

Establishment and Optimization of RNA Interference Technology in Platelets

Yuting WU¹^,²(), Wei LIANG¹^,², Hongtao WANG¹^,², Cuicui LIU¹^,²(), Jiaxi ZHOU¹^,²()

^1.State Key Laboratory of Experimental Hematology，National Clinical Research Center for Blood Diseases，Haihe Laboratory of Cell Ecosystem，Institute of Hematology Blood Diseases Hospital，Chinese Academy of Medical Sciences Peking Union Medical College，Tianjin 300020，China
^2.Tianjin Institutes of Health Science，Tianjin 301600，China

Received:2026-02-10 Accepted:2026-03-31 Online:2026-05-25 Published:2026-07-14
Contact: Cuicui LIU,Jiaxi ZHOU

摘要/Abstract

摘要：

血小板是血液中最小的无核血细胞，其中丰富的RNA和蛋白质是发挥凝血、免疫等功能的分子基础。RNA干扰是研究血小板基因功能的有力工具，但相关研究还处于初步探索阶段，目前尚无有效、稳定的血小板RNA干扰技术。为了建立高效、稳定的血小板RNA干扰技术，以小鼠外周血血小板为研究对象，分别从转染试剂、干扰RNA类型等方面进行体系优化。通过监测核酸转染效率、维持时间、血小板存活状态、基因干扰效率等进行系统评价，最终建立了以Mate Plus脂质纳米颗粒代替经典阳离子脂质体转染试剂Lipofectamine 2000、单链反义寡核苷酸代替传统双链小干扰RNA的高效RNA干扰体系，将干扰RNA的转染效率从10%以下提高至70%以上，且维持稳定转染48 h以上。在此基础上，以血小板中表达丰度极高的胸腺素β4（thymosin β4，Tmsb4x）基因为例，成功利用ASO/Mate-Plus体系实现Tmsb4x基因的有效敲降，其RNA水平降低30%以上。但是，Tmsb4x基因的敲降并不影响其蛋白表达，提示其可用于特异性研究血小板中RNA的功能。同时，仅Tmsb4x RNA的敲降可显著影响活化血小板的黏附铺展功能，提示该基因可能存在独立于蛋白外的RNA功能。综上，建立与优化的血小板RNA干扰技术，为研究血小板特定RNA功能以及血小板快速功能响应的分子基础提供了有力的工具。

关键词: 血小板, RNA干扰, 小干扰RNA, 反义寡核苷酸, 胸腺素β4

Abstract:

Platelets are the smallest anucleate blood cells in circulation， their rich and diverse RNA and protein contents constitute the molecular basis underlying their hemostatic， immune， and inflammatory functions. RNA interference represents a powerful tool for investigating platelet gene function， however， current studies remain at an early exploratory stage， and efficient and stable RNA interference approaches for platelets are still lacking. To establish a robust and effective platelet RNA interference platform， murine peripheral blood platelets were used as the experimental model， and key parameters—including transfection reagents and RNA modalities—were systematically optimized. Transfection efficiency， intracellular retention duration of nucleic acids， platelet viability， and gene-silencing efficacy were comprehensively evaluated. Through this optimization， we developed a highly efficient RNA interference platform by replacing the conventional cationic liposomal transfection reagent Lipofectamine 2000 with Mate Plus lipid nanoparticles， and substituting traditional double-stranded small interfering RNA （siRNA） with single-stranded antisense oligonucleotides. This strategy increased oligonucleotide transfection efficiency from 10% to 70% and sustained stable intracellular delivery for more than 48 h. Using the highly abundant platelet transcript thymosin β4 （Tmsb4x） as a proof-of-concept target， we successfully achieved effective knockdown of Tmsb4x mRNA via the ASO/Mate-Plus platform， reducing Tmsb4x mRNA levels by 30%. Notably， suppression of Tmsb4x RNA did not alter its corresponding protein expression， indicating that this system enables selective interrogation of platelet RNA function independent of protein abundance. Moreover， knockdown of Tmsb4x RNA alone significantly impaired adhesion and spreading of activated platelets， suggesting a potential protein-independent regulatory role of Tmsb4x RNA in platelet cytoskeletal dynamics. In summary， this study established and optimized an efficient platelet RNA interference strategy， providing a powerful experimental tool for dissecting platelet-specific RNA functions and elucidating the molecular basis of rapid platelet functional responses.

Key words: platelets, RNA interference, small interfering RNA, antisense oligonucleotide, thymosin β4

中图分类号:

Q786

伍宇婷, 梁炜, 王洪涛, 刘翠翠, 周家喜. 血小板RNA干扰技术的建立与优化[J]. 生物技术进展, 2026, 16(3): 665-676.

Yuting WU, Wei LIANG, Hongtao WANG, Cuicui LIU, Jiaxi ZHOU. Establishment and Optimization of RNA Interference Technology in Platelets[J]. Current Biotechnology, 2026, 16(3): 665-676.

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链接本文: https://swjsjz.magtechjournal.com/CN/10.19586/j.2095-2341.2026.0036

https://swjsjz.magtechjournal.com/CN/Y2026/V16/I3/665

图/表 5

表1 RT-qPCR引物序列

Table 1 RT-qPCR primer sequences

引物名称	引物序列（5'→3'）
m-Tmsb4x-F	5'-GCGAATCGTAATGAGGCGAG-3'
m-Tmsb4x-R	5'-TGATGTGAAAGGGGCAGCAC-3'
m-Pf4-F	5'-GAGGTGATCAAGGCAGGACG-3'
m-Pf4-R	5'-TATAGGGGTGCTTGCCGGTC-3'

图1 转染试剂M-Plus相较Lipo2000可高效介导血小板siRNA转染A：血小板siRNA转染实验示意图；B：免疫荧光染色显示siRNA在血小板中的定位；C：流式细胞术显示空白对照及2种转染试剂介导的血小板siRNA转染效率；D：统计图显示Lipo2000和M-Plus转染试剂介导的血小板siRNA转染效率；E：统计图显示空白对照及2种转染试剂处理下的血小板计数；F：流式检测空白对照及2种转染试剂处理下CD62P+活化血小板的比例。**、****表示差异在P0.01、P0.000 1水平上具有统计学意义；ns表示无统计学意义。

Fig. 1 M-Plus efficiently mediate siRNA transfection in platelets compared with Lipo2000

图2 ASO相较siRNA可显著提升血小板RNA转染的时效性A：流式检测48 h内无核血小板与有核细胞的siRNA转染率；B：血小板ASO和siRNA转染实验示意图；C：流式动态监测48 h内血小板siRNA和ASO转染效率；D：折线图显示48 h内血小板siRNA和ASO转染效率变化；E：统计图显示48 h内siRNA和ASO转染组的血小板计数变化；F：统计图显示48 h内siRNA和ASO转染组CD62P+活化血小板的比例变化。**、****表示差异在P0.01、P0.000 1水平上具有统计学意义；ns表示无统计学意义。

Fig. 2 ASO enhance the timeliness of RNA transfection in platelets compared with siRNA

图3 利用ASO/M-Plus转染系统可有效敲降血小板高丰度基因Tmsb4x的表达A：血小板中Tmsb4x基因靶向干扰实验示意图；B：流式检测转染24 h血小板中ASO-NC和ASO-Tmsb4x的转染率；C：统计显示ASO-NC和ASO-Tmsb4x转染组血小板的数量；D：RT-qPCR检测转染24 h 2组血小板中Tmsb4x的RNA表达；E：胞内流式检测转染24 h 2组血小板中TMSB4X蛋白表达；F：定量统计显示转染24 h 2组血小板中TMSB4X蛋白表达水平；G：胞内流式检测转染48 h 2组血小板中TMSB4X蛋白表达；H：定量统计显示转染48 h 2组血小板中TMSB4X蛋白表达水平

Fig. 3 The expression of the highly abundant platelet gene Tmsb4x could be effectively knocked down by using the ASO/M-Plus transfection system

图4 特异性敲降Tmsb4x RNA可显著降低血小板铺展面积A：设计探究敲降Tmsb4x对血小板的功能影响；B：流式统计激动剂凝血酶刺激前后2组血小板的CD62P阳性率；C：免疫荧光染色显示激动剂凝血酶刺激后2组血小板的黏附铺展形态；D：激动剂凝血酶刺激后2组血小板的铺展面积统计图。****表示差异在P0.000 1水平上具有统计学意义；ns表示无统计学意义。

Fig. 4 Specific knockdown of Tmsb4x RNA significantly reduce the platelet spreading area

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血小板RNA干扰技术的建立与优化

Establishment and Optimization of RNA Interference Technology in Platelets

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