药学学报

Plant	Isolation method	Size/nm	Zeta potential/mV	Animal model and administration route	Distribution	Ref
Grape	Differential ultracentrifugation and density gradient ultracentrifugation	~380	~-26	Healthy mice, p.o.	Intestinal stem cells	^[26]
Ginger	Differential ultracentrifugation and density gradient ultracentrifugation	102-998	-29 - -24	Healthy mice, p.o.	Liver, mesenteric, lymph nodes	^[27]
	Differential ultracentrifugation and density gradient ultracentrifugation	~231	~-12	Healthy mice, p.o.	Colon	^[10]
Ginseng	Differential ultracentrifugation and density gradient ultracentrifugation	~344	~-25	Healthy mice, i.p.; i.v. healthy mice, p.o.	Liver, spleen Stomach, gut	^[28]
Broccoli	Differential ultracentrifugation and density gradient ultracentrifugation	18-118	~-39	Colitismice, p.o.	Colon, mesenteric, lymph nodes, spleen	^[29]
Grapefruit	Differential ultracentrifugation and density gradient ultracentrifugation	~210	~-49	Healthy mice, p.o.	Middle and distal part of small intestine, cecum, colon	^[30]
Lemon	Differential ultracentrifugation and density gradient ultracentrifugation	50-70	~-22^a	Tumor-bearingmice, i.p.	Tumor, liver, spleen, kidney	^[31]
Carrot	Differential ultracentrifugation and density gradient ultracentrifugation			C57BL/6 mice or knockout mice, p.o.	Small and large intestine macrophages, intestinal stem cells	^[32]

Plant	Isolation method	Size/nm	Zeta potential/mV	Animal model and administration route	Distribution	Ref
Grape	Differential ultracentrifugation and density gradient ultracentrifugation	~380	~-26	Healthy mice, p.o.	Intestinal stem cells	^[26]
Ginger	Differential ultracentrifugation and density gradient ultracentrifugation	102-998	-29 - -24	Healthy mice, p.o.	Liver, mesenteric, lymph nodes	^[27]
	Differential ultracentrifugation and density gradient ultracentrifugation	~231	~-12	Healthy mice, p.o.	Colon	^[10]
Ginseng	Differential ultracentrifugation and density gradient ultracentrifugation	~344	~-25	Healthy mice, i.p.; i.v. healthy mice, p.o.	Liver, spleen Stomach, gut	^[28]
Broccoli	Differential ultracentrifugation and density gradient ultracentrifugation	18-118	~-39	Colitismice, p.o.	Colon, mesenteric, lymph nodes, spleen	^[29]
Grapefruit	Differential ultracentrifugation and density gradient ultracentrifugation	~210	~-49	Healthy mice, p.o.	Middle and distal part of small intestine, cecum, colon	^[30]
Lemon	Differential ultracentrifugation and density gradient ultracentrifugation	50-70	~-22^a	Tumor-bearingmice, i.p.	Tumor, liver, spleen, kidney	^[31]
Carrot	Differential ultracentrifugation and density gradient ultracentrifugation			C57BL/6 mice or knockout mice, p.o.	Small and large intestine macrophages, intestinal stem cells	^[32]

Plant origin	Functionality	Mechanism	Ref
Ginger	Treatment of IBD	Itinhibits the expression of TNF-α, IL-6 and IL-1β and increases the expression of IL-10 and IL-22	^[10]
	Prevent chronicperiodontitis	It binds to the HBP35 protein on the surface of Porphyromonas gingivalis, inhibits the expression of bacteria-related mRNA, inhibits the transfer of bacteria to normal cells, and reduces the expression of protein and mRNA of FimA	^[51]
	Protect against alcoholinduced liver damage	It induces the expression of Nrf2, inhibits the production of ROS and LPS-induced inflammation by TLRs mediated pathway	^[27]
Broccoli	Treatment of IBD	It increases the expression of IL-10, reduces the IFN-γ, IL-17A and TNF-α in colon tissue; mediates activation of AMPK in DCs	^[29]
Ginseng	Inhibit melanoma growth	It induces the polarization of M1-like macrophages by TLR-4 and MyD88 signaling pathways, promotes the production of ROS and significantly increases M1-related cytokines and chemokines (CCL5, IL-6, MCP-1, TNF-α, IL-1α, IL-12)	^[28]
Grape	Treatment of colitis	It targets Lgr5⁺ intestinal stem cells, enables intestinal epithelial cell proliferation and directly promotes the generation of stem cell-like organs	^[26]
Dendropanaxmor bifera	Whitening	It reduces the activity of tyrosinase and inhibits the expression of melanin-producing related enzymes (TYR, TRP-1, TRP-2)	^[52]
Lemon	Antitumor	It increases the expression of pro-apoptotic gene, inhibits the expression of antiapoptotic gene, activates a TRAIL-mediated apoptotic cell process, and inhibits the secretion of pro-angiogenic factors (VEGF-A, IL-6, and IL-8)	^[31]
Carrot	Treatment of colitis	It increases nuclear translocation of Nrf2 and induces macrophage to produce IL-10	^[32]
Rhodiolacrenulata	Treatment of pulmonary fibrosis	Lipids facilitates cellular uptake of HJT-sRNA-m7, HJT-sRNA-m7 reduces the expression of α-SMA fibronectin and COL3A1	^[53]

Plant origin	Functionality	Mechanism	Ref
Ginger	Treatment of IBD	Itinhibits the expression of TNF-α, IL-6 and IL-1β and increases the expression of IL-10 and IL-22	^[10]
	Prevent chronicperiodontitis	It binds to the HBP35 protein on the surface of Porphyromonas gingivalis, inhibits the expression of bacteria-related mRNA, inhibits the transfer of bacteria to normal cells, and reduces the expression of protein and mRNA of FimA	^[51]
	Protect against alcoholinduced liver damage	It induces the expression of Nrf2, inhibits the production of ROS and LPS-induced inflammation by TLRs mediated pathway	^[27]
Broccoli	Treatment of IBD	It increases the expression of IL-10, reduces the IFN-γ, IL-17A and TNF-α in colon tissue; mediates activation of AMPK in DCs	^[29]
Ginseng	Inhibit melanoma growth	It induces the polarization of M1-like macrophages by TLR-4 and MyD88 signaling pathways, promotes the production of ROS and significantly increases M1-related cytokines and chemokines (CCL5, IL-6, MCP-1, TNF-α, IL-1α, IL-12)	^[28]
Grape	Treatment of colitis	It targets Lgr5⁺ intestinal stem cells, enables intestinal epithelial cell proliferation and directly promotes the generation of stem cell-like organs	^[26]
Dendropanaxmor bifera	Whitening	It reduces the activity of tyrosinase and inhibits the expression of melanin-producing related enzymes (TYR, TRP-1, TRP-2)	^[52]
Lemon	Antitumor	It increases the expression of pro-apoptotic gene, inhibits the expression of antiapoptotic gene, activates a TRAIL-mediated apoptotic cell process, and inhibits the secretion of pro-angiogenic factors (VEGF-A, IL-6, and IL-8)	^[31]
Carrot	Treatment of colitis	It increases nuclear translocation of Nrf2 and induces macrophage to produce IL-10	^[32]
Rhodiolacrenulata	Treatment of pulmonary fibrosis	Lipids facilitates cellular uptake of HJT-sRNA-m7, HJT-sRNA-m7 reduces the expression of α-SMA fibronectin and COL3A1	^[53]

植物来源囊泡及其生物医学应用研究进展

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赵梦 ¹ , 李思敏 ¹ , 张蕾 ¹ , 丛明慧 ¹ , 胡立宏 ¹ , 乔宏志 ¹^,²^,^*

药学学报 | 专题报道：结构中药学 2021,56(8): 2039-2047

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药学学报 | 专题报道：结构中药学 2021, 56(8): 2039-2047

植物来源囊泡及其生物医学应用研究进展

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赵梦¹, 李思敏¹, 张蕾¹, 丛明慧¹, 胡立宏¹, 乔宏志¹^,²^,^*

作者信息

1.南京中医药大学药学院, 江苏省中药功效物质重点实验室, 江苏南京 210023

2.江苏省中药高效给药系统工程技术研究中心, 江苏南京 210023

通讯作者:

*乔宏志, Tel: 86-25-85811050, E-mail: qiaohz@njucm.edu.cn

Research progress of plant-derived vesicles and their biomedical applications

Meng ZHAO¹, Si-min LI¹, Lei ZHANG¹, Ming-hui CONG¹, Li-hong HU¹, Hong-zhi QIAO¹^,²^,^*

Affiliations

1. Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

2. Jiangsu Engineering Research Center for Efficient Delivery System of TCM, Nanjing 210023, China

出版时间: 2021-08-12 doi: 10.16438/j.0513-4870.2020-1514

文章导航

摘要

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植物细胞外囊泡（extracellular vesicles，EVs）是一种由植物细胞分泌的，以脂质双层为基本骨架、包裹各种蛋白和核酸等活性物质的膜性小泡，对植物的生长发育、组织修复和自体防御等过程起重要作用。近年来，参照EVs的分离方法，从植物样本中制备得到细胞外囊泡样纳米粒（extracellular vesicle-like nanoparticles，EVNs）具有与EVs相似的结构组成，也显示出独特的活性功能。本文将上述结构统称为植物来源囊泡（plant-derived vesicles，PDVs），系统介绍了PDVs生源途径、分离表征方法和体内外性质，讨论其作为天然治疗剂和药物功能载体等方面的生物医学应用，最后对该领域存在的问题及未来发展方向提出观点和建议。

关键词

细胞外囊泡 / 外泌体 / 纳米粒 / 植物 / 中药 / 药理作用 / 药物递送 / 结构中药学 / 物质基础

Abstract

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Plant-derived extracellular vesicles (EVs) are membranous vesicles secreted by plants, which include lipid bilayer as the basic framework and encapsulate various proteins, nucleic acid and other active substances. They play an important role in plant growth and development, tissue repair and self-defense. In recent years, extracellular vesicle-like nanoparticles (EVNs) are prepared from plant samples referring to the separation method of EVs and show unique functions. In this review, the above structures are collectively called plant-derived vesicles (PDVs). The biogenesis, separation and characterization methods, in vivo and in vitro properties of PDVs have been reviewed. The biomedical applications of PDVs as natural therapeutic agents and functional drug carriers are described, and finally some opinions on the existing problems and future prospect in this field are put forward.

Key words

extracellular vesicle / exosome / nanoparticle / plant / Chinese medicine / pharmacological action / drug delivery / structural Chinese medicine / pharmacodynamic substance basis

引用本文

赵梦, 李思敏, 张蕾, 丛明慧, 胡立宏, 乔宏志. 植物来源囊泡及其生物医学应用研究进展. 药学学报, 2021 , 56 (8) : 2039 -2047 . DOI: 10.16438/j.0513-4870.2020-1514

Meng ZHAO, Si-min LI, Lei ZHANG, Ming-hui CONG, Li-hong HU, Hong-zhi QIAO. Research progress of plant-derived vesicles and their biomedical applications[J]. Acta Pharmaceutica Sinica, 2021 , 56 (8) : 2039 -2047 . DOI: 10.16438/j.0513-4870.2020-1514

正文

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1 植物来源囊泡概述

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2013年诺贝尔生理学或医学奖授予3位科学家以表彰他们在囊泡传输系统领域做出的贡献。事实上, 不仅是哺乳动物, 植物细胞也能分泌囊泡, 甚至比哺乳动物囊泡发现得更早。20世纪60年代, 研究人员首次用电镜观察到胡萝卜细胞可以分泌囊泡^[1]。后来的几十年, 不断有从各种植物中发现细胞外囊泡(extracellular vesicles, EVs) 的报道。2008年, Gonorazky等^[2]发现番茄细胞的悬浮液中含有泡状结构, 并证明其所含磷脂与胞内磷脂不同。同年, Regente等^[3]研究了向日葵种子细胞外液, 揭示Rab蛋白参与了向日葵细胞多泡体(multivesicular body, MVB) 的分泌。MVB是真核细胞蛋白运输和分拣中心^[4], 一部分与溶酶体融合; 另一部分通过与细胞膜融合释放到胞外基质中, 这被认为是EVs可能的分泌途径。

目前普遍认为EVs是一种以脂质双层为基本骨架、包裹各种蛋白和核酸等活性物质的膜性小泡, 对植物的生长发育、组织修复和自体防御等过程起重要作用^[5]。Zhao等^[6]从成熟和未成熟的椰子汁中分离到EVs, 通过RNA测序发现EVs参与了椰子的成熟过程。Rutter等^[7]用致病菌Pseudomonas syringae感染拟南芥后发现EVs的分泌量明显升高, 证明EVs在植物免疫应答中起重要作用。Cai等^[8]发现拟南芥细胞分泌的EVs可以递送sRNA至灰霉菌Botrytis cinerea体内, 并使其关键致病基因沉默。

近年来, 有学者参考EVs的分离方法, 从植物样本中制备得到细胞外囊泡样纳米粒(extracellular vesiclelike nanoparticles, EVNs), 这些EVNs具有与EVs相似的结构组成, 并在动物和人体功能调控方面有重要作用。尽管上述囊泡的来源不尽相同, 但是在分离表征方法和生物医学应用等方面的研究具有相似性。因此, 本文将这些囊泡统称为植物来源囊泡(plant-derived vesicles, PDVs), 对其研究进展进行综述, 以期为不同领域科研人员提供参考(图 1)。

2 PDVs的分离纯化方法

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2.1 差速离心法

差速离心法利用不同离心力实现PDVs与其他物质的分离, 是目前PDVs最常用的分离方法^[9]。将榨好的姜汁依次经过3 000 ×g离心20 min、10 000 ×g离心40 min和150 000 ×g离心2 h, 可得到较纯的生姜PDVs^[10]。但该法对于黏性较大或含杂质颗粒较多的植物样本如葛根和山药等的分离效率较低。

2.2 密度梯度离心法

密度梯度离心法是指具有不同沉降系数的各成分在离心力作用下各自以一定速度沉降, 在不同的密度梯度区域形成条带的方法。常用的梯度构建物质是蔗糖和碘克沙醇等。将差速离心后的PDVs经密度梯度离心纯化, 可得高纯度的PDVs^[10], 但本法在规模化制备方面比较受限。

2.3 超滤/过滤法

超滤是利用超滤膜的微孔结构, 在一定压力下实现不同尺度样品分离的方法^[11]。由于超滤膜已经实现商业化且规格选用较方便, 所以本法可考虑作为超高速离心的替代方案。过滤法是基于分子质量和尺寸分离PDVs的一种方法, 通常采用纤维束过滤器, 多与超滤法联用。与差速离心法相比, 过滤法的压力小, 净化效果较好, 但由于挤压效应, 超滤/过滤过程可能会改变PDVs的结构, 且滤膜可能存在堵塞和污染等问题^[12]。

2.4 免疫磁珠法

免疫磁珠法利用磁珠表面功能基团特异性识别PDVs表面蛋白, 在磁场作用下实现PDVs的分离。该法在满足PDVs表面蛋白特异性识别和有效解离的情况下, 可以实现对特定PDVs及其亚型的分离, 外加磁场易于调节且具有较好的富集效果^[13]。

2.5 聚合物沉淀法

聚合物沉淀法利用聚合物竞争性结合水分子, 使PDVs从溶液中析出达到分离目的^[9]。通常在含PDVs的液体样品中加入聚乙二醇(polyethylene glycol, PEG), 再经低速离心得到PDVs^[14]。Ren等^[15]利用差速离心分离得到地黄PDVs后, 又采用PEG沉淀法对PDVs进一步纯化。目前已有基于此方法的试剂盒上市。本法操作简便, 但提取样品纯度不高。残留的PEG可增加样品黏度, 同时可能对后续实验产生影响。

2.6 尺寸排阻色谱法

尺寸排阻色谱法基于样本尺度的差异实现分离。将样本通过多孔聚合物微球, 小尺寸物质向孔中扩散, 洗脱较慢; 大颗粒从微球间通过, 被直接洗脱^[16]。该法可以获得较纯的PDVs, 但耗时较长, 并且含PDVs的流体通过填料时可能被挤压变形, 完整性受到一定影响。

2.7 场流分离法

场流分离法的原理是样品流过扁平通道同时受到水平和垂直两个方向的流场作用; 尺寸小的分子, 受垂直方向作用力小, 向侧壁平移小; 尺寸较大的分子, 受垂直方向作用力大, 更靠近侧壁, 从而在垂直方向形成尺寸梯度。由于越靠近中心, 流速越快, 先流出; 越靠近侧壁流速越均匀缓慢, 后流出^[17]。本法与传统尺寸排阻色谱相比无固定相, 因此系统压力较低, 剪切效应小, 对PDVs的影响更小。Zhang等^[18]采用本法从黑色素瘤细胞(B16F10) 的EVs中分离出不同亚型, 发现具有不同的生物学特性, 表明该法在PDVs细分领域可能具有一定优势。

2.8 其他方法

近年来, 随着新兴分离技术的发展, 开发出一些新的EVs分离方法, 如微流控利用微管道处理或操纵微小流体, 借助免疫亲和力或物理场实现EVs的分离^{[19, 20]}, 具有样品量少、低成本、高通量和高精度等优点; Morales-Kastresana等^[21]还开发了纳米流式细胞术, 实现了对免疫细胞系及肿瘤细胞株来源EVs的分离。相关分离技术的发展可为PDVs的研究提供借鉴, 但不同方法各有优势, 需根据研究对象和目的进行选择和优化。

3 PDVs的物理表征

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3.1 粒径与电位

不同PDVs的粒径和电位差别较大, 平均粒径从30 nm到400 nm不等, 电位也从近中性到约-50 mV, 这可能与不同种属PDVs所含成分不同或生源差异有关, 当然分离工艺的不同对粒度也有影响。目前文献报道的关于EVs粒径测定的方法主要有动态光散射(DLS)、纳米颗粒跟踪分析技术(NTA)^[22]及可调电阻式脉冲传感技术^[23]等, 其中前二者使用较多, 但测试原理和输出结果不同。DLS是基于粒子衍射的光强波动计算的平均粒径, 对于单分散体系测试结果较准确。NTA则是对单个粒子的抓取和统计, 能够反映颗粒的真实状态并能提供浓度数据, 比较适合多相复杂体系的粒径和浓度测算。另外, 利用NTA荧光模式可以跟踪特定表型EVs, 有利于排除背景干扰^{[24, 25]}。由于不同仪器存在感知机制上的差异, 一般推荐采用包括电镜在内的多平台手段阐述EVs的特性(表 1^{[10, 26-32]})。

3.2 形貌与性能

PDVs的形貌特征一般借助电镜技术表征, 常用的包括扫描电子显微镜(scanning electron microscope, SEM)、透射电子显微镜(transmission electron micro‐ scope, TEM)、冷冻电镜(cryo-electron microscopy, Cyro-EM) 和原子力显微镜(atomic force microscopy, AFM)。不同电镜获得的PDVs信息不尽相同。SEM主要用于获得样品表面结构。TEM可以观察到粒子的内部结构和形态^[17], 但制样时的脱水操作可能引起样品收缩而改变实际形貌。Cyro-EM可以在低温下分析样品的原始形态, 避免脱水和固定对样品的影响^[33]。AFM分辨率高, 可以观察到粒子的三维形态, 也可以获得囊泡膜结构的黏弹性和刚性等物理参数^[34], 但对样品的要求较高, 测试过程也比较复杂。根据目前报道的电镜图像, 一般认为PDVs与人源或动物来源囊泡相似, 为近球形或茶托状形貌, 其表面存在的凹陷多与制样过程有关, Cyro-EM镜下则呈现球形。

4 PDVs的成分分析

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4.1 脂质

脂质是PDVs的主要组成成分, 对PDVs结构的维持和功能发挥起重要作用。各种PDVs的脂质种类和含量存在较大差异, 一般可通过薄层色谱和质谱进行分析。研究表明, 葡萄PDVs中含量最多的是磷脂酸(phosphatidic acid, PA), 在细胞增殖及信号转导方面发挥作用; 其次为磷脂酰乙醇胺(phosphatidylethanol‐ amine, PE), 它可以调节细胞膜的弯曲, 对细胞膜的分裂和融合起关键作用^[26]。生姜囊泡中除PA外, 还含有双半乳糖基甘油二酯(digalactosyl diglyceride, DGDG) 和单半乳糖基二酰甘油, 其余则为少量磷脂酰甘油和PE等, 其中DGDG与植物叶绿体中类囊体的生成有关^[35]。

4.2 蛋白质

各种PDVs所含蛋白种类和含量存在较大差异, 一般采用SDS-PAGE凝胶电泳、液质联用技术(LCMS/MS) 等方法进行分析。Rutter等^[7]发现拟南芥叶片的囊泡中含有许多生物和非生物胁迫反应相关蛋白。Pocsfalvi等^[36]采用无标签鸟枪法对4种不同种属柑橘来源囊泡的蛋白进行定量, 检测到高表达的共有蛋白包括patellin3蛋白、网格重链蛋白和热休克蛋白等。Pérez-Bermúdez等^[37]通过LC-MS/MS在葡萄来源囊泡中鉴定出121个蛋白对应的246个多肽, 这些蛋白很多是参与糖代谢的酶。生姜来源囊泡中的蛋白含量较低, 以胞质蛋白为主, 如肌动蛋白, 还有少量膜通道或转运体蛋白。综上所述, PDVs中的蛋白可能参与细胞骨架形成和膜转运过程, 在生理代谢和信号传导等方面发挥重要作用, 但与人源囊泡不同, 目前PDVs尚缺乏共性或专属性蛋白标志物, 这给生源鉴定、纯度检测和生物医学功能的评价带来一定难度。

4.3 核酸

PDVs中含有多种核酸成分, 如miRNA、sRNA、DNA和其他非编码RNA等^[35], 在植物体生长发育、环境胁迫和免疫应答等过程中发挥重要作用。有研究者采用RNA测序技术分析了蓝莓、椰汁、生姜、猕猴桃、橙子和番茄等11种PDVs的miRNA, 发现含有大量20~22 nt的miRNA^[38]。近年来的研究表明, PDVs广泛参与miRNA的跨界调控, 这不仅与植物自身防御过程有关, 还涉及到PDVs进入动物或人体内后参与相关生理病理过程的调节。有研究表明, 大丽轮枝菌侵染棉花后可以诱导植物内源性miRNA的表达, 这些miRNA能够转运到病菌细胞中, 可降解致病菌基因^[39]。拟南芥感染灰霉菌的过程中, 细胞会将sRNA包裹在外泌体内, 从植物细胞中分泌, 并在灰霉菌感染部位聚集, 使得宿主sRNA抑制灰霉菌基因的表达^[8]。患有炎性肠病的小鼠摄入生姜后, 其囊泡携带的miRNA可靶向调控肠道鼠李糖乳酸杆菌的多个基因, 引起吲哚-3-甲醛(indole-3-carboxaldehyde, Ⅰ3A) 分泌增加, 升高白细胞介素-22 (IL-22) 水平, 起到修复肠黏膜损伤的作用^[40]。Zhou等^[41]发现金银花药液中含有miR2911, 给小鼠口服后可直接靶向多种流感病毒(H1N1、H5N1和H7N9), 抑制病毒的复制。

4.4 小分子化合物

除了上述生物大分子, PDVs中还检测到同源植物的活性小分子成分, 如西柚来源囊泡可检测到柚皮苷和柚皮素^[30], 地黄来源囊泡可检测到梓醇, 柠檬来源囊泡可检测到柠檬酸和柠檬苦素等^[42], 生姜来源囊泡可检测到姜烯酚和辣椒素^[27], 西兰花来源囊泡中可检测到莱菔硫烷^[29]。这表明PDVs的传递伴随小分子化合物的转运, 同时由于PDVs具有的天然载体属性, 提高了小分子化合物的水溶性和跨膜能力, 这对于揭示活性小分子在生物体内的起效机制具有重要的启示意义。

5 PDVs的稳定性

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根据目前的研究结果, 多数PDVs在体外模拟生理条件和4 ℃冷藏条件下的稳定性较好^[43]。作者前期测定了生姜和地黄来源的囊泡在4 ℃条件下的稳定性, 发现囊泡的尺寸可维持半月以上未发生明显变化。反复冻融过程和冷冻(-80 ℃) 条件对PDVs稳定性的影响尚有不同观点^{[44, 45]}, 因该过程涉及脂膜破坏和重排, 对PDVs结构和功能的影响需要更多的实验评价。PDVs对酸碱和模拟胃肠道环境的耐受情况也有所不同。例如西柚来源PDVs的粒径在酸性溶液中比碱性溶液更均一^[29]。Zhuang等^[27]将生姜来源囊泡置于模拟胃液(pH 2.0) 和模拟肠液(pH 6.5) 中, 发现生姜囊泡在模拟胃液中粒径增大, 表面电荷由负变正, 随后在模拟肠溶液中继续作用, 粒径进一步增大, 表面电荷由正变负。应该注意到, 由于PDVs含有蛋白和脂质等多种天然聚电解质, 其电离水平受环境pH值影响较大, 同时电荷的高低与分布影响分子间作用力及颗粒聚集尺度, 因此单纯凭借电荷和粒径的数据变化来判断PDVs的稳定性是不可靠的, 还需要结合标志物的鉴定及活性评价进行综合分析。

6 PDVs的转运与分布

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自然界存在的很多植物是动物、人类的食物或药物的来源。PDVs进入生物体后的转运行为与PDVs的来源、给药方式和实验模型有关(表 1)。Mu等^[32]给小鼠口服葡萄、西柚、胡萝卜和生姜来源的PDVs, 发现肠道巨噬细胞和干细胞可以摄取这些PDVs。Teng等^[40]发现西柚来源囊泡经小鼠口服后趋向于向肝脏转移, 生姜来源囊泡则更多停留在肠道内。Cao等^[28]将人参来源囊泡经静脉和腹腔注射后, 主要分布在肝脏和脾脏, 而口服后, 囊泡主要分布在胃和肠道。Zhuang等^[27]通过标记肠内皮细胞和毛细淋巴血管发现, 生姜来源囊泡口服后沿肠内皮血管分布, 通过血管从肠道进入肝脏, 而在毛细淋巴管中未见明显的荧光, 表明生姜囊泡吸收后主要进入血液循环。目前关于不同PDVs的体内分布行为及机制还缺乏系统的研究, 笔者认为这一领域可以借鉴人源囊泡的相关报道。如Hoshino等^[46]将6种肿瘤细胞外泌体注射至小鼠体内, 发现这些外泌体倾向于分布到源肿瘤转移的器官, 并证明外泌体表面的整合素类型决定着肿瘤的转移部位。如整合素α₆β₄和α₆β₁与肿瘤的肺转移有关, 整合素α_vβ₅与肝转移有关。这对PDVs的分布行为和规律探究有一定的启发意义。

7 PDVs的功能和生物医学应用

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PDVs的功能主要体现在两个维度。一方面, 作为植物体的组成部分, 对植物自身生理功能维持、物质代谢和防御等有重要意义; 另一方面, 植物作为哺乳动物和人类的食物或药物来源, 在营养供给、防病治病等环节有重要作用和应用价值。

7.1 对植物自身的功能

7.1.1 维持正常生理和代谢

如前文所述, PDVs可能通过分泌细胞壁相关蛋白维持细胞的结构和功能, 并且还能清除细胞中的有害产物以及参与免疫监视等过程。对向日葵种子囊泡的蛋白质组学分析结果显示, PDVs与修饰细胞壁的酶的分泌有关^[47]。此外, PDVs还可参与细胞增殖、分化和对压力等刺激的响应过程^[48]。

7.1.2 参与植物体自身防御

研究表明, PDVs广泛参与自身防御反应, 尤其是在诱导型防御机制中扮演重要角色^[49]。Cai等^[8]发现, 拟南芥在受到灰葡萄球菌感染时感染部位会分泌PDVs, 并将携带的sRNA导入真菌体内, 沉默关键致病基因, 发挥跨物种防御调节作用。Rutter等^[7]发现感染了丁香假单胞菌的拟南芥PDVs分泌会增加, 类似地, 用水杨酸处理后的PDVs分泌也增多, 表明PDVs参与了植物的免疫防御反应。

7.2 作为治疗剂

7.2.1 治疗炎性肠病

炎性肠病(inflammatory bowel disease, IBD) 是一种慢性易复发的炎症性疾病, 主要包括溃疡性结肠炎和克罗恩病, 目前还没有能彻底治愈IBD的药物^[50]。Zhang等^[10]将提取的生姜来源囊泡给正常小鼠口服后发现主要分布于结肠部位, 由肠上皮细胞和巨噬细胞摄取。应用于肠炎模型小鼠后可明显下调炎性细胞因子肿瘤坏死因子-α (TNF-α)、IL-6和IL-1β, 并上调抗炎因子IL-10和IL-22, 促进损伤黏膜的修复。除生姜外, 其他PDVs也有类似效果。Ju等^[26]将葡萄来源囊泡用于结肠炎小鼠后发现能诱导Lgr5⁺肠干细胞增殖, 通过Wnt信号通路调节促使肠干细胞生长和增殖的基因的表达, 使治疗组小鼠的死亡率明显降低, 肠长度趋于正常等。西兰花来源囊泡可显著降低结肠组织中干扰素γ (IFN-γ)、IL-17A和TNF-α的表达, 升高IL-10水平, 有效缓解结肠炎小鼠的症状^[29]。这些发现为结肠炎等肠道疾病的防治提供了新思路。

7.2.2 治疗牙周炎

研究表明, 生姜来源囊泡对牙龈卟啉单胞菌有抑制作用, 其作用机制是囊泡中特定比例的PA可与细菌表面的HBP35蛋白结合, 抑制细菌生长。因此, 生姜来源囊泡有望开发为防治慢性牙周炎的药物^[51]。

7.2.3 抗肿瘤

有研究报道, 一些PDVs能有效抑制肿瘤生长。Raimondo等^[31]研究发现, 柠檬来源囊泡可以特异性靶向肿瘤部位, 通过激活TRAIL信号通路介导的肿瘤细胞凋亡过程, 抑制多发性骨髓瘤的生长。Cao等^[28]发现人参来源囊泡与巨噬细胞接触后会被迅速识别、内化, 诱导巨噬细胞M1型极化, 促进活性氧(reactive oxygen species, ROS) 产生, 促使小鼠黑色素瘤细胞凋亡, 抑制小鼠肿瘤生长。

7.2.4 治疗酒精性肝损伤

Zhuang等^[27]将生姜来源囊泡给正常小鼠口服, 发现主要在肝脏和肠系膜淋巴结中累积, 作用于肝细胞时可抑制ROS产生。囊泡处理后的酒精性肝损伤小鼠肝脏中的脂滴减少, 甘油三酯水平下降, 肝脏的重量减轻, 明显优于未给药模型组, 表明生姜囊泡具有预防和治疗酒精性肝损伤的潜力。

7.2.5 美白作用

Lee等^[52]发现黄漆木茎叶来源囊泡有抗黑色素生成的作用。将黄漆木茎叶来源的囊泡作用于B16BL6黑色素瘤细胞能显著降低黑色素含量和酪氨酸酶(tyrosinase, TYR) 活性, 叶源性囊泡可以抑制黑色素生成基因及酶的表达。其美白活性优于熊果苷的阳性对照组, 且无明显毒性。类似结果在人表皮模型中也得到证实。

7.2.6 抗纤维化

Du等^[53]发现, 中药红景天中含有sRNA (HJT-sRNA-m7) 可以靶向肺纤维化相关蛋白, 降低纤维化因子表达, 改善肺纤维化症状。而红景天汤药中的大部分脂质成分也存在于穿心莲、蒲公英和金银花中, 并证明脂质形成的囊泡可以促进sRNA的吸收入血。尽管该研究未强调囊泡结构及其与RNA的关系, 但药用植物煎煮形成纳米粒的现象非常普遍^[54], 且分离和表征手段也与榨汁得到的PDVs相似, 本质上都是植物样本经过不同的加工形式得到的包含多重活性成分的纳米结构(表 2)。

7.3 作为药物载体

PDVs具有蛋白脂膜结构, 膜材包裹的囊腔也为活性分子的装载提供了空间。由于PDVs天然具有装载货物和远程递送的职能, 因此可用于携带治疗剂, 发挥纳米载体的作用^[55]。PDVs具有广泛的运载能力, 可装载亲疏水药物和核酸药物等^{[56, 57]}。另外, 借助其纳米级特性, PDVs也可提高药物的病灶靶向性和组织渗透性。目前PDVs作为载体一般通过两种方法, 一种是将分离到的PDVs直接用于载药; 另一种是提取PDVs中的部分组分, 重新组装后作为脂质载体或插入功能基团后作为复合载体。

7.3.1 PDVs直接作为药物载体

Wang等^[30]将甲氨蝶呤载入西柚来源囊泡, 发现甲氨蝶呤的毒副作用显著降低, 提高了对DSS诱导的小鼠结肠炎的治疗作用。

7.3.2 PDVs重组装后作为药物载体

Zhang等^[58]将多柔比星载入生姜囊泡脂质提取物, 可被结肠癌细胞摄取。与游离药物相比, 载药PDVs的肿瘤靶向性增强, 能更好地抑制肿瘤生长。Wang等^[57]将西柚来源囊泡含有的脂质提取出来, 合成了新的纳米粒, 该粒子平均粒径为180 nm, 有很好的细胞摄取能力和组织趋向性, 载入抗肿瘤药物JSI-124后有明显的肿瘤抑制作用。Teng等^[56]发现, 西柚囊泡的脂质提取物包裹miR-18a后可以诱导M1巨噬细胞, 并激活自然杀伤细胞和自然杀伤T细胞, 抑制结肠癌的肝转移。同样, Zhuang等^[59]将西柚囊泡用叶酸修饰后包裹聚乙烯亚胺, 发现不仅可以提高RNA的携带能力, 并且消除了聚乙烯亚胺的毒性, 将西柚囊泡修饰并包裹miR17后经鼻腔给药, 可以透过血脑屏障, 被GL-26肿瘤细胞选择性地摄取入脑内, 延缓小鼠脑肿瘤的生长^[57]。

8 总结与展望

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植物囊泡虽然发现较早, 但相比哺乳动物和人源囊泡, 研究还不够深入, 特别是在生物医学领域大部分处于实验室研究阶段。近年来, 哺乳动物和人体来源囊泡研究取得巨大进展, 这为PDVs的研究提供了可借鉴的思路和方法。然而, 植物与动物、人类分属于不同物种, 且亲缘关系较远, 从中获得的PDVs往往存在较大差异, 探究PDVs结构和功能的独特性是未来该领域需要深入开展的工作。此外, 植物存在地域分布和季节性差异, 不同地区和时令下获得的PDVs是否稳定一致也需要系统评价。特别是作为治疗剂或药物载体用于生物医学领域时, 产品的安全、有效、稳定和可控是必须满足的金标准。再者, 目前很多PDVs来源于鲜品榨汁, 提取的PDVs如何保存是决定其是否适合规模化加工的制约因素, 这方面的研究目前还比较缺乏, 有些报道甚至存在相反之处, 其内在原因还需深入研究。不过, 有报道^[60]从烟草、小曼长春花和槲寄生3种植物的干品中也分离到PDVs, 证明PDVs可以耐受干燥过程, 这为其规模化加工储存提供了条件。

与哺乳动物和人体来源囊泡存在的问题类似, 产率和纯度仍是实现PDVs临床转化的前提, 因为低产率和低纯度意味着更高的成本, 很难得到工业界的青睐, 也不符合临床应用的要求。但应该看到, 随着细胞工程、生物工程和发酵技术的发展, 以及无土栽培技术的不断成熟, 未来有望将植物从田间栽培引向工业生产, 这或许是该领域的未来趋势。

作者贡献: 赵梦负责文章撰写及部分资料搜集; 李思敏和张蕾负责部分资料搜集; 丛明慧负责文章资料搜集及图片制作; 胡立宏对文章提供指导性建议; 乔宏志拟定文章框架并审校全文。

利益冲突: 所有作者声明本文不存在任何利益冲突。

基金

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中国科协青年人才托举工程项目(2017QNRC001)
中央本级重大增减支项目(2060302-1907-04)
江苏省高等学校自然科学研究重大项目(20KJA360004)
江苏省六大人才高峰项目(SWYY-057)
江苏省青蓝工程项目
江苏省研究生创新工程项目(SJCX20_0547)
江苏省研究生创新工程项目(SJCX20_0546)
江苏省研究生创新工程项目(KYCX20_1497)

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2021年第56卷第8期

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doi: 10.16438/j.0513-4870.2020-1514

接收时间：2020-09-18
首发时间：2025-12-18
出版时间：2021-08-12

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收稿日期：2020-09-18
修回日期：2020-11-16

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中国科协青年人才托举工程项目(2017QNRC001)

中央本级重大增减支项目(2060302-1907-04)

江苏省高等学校自然科学研究重大项目(20KJA360004)

江苏省六大人才高峰项目(SWYY-057)

江苏省青蓝工程项目

江苏省研究生创新工程项目(SJCX20_0547)

江苏省研究生创新工程项目(SJCX20_0546)

江苏省研究生创新工程项目(KYCX20_1497)

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1.南京中医药大学药学院, 江苏省中药功效物质重点实验室, 江苏南京 210023

2.江苏省中药高效给药系统工程技术研究中心, 江苏南京 210023

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*乔宏志, Tel: 86-25-85811050, E-mail: qiaohz@njucm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Plant	Isolation method	Size/nm	Zeta potential/mV	Animal model and administration route	Distribution	Ref
Grape	Differential ultracentrifugation and density gradient ultracentrifugation	~380	~-26	Healthy mice, p.o.	Intestinal stem cells	^[26]
Ginger	Differential ultracentrifugation and density gradient ultracentrifugation	102-998	-29 - -24	Healthy mice, p.o.	Liver, mesenteric, lymph nodes	^[27]
	Differential ultracentrifugation and density gradient ultracentrifugation	~231	~-12	Healthy mice, p.o.	Colon	^[10]
Ginseng	Differential ultracentrifugation and density gradient ultracentrifugation	~344	~-25	Healthy mice, i.p.; i.v. healthy mice, p.o.	Liver, spleen Stomach, gut	^[28]
Broccoli	Differential ultracentrifugation and density gradient ultracentrifugation	18-118	~-39	Colitismice, p.o.	Colon, mesenteric, lymph nodes, spleen	^[29]
Grapefruit	Differential ultracentrifugation and density gradient ultracentrifugation	~210	~-49	Healthy mice, p.o.	Middle and distal part of small intestine, cecum, colon	^[30]
Lemon	Differential ultracentrifugation and density gradient ultracentrifugation	50-70	~-22^a	Tumor-bearingmice, i.p.	Tumor, liver, spleen, kidney	^[31]
Carrot	Differential ultracentrifugation and density gradient ultracentrifugation			C57BL/6 mice or knockout mice, p.o.	Small and large intestine macrophages, intestinal stem cells	^[32]

Plant

Isolation method

Size/nm

Zeta potential/mV

Animal model and administration route

Distribution

Ref

Grape

Differential ultracentrifugation and density gradient ultracentrifugation

~380

~-26

Healthy mice, p.o.

Intestinal stem cells

^[26]

Ginger

Differential ultracentrifugation and density gradient ultracentrifugation

102-998

-29 - -24

Healthy mice, p.o.

Liver, mesenteric, lymph nodes

^[27]

Differential ultracentrifugation and density gradient ultracentrifugation

~231

~-12

Healthy mice, p.o.

Colon

^[10]

Ginseng

Differential ultracentrifugation and density gradient ultracentrifugation

~344

~-25

Healthy mice, i.p.; i.v. healthy mice, p.o.

Liver, spleen Stomach, gut

^[28]

Broccoli

Differential ultracentrifugation and density gradient ultracentrifugation

18-118

~-39

Colitismice, p.o.

Colon, mesenteric, lymph nodes, spleen

^[29]

Grapefruit

Differential ultracentrifugation and density gradient ultracentrifugation

~210

~-49

Healthy mice, p.o.

Middle and distal part of small intestine, cecum, colon

^[30]

Lemon

Differential ultracentrifugation and density gradient ultracentrifugation

50-70

~-22^a

Tumor-bearingmice, i.p.

Tumor, liver, spleen, kidney

^[31]

Carrot

Differential ultracentrifugation and density gradient ultracentrifugation

C57BL/6 mice or knockout mice, p.o.

Small and large intestine macrophages, intestinal stem cells

^[32]

Plant origin	Functionality	Mechanism	Ref
Ginger	Treatment of IBD	Itinhibits the expression of TNF-α, IL-6 and IL-1β and increases the expression of IL-10 and IL-22	^[10]
	Prevent chronicperiodontitis	It binds to the HBP35 protein on the surface of Porphyromonas gingivalis, inhibits the expression of bacteria-related mRNA, inhibits the transfer of bacteria to normal cells, and reduces the expression of protein and mRNA of FimA	^[51]
	Protect against alcoholinduced liver damage	It induces the expression of Nrf2, inhibits the production of ROS and LPS-induced inflammation by TLRs mediated pathway	^[27]
Broccoli	Treatment of IBD	It increases the expression of IL-10, reduces the IFN-γ, IL-17A and TNF-α in colon tissue; mediates activation of AMPK in DCs	^[29]
Ginseng	Inhibit melanoma growth	It induces the polarization of M1-like macrophages by TLR-4 and MyD88 signaling pathways, promotes the production of ROS and significantly increases M1-related cytokines and chemokines (CCL5, IL-6, MCP-1, TNF-α, IL-1α, IL-12)	^[28]
Grape	Treatment of colitis	It targets Lgr5⁺ intestinal stem cells, enables intestinal epithelial cell proliferation and directly promotes the generation of stem cell-like organs	^[26]
Dendropanaxmor bifera	Whitening	It reduces the activity of tyrosinase and inhibits the expression of melanin-producing related enzymes (TYR, TRP-1, TRP-2)	^[52]
Lemon	Antitumor	It increases the expression of pro-apoptotic gene, inhibits the expression of antiapoptotic gene, activates a TRAIL-mediated apoptotic cell process, and inhibits the secretion of pro-angiogenic factors (VEGF-A, IL-6, and IL-8)	^[31]
Carrot	Treatment of colitis	It increases nuclear translocation of Nrf2 and induces macrophage to produce IL-10	^[32]
Rhodiolacrenulata	Treatment of pulmonary fibrosis	Lipids facilitates cellular uptake of HJT-sRNA-m7, HJT-sRNA-m7 reduces the expression of α-SMA fibronectin and COL3A1	^[53]

Plant origin

Functionality

Mechanism

Ref

Ginger

Treatment of IBD

Itinhibits the expression of TNF-α, IL-6 and IL-1β and increases the expression of IL-10 and IL-22

^[10]

Prevent chronicperiodontitis

It binds to the HBP35 protein on the surface of Porphyromonas gingivalis, inhibits the expression of bacteria-related mRNA, inhibits the transfer of bacteria to normal cells, and reduces the expression of protein and mRNA of FimA

^[51]

Protect against alcoholinduced liver damage

It induces the expression of Nrf2, inhibits the production of ROS and LPS-induced inflammation by TLRs mediated pathway

^[27]

Broccoli

Treatment of IBD

It increases the expression of IL-10, reduces the IFN-γ, IL-17A and TNF-α in colon tissue; mediates activation of AMPK in DCs

^[29]

Ginseng

Inhibit melanoma growth

It induces the polarization of M1-like macrophages by TLR-4 and MyD88 signaling pathways, promotes the production of ROS and significantly increases M1-related cytokines and chemokines (CCL5, IL-6, MCP-1, TNF-α, IL-1α, IL-12)

^[28]

Grape

Treatment of colitis

It targets Lgr5⁺ intestinal stem cells, enables intestinal epithelial cell proliferation and directly promotes the generation of stem cell-like organs

^[26]

Dendropanaxmor bifera

Whitening

It reduces the activity of tyrosinase and inhibits the expression of melanin-producing related enzymes (TYR, TRP-1, TRP-2)

^[52]

Lemon

Antitumor

It increases the expression of pro-apoptotic gene, inhibits the expression of antiapoptotic gene, activates a TRAIL-mediated apoptotic cell process, and inhibits the secretion of pro-angiogenic factors (VEGF-A, IL-6, and IL-8)

^[31]

Carrot

Treatment of colitis

It increases nuclear translocation of Nrf2 and induces macrophage to produce IL-10

^[32]

Rhodiolacrenulata

Treatment of pulmonary fibrosis

Lipids facilitates cellular uptake of HJT-sRNA-m7, HJT-sRNA-m7 reduces the expression of α-SMA fibronectin and COL3A1

^[53]