Laboratory Animal Center, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, People's Republic of China.
Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an, Shaanxi, People's Republic of China.
Int J Nanomedicine. 2022 May 19;17:2301-2318. doi: 10.2147/IJN.S354003. eCollection 2022.
Extracellular vesicles (EVs), as a promising platform for drug delivery, have attracted much attention. Degradation and regeneration of EVs maintain their homeostasis in vivo, but this regeneration is missing in the in vitro culture (IVC) system, which is likely to lead to negative effects. It is particularly concerning that most studies involving the addition of EVs in IVC seem to overlook this point.
We used rabbit embryos and oviduct fluid EVs as a model of embryo development to examine the effect of loss or gain of EV functionality in an IVC system. Embryonic development ratios were determined in each group. Malondialdehyde and ammonium ions in the culture medium were measured. RNA-seq, reactive oxygen species (ROS) staining, immunofluorescence of LC3 and H3K36me3, and qPCR of oxidative stress-related genes and autophagy-related genes of blastocysts in the in vivo group, non-EVs group, con-EVs group, and R-EVsM group was implemented.
Incubation of embryos with 9.1×10 EV particles/mL had a positive effect at 48 h and 72 h, which disappeared by 96 h, however. EVs at a concentration of 9.1×10 particles/mL even showed a negative effect at 96 h. As culture time in the IVC system was increased, the amount of malondialdehyde and ammonium ions in the culture medium was increased, and there was a decrease in embryonic development activity of EVs. Lack of EV renewal in the IVC system impaired embryonic development competence, while replacement of EVs and medium during IVC could sustain embryonic development. Loss or gain of renewal in the IVC system affected EVs' influence on embryo transcriptome, embryonic ROS, autophagy, epigenetic state and apoptosis.
Loss of renewal in the IVC system affected EVs' role in embryonic development by causing an imbalance in ROS, autophagy, abnormal H3K36me3 levels and apoptosis, while gain of renewal in the IVC system reduced these adverse effects and ensured the beneficial function of EVs.
细胞外囊泡 (EVs) 作为一种有前途的药物传递平台，引起了广泛关注。EVs 的降解和再生维持了其在体内的内稳态，但在体外培养 (IVC) 系统中这种再生是缺失的，这可能会导致负面效应。特别值得关注的是，大多数涉及在 IVC 中添加 EV 的研究似乎都忽略了这一点。
我们使用兔胚胎和输卵管液 EVs 作为胚胎发育模型，研究 IVC 系统中 EV 功能丧失或获得对胚胎发育的影响。在每组中确定胚胎发育比例。测量培养基中的丙二醛和铵离子。对体内组、非 EVs 组、对照 EVs 组和 R-EVsM 组的囊胚的 RNA-seq、活性氧 (ROS) 染色、LC3 和 H3K36me3 的免疫荧光、氧化应激相关基因和自噬相关基因的 qPCR 进行了研究。
在 48 h 和 72 h 时，胚胎孵育 9.1×10 EV 颗粒/ml 有积极作用，但在 96 h 时消失。浓度为 9.1×10 颗粒/ml 的 EVs 甚至在 96 h 时显示出负面作用。随着 IVC 系统培养时间的增加，培养基中丙二醛和铵离子的含量增加，EV 对胚胎发育活性降低。IVC 系统中缺乏 EV 更新会损害胚胎发育能力，而在 IVC 中更换 EV 和培养基可以维持胚胎发育。IVC 系统中更新的缺失或获得会影响 EV 对胚胎转录组、胚胎 ROS、自噬、表观遗传状态和细胞凋亡的影响。
IVC 系统中更新的缺失通过导致 ROS、自噬、异常 H3K36me3 水平和细胞凋亡失衡，影响 EVs 在胚胎发育中的作用，而 IVC 系统中更新的获得减少了这些不利影响，并确保了 EVs 的有益功能。
