Exosome Research Articles (Page 1)

The effect of electric field microenvironment on the exosome secretion of PC12 cells by chitosan carbon dots.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell-cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation-contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (-25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

BackgroundNeuroinflammation is a characteristic pathological change of Alzheimer’s Diseases (AD). Microglia have been reported to participate in inflammatory responses within the central nervous system. However, the mechanism of microglia released exosome (EXO) contribute to communication within AD microenvironment remains obscure.MethodsThe interaction between microglia and AD was investigated in vitro and in vivo. RNA-binding protein immunoprecipitation (RIP) was used to investigate the mechanisms of miR-223 and YB-1. The association between microglia derived exosomal YB-1/miR-223 axis and nerve cell damage were assessed using Western blot, immunofluorescence, RT-PCR, ELISA and wound healing assay.ResultsHere, we reported AD model was responsible for the M1-like (pro-inflammatory) polarization of microglia which in turn induced nerve cell damage. While M2-like (anti-inflammatory) microglia could release miR-223-enriched EXO which reduced neuroinflammation and ameliorated nerve damage in AD model in vivo and in vitro. Moreover, YB-1 directly interacted with miR-223 both in cell and EXO, and participated in microglia exosomal miR-223 loading.ConclusionThese results indicate that anti-inflammatory microglia-mediated neuroprotection form inflammatory damage involves exporting miR-223 via EXO sorted by YB-1. Consequently, YB-1-mediated microglia exosomal sorting of miR-223 improved the nerve cell damage repair, representing a promising therapeutic target for AD.

Improvement of biomedical properties of PEO-treated titanium with flurbiprofen and exosome conjugation

Translational strategies to characterize and monitor extracellular vesicles such as exosome (EX) shedding and the clinical impact of this data within hepatocellular carcinoma (HCC) remains unclear. In this study, EX shedding was assessed in early-stage HCC and evaluated as a stratification factor for time to progression (TTP) following first-cycle liver-directed therapy (LDT). Plasma EXs were isolated from HCC patients undergoing LDT using ultracentrifugation. Purified EXs were stained using markers CD9 and CD63 and quantified using an ImageStreamX flow cytometer. Circulating EXs expressing CD9 were isolated at 10-fold higher levels compared to CD63. The intensity of CD9+ EX shedding following LDT was positively correlated with treatment response. High post-LDT CD9+ EX shedding stratified TTP risk with a 30% lower frequency of disease progression at 1 year following LDT. Post-LDT high CD9+ EX shedding was observed in 100% (10/10) of patients successfully bridged to liver transplantation while only 22% (2/9) of patients with tumor progression had high CD9+ EX shedding post-LDT. CD9+ EX shedding also stratified TTP risk within the first cycle objective response rate (ORR) group, identifying patients still at higher disease progression. EX shedding was concordant with imaging response rate, stratified TTP in early-stage HCC, and may have important implications for assessing post-LDT viable, biologically aggressive HCC.

Simple SummaryWith the aim of improving the therapeutic potential of mesenchymal stromal/stem cells (MSCs), we analyzed miRNA expression to investigate the effects of priming on intracellular and exosome (EXO)-derived miRNAs of MSCs. We primed MSCs with 3D culture (3D MSCs) or IFN-γ treatment (γ-MSCs), and EXOs were isolated from the conditioned medium. The miRNA analysis revealed similar expression patterns in intracellular miRNAs among biological replicates, while we observed noticeable variability in EXO miRNAs released even with the same priming condition. Although the MSCs and their EXOs exhibited distinct miRNA profiles following each priming treatment, we found deregulated miRNAs in common between the two sample types. The gene ontology of the deregulated miRNAs obtained after priming showed that MSC and EXO-derived miRNAs were functionally associated with tissue repair/regeneration. Specifically, γ-MSCs, 3D MSC EXOs, and γ-MSC EXOs contained more enriched miRNAs related to immunomodulation compared with 3D MSCs. Moreover, compared with IFN-γ treatment, both cells and EXOs derived from the 3D culture had more enriched miRNAs targeting genes involved in angiogenesis. Our study demonstrates that both 3D culture and IFN-γ treatment are able to modify intracellular and exosomal miRNAs, and our findings might contribute to a better understanding of the molecular mechanisms underlying the miRNA-mediated beneficial effects of MSCs.Mesenchymal stromal/stem cells (MSCs) have emerged as a therapeutic tool in regenerative medicine. Recent studies have shown that exosome (EXO)-derived microRNAs (miRNAs) play a crucial role in mediating MSC functions. Additionally, intracellular miRNAs have been found to regulate MSC therapeutic capacities. However, the molecular mechanisms underlying miRNA-mediated MSC effects are not fully understood. We used 3D culture and IFN-γ to prime/enhance the MSC therapeutic effects in terms of functional miRNAs. After priming, our analysis revealed stable variations in intracellular miRNA among the MSC biological replicates. Conversely, a significant variability of miRNA was observed among EXOs released from biological replicates of the priming treatment. For each priming, we observed distinct miRNA expression profiles between the MSCs and their EXOs. Moreover, in both types of priming, gene ontology (GO) analysis of deregulated miRNAs highlighted their involvement in tissue repair/regeneration pathways. In particular, the 3D culture enhanced angiogenic properties in both MSCs and EXOs, while IFN-γ treatment enriched miRNAs associated with immunomodulatory pathways. These findings suggest that 3D culture and IFN-γ treatment are promising strategies for enhancing the therapeutic potential of MSCs by modulating miRNA expression. Additionally, the identified miRNAs may contribute to understanding the molecular mechanisms underlying the miRNA-mediated therapeutic effects of MSCs.

ABSTRACT Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA (76.2% of total reads on average), whereas EV-D was notably enriched in human RNA (70.3% of total reads on average). As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV, P < 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest.

Introduction: The current study aimed to construct a circNR4A1 loaded exosome (EXO) nano system for the treatment of oral squamous cell carcinoma (OSCC) and elucidate the related regulation mechanism in chemoresistance and tumor biology. Methods: Exosomes were isolated from the HEK293 cells by the ultracentrifugation method. Then, the purified nanoproduction was characterized and identified by transmission electron microscopy, dynamic light scattering, and Western blot. The hydrophobic circNR4A1 was then loaded into exosomes by the coincubation method. The effects of circNR4A1 on chemotherapy and apoptosis were evaluated in three multiresistant OSCC cell lines. Results: The range of size distribution of the exosomes was found to be 40-170 nm. Positive TSG101 and CD63 expressions were observed, and the expression of calnexin was negative. In the cisplatin group, circNR4A1 could sensitize 67% of cell lines, while circNR4A1-EXO could sensitize 100% of cell lines. In the 5FU group, circNR4A1 could only sensitize 33% of cell lines, while circNR4A1-EXO could sensitize 67% of cell lines. circNR4A1-EXO is involved in multiple signaling pathways, which can not only activate K-RAS/ERK and MDR-1 signaling pathways but activate DNMT-1 gene expression simultaneously. Conclusion: circNR4A1-EXO can increase the sensitivity of OSCC to anticancer drugs, which may be due to the regulation of the K-RAS/ERK and p53 signaling pathway.

A significant number of cancer-related deaths are recorded globally each year, despite attempts to cure this illness. Medical science is working to develop new medication therapies as well as to find ways to identify this illness as early as possible, even using noninvasive techniques. Early detection of cancer can greatly aid its treatment. Studies into cancer diagnosis and therapy have recently shifted their focus to exosome (EXO) biomarkers, which comprise numerous RNA and proteins. EXOs are minuscule goblet vesicles that have a width of 30 to 140 nm and are released by a variety of cells, including immune, stem, and tumor cells, as well as bodily fluids. According to a growing body of research, EXOs, and cancer appear to be related. EXOs from tumors play a role in the genetic information transfer between tumor and basal cells, which controls angiogenesis and fosters tumor development and spread. To identify malignant activities early on, microRNAs (miRNAs) from cancers can be extracted from circulatory system EXOs. Specific markers can be used to identify cancer-derived EXOs containing miRNAs, which may be more reliable and precise for early detection. Conventional solid biopsy has become increasingly limited as precision and personalized medicine has advanced, while liquid biopsy offers a viable platform for noninvasive diagnosis and prognosis. Therefore, the use of body fluids such as serum, plasma, urine, and salivary secretions can help find cancer biomarkers using technologies related to EXOs.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected with current clinical tools. Herein, an exosome (EX)-based imaging tool is developed for lung micrometastasis by positron emission tomography (PET) using osteosarcoma-derived EXs as natural nanocarriers of the positron-emitter copper-64 (64 Cu). Exosomes are isolated from metastatic osteosarcoma cells and functionalized with the macrocyclic chelator NODAGA for complexation with 64 Cu. Surface functionalization has no effect on the physicochemical properties of EXs, or affinity for donor cells and endows them with favorable pharmacokinetics for in vivo studies. Whole-body PET/magnetic resonance imaging (MRI) images in xenografted models show a specific accumulation of 64 Cu-NODAGA-EXs in metastatic lesions as small as 2-3mm or in a primary tumor, demonstrating the exquisite tropism of EXs for homotypic donor cells. The targetability for lung metastasis is also observed by optical imaging using indocyanine green (ICG)-labeled EXs and D-luciferin-loaded EXs. These findings show that tumor-derived EXs hold great potential as targeted imaging agents for the noninvasive detection of small lung metastasis by PET. This represents a step forward in the biomedical application of EXs in imaging diagnosis with increased translational potential.

Exosomes of human cardiosphere-derived cells (CDCs) are very promising for treating cardiovascular disorders. However, the current challenge is inconvenient delivery methods of exosomes for clinical application. The present study aims to explore the potential to enhance the therapeutic effect of exosome (EXO) from human CDCs to myocardial hypertrophy. A heart homing peptide (HHP) was displayed on the surface of exosomes derived from CDCs that were forced to express the HHP fused on the N-terminus of the lysosomal-associated membrane protein 2b (LAMP2b). The cardiomyocyte-targeting capability of exosomes were analyzed and their therapeutic effects were evaluated in a mouse model of myocardial hypertrophy induced by transverse aorta constriction (TAC). The molecular mechanisms of the therapeutic effects were dissected in angiotensin II-induced neonatal rat cardiomyocyte (NRCMs) hypertrophy model using a combination of biochemistry, immunohistochemistry and molecular biology techniques. We found that HHP-exosomes (HHP-EXO) accumulated more in mouse hearts after intravenous delivery and in cultured NRCMs than control exosomes (CON-EXO). Cardiac function of TAC mice was significantly improved with intravenous HHP-EXO administration. Left ventricular hypertrophy was reduced more by HHP-EXO than CON-EXO via inhibition of β-MHC, BNP, GP130, p-STAT3, p-ERK1/2, and p-AKT. Similar results were obtained in angiotensin II-induced hypertrophy of NRCMs, in which the beneficial effects of HHP-EXO were abolished by miRNA-148a inhibition. Our results indicate that HHP-EXO preferentially target the heart and improve the therapeutic effect of CDCs-exosomes on cardiac hypertrophy. The beneficial therapeutic effect is most likely attributed to miRNA-148a-mediated suppression of GP130, which in turn inhibits STAT3/ERK1/2/AKT signaling pathway, leading to improved cardiac function and remodeling.

Mesenchymal stem cells derived from adipose tissue (ADMSCs) are being increasingly considered in regenerative medicine-based clinical applications. Apart from possessing therapeutic applications themselves, ADMSCs also secrete a myriad of soluble factors which are promising candidates for treating several degenerative diseases such as osteoarthritis and neurodegenerative diseases, wound repair as well as for cosmeceutical purposes. In our research study, we successfully isolated ADMSCs in-house, now called CKC-Endeavour-1 from the lipoaspirate sample of a patient who underwent liposuction. The subsequent expansion of cells was performed in xeno-free and serum-free conditions and their characterisation was performed using tri-lineage differentiation studies. The levels of differentiation were assessed by staining and gene expression which was observed to be comparable between the in-house developed ADMSC cell line and the commercially purchased ADMSCs. Following characterisation, the secretory components from these MSCs, namely, conditioned media (ADMSC-CM) and exosomes (ADMSC-EXO) were harvested from CKC-Endeavour-1 under xeno-free, serum-free, and supplement-free conditions followed by lyophilisation in order to attempt to prolong its shelf-life. The comprehensive analysis of the secretome profile of ADMSC-CM using carried out using cytokine array and demonstrated the presence of 105 cytokines and growth factors. Also, clinical grade Izon columns were used to isolate the exosomes from ADMSC-CM obtaining exosomes in the size range of <200nm, analysed using nanoparticle tracking analysis. Overall, our study developed an ADMSC cell line, CKC-Endeavour-1, along with their CM and exosome (EXO) products under clinically safe conditions. Additionally, we have obtained a comprehensive understanding of the secreted factors present in the ADMSC-CM which could be further explored in detail to tap the best therapeutic benefits from them.

Extracellular vesicles (EVs) are vesicles secreted by cells into the extracellular space, first discovered in 1967 as platelet dust. In recent years, the idea that EVs can treat various diseases has emerged in studies to understand these vesicles' origin and biological functions. According to their size, biogenesis, content, release pathways and function, EVs have three main subtypes: microvesicle (MV), exosome (EX) and apoptotic body. EVs are found in all body fluids, including urine, plasma, and physiological fluids such as bronchial lavage. In addition, it is secreted by many cell types such as dendritic cells, B cells, T-cells, mast cells, tumour cells, and sperm. This literature article reviewed studies using stem cell-derived EVs in numerous clinical and preclinical studies.

Mesenchymal stem cells (MSCs) are crucial cells that play an essential role in the maintenance, self-renewal, and proliferation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow niche. It has been proven that MSCs can be used as a feeder layer for the proliferation of HSCs to enhance the number of HPCs and HSCs. Recently, it has been demonstrated that MSC-derived exosome (MSC-DE) has critical roles in different biological processes in bone marrow (BM). In the current research, we examined the importance of hypoxia-preconditioned MSC-derived exosomes (HP-MSC-DE) and normoxia-preconditioned MSC-derived exosomes (NP-MSC-DE) in the self-renewal and long-term clonogenic potential of umbilical cord blood hematopoietic stem cells (UCB-HSCs). We showed that the secretion rate and component of the exosome (EXO) were changed in HP-MSC-DE compared to NP-MSC-DE. Notably, the Jagged-1 (Notch ligand) content of EXO was much more plentiful in HP-MSC-DE compared to NP-MSC-DE. The addition of HP-MSC-DE enriched by Jagged-1 to the co-culture system stimulates the Notch pathway on the membrane of UCB-HSCs CD133+ and enhances proliferation. HP-MSC-DE induction using an anti-Jagged-1 antibody suppresses all biological functions of the Jagged-1 protein. Importantly, HP-MSC-DE containing Jagged-1 could change the biology of HSCs CD133+ and increase the self-renewal capacity, quiescence, and clonogenic potential of CD133+ cells. Moreover, they support generating a large number of primitive cells. Our study signified the importance of HP-MSC-DE in the proliferation of UCB-HSCs CD133+, which manifested therapeutic applications of EXO in the enhanced number of HSCs and subsequently alleviated bone marrow transplantation.

Radiotherapy (RT) is a standard treatment strategy for many cancer types, but the need to frequently apply high doses of ionizing radiation in order to achieve therapeutic efficacy can cause severe harm to healthy tissues, leading to adverse patient outcomes. In an effort to minimize these toxic side effects, we herein sought to design a novel approach to the low-dose RT treatment of hypoxic tumors using a Tirapazamine (TPZ)-loaded exosome (EXO) nanoplatform (MT). This MT platform was synthesized via loading EXOs with TPZ, which is a prodrug that is activated when exposed to hypoxic conditions. MT application was able to achieve effective tumor inhibition at a relatively low RT dose (2 Gy) that was superior to standard high-dose (6 Gy) RT treatment with specific targeting to the hypoxic region of tumor. RT-mediated oxygen consumption further aggravated hypoxic conditions to improve TPZ activation and treatment efficacy. Together, our findings demonstrate the clinical promise of this MT platform as a novel tool for the efficient radiosensitization and treatment of cancer patients.

Development of Lectin-immobilized Spongy Monoliths for Sub-classification of Exosome

IntroductionTRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can induce apoptosis in a variety of cancer cells. However, drug resistance of tumor and short half-life seriously affects its clinical targeted therapy. Cabazitaxel (CTX) is a taxane drug, which can induce apoptosis or autophagy by inhibiting the phosphorylation of PI3K/Akt/mTOR and sensitive to some drug-resistant tumors. Therefore, we explored the possibility of developing a mesenchymal stem cell-derived exosomes (MSC-EXO) vector for oral squamous cell carcinoma (OSCC) to deliver CTX/TRAIL combinations.MethodsAfter ultracentrifugation and dialysis, CTX/TRAIL loaded exosomes transfected MSC (MSCT)-derived exosome (EXO) (MSCT-EXO/CTX) were isolated and purified. The expression of CD63, CD9 and TRAIL was detected by BCA to confirm the origin of EXO. High-performance liquid chromatography (HPLC) was used to determine the drug loading of VPF and draw the in vitro release profile. MTT assay, flow cytometry and Western blot were used to detect the antitumor effect of MSCT-EXO/CTX in vitro. Subsequently, the antitumor effect of MSCT-EXO/CTX in vivo was verified by mouse model.ResultsThe diameter of the membrane particles was about 60–150 nm. We have proved that the incorporation and release of CTX in MSCT-EXO can inhibit the activation of PI3K, Akt and mTOR, which is a possible synergistic mechanism of CTX. MSCT-EXO and CTX can induce the apoptosis of SCC25 tumor cells in a dose-dependent manner and exert a good synergistic effect in the proportion range of 10:1–5:1. The inherent activity of MSCT-EXO and the direct effect of MSCT-EXO/CTX on OSCC confirm that MSCT-EXO/CTX makes MSCT-EXO and CTX have an efficient synergistic effect and a highly effective pharmacological inhibition on cancer cells, as verified by the subsequent mouse model. MSCT-EXO/CTX showed the lowest relative tumor volume and the highest tumor inhibition rate (P<0.05) in vivo.ConclusionAn MSCT-EXO-based CTX delivery system might be an effective anticancer method.

Considering the significance of effective antigen presentation for boosting immune responses, it is essential to develop delivery systems for antigen presenting cells (APCs; dendritic cells and macrophages). As a simple and facile way for improving delivery efficiency of PLGA microspheres (MS) into APCs, we fabricated exosome-conjugated PLGA MS via polydopamine coating in this study. Spherical micro-sized particles were first prepared by conventional water-in oil-in water (W1/O/W2) double emulsion and solvent evaporation methods and were observed by scanning electron microscopy (SEM). With increasing model protein (ovalbumin)/MS weight ratios, higher amounts of ovalbumin (OVA) were immobilized onto MS. After exosome (EXO) conjugation to MS via polydopamine coating, the amount of nitrogen was significantly increased on the surface of MS, indicating that EXO were successfully conjugated onto MS. EXO-coated dopamine MS (EXO-Dopa MS) exhibited significantly improved delivery into DC2.4 cells and RAW264.7 cells, compared with bare MS and Dopa MS. Therefore, EXO-Dopa MS could be used as effective carriers of immune stimulating biomolecules into APCs for cancer immunotherapy.

Combat or burn injuries are associated with a series of risks, such as microbial infection, an elevated level of inflammatory response, and pathologic scar tissue formation, which significantly postpone wound healing and also lead to impaired repair. Skin engineering for wound healing requires a biomimetic dressing substrate with ideal hydrophilicity, holding antioxidant and antimicrobial properties. In addition, available bioactive specification is required to reduce scar formation, stimulate angiogenesis, and improve wound repair. In this study, we successfully fabricated chitosan (Ch)-based hydrogel enriched with isolated exosome (EXO) from easy-accessible stem cells, which could promote fibroblast cell migration and proliferation in vitro. Full-thickness excisional wound model was used to investigate the in vivo dermal substitution ability of the fabricated hydrogel composed Ch and EXO substrates. Our finding confirmed that the wounds covered with Ch scaffold containing isolated EXO have nearly 83.6% wound closure ability with a high degree of re-epithelialization, whereas sterile gauze showed 51.5% of reduction in wound size. In summary, obtained results imply that Ch-glycerol-EXO hydrogel construct can be utilized at the full-thickness skin wound substitution and skin tissue engineering.

Abstract The Latent infection cansed by Epstein-Barr virus (EBV) closely relates with the occurrence and development of several kinds of lymphoma. Exosome (EXO) is functional bilayer membrane structural corpuscles which are secreted by cells contain proteins, lipids and nucleic acids. In recent years, researches showed that EXO play an important role in the occurrence and development of tumors. Therefore, the resenrches which compare the differences in quantity and contents of EXO secreted by cells between EBV negative lymphoma and EBV positive lymphoma and the clarify the influence of EXO on biological behaviors of lymphoma cells and immune cells have the important, significance for understanding the mechanisms related with effect of latent EBV on the occurrence and development of lymphoma by exosome pathway. This review focuses on research progress about the effect of latent EBV on amounts, contents and functions of EXO secreted by lymphoma cells.EB病毒潜伏感染致淋巴瘤细胞外泌体分泌和功能异常的研究进展.EB病毒(EBV)潜伏感染与多种淋巴系统肿瘤发生发展有着密切关系。外泌体(EXO)是细胞分泌的包含蛋白质、脂质、核酸等的功能性双层膜结构小体。近几年来有研究表明，EXO在肿瘤发生发展过程中起着重要作用，故对比EBV阴性和EBV阳性淋巴瘤细胞在EXO分泌数量与内容物方面的差异，了解EXO对淋巴瘤细胞和免疫细胞生物学行为的影响，对EBV潜伏感染通过外泌体途径影响淋巴瘤发生发展机制的研究具有重要意义。本综述重点阐述EBV潜伏感染对淋巴瘤细胞EXO分泌数量、内容物及其功能的影响.