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CytoFLEX nano精準(zhǔn)解析外泌體表型，賦能骨再生前沿研究與應(yīng)用
近期，中國(guó)醫(yī)科大學(xué)團(tuán)隊(duì)在《Materials Today Bio》上發(fā)表了一項(xiàng)創(chuàng)新研究，探討了黃芪修飾的硅酸鈣（AstCS）刺激M2型巨噬細(xì)胞來(lái)源的細(xì)胞外囊泡（AstCSM2EVs）在骨再生中的治療潛力。
研究發(fā)現(xiàn)，AstCSM2EVs能有效促使巨噬細(xì)胞向M2表型極化，表現(xiàn)出優(yōu)異的免疫調(diào)節(jié)能力：促炎因子（IL-1β、TNF-α）顯著下調(diào)，而抗炎介質(zhì)（IL-4、IL-10）則明顯上調(diào)。此外，AstCSM2EVs還展現(xiàn)出增強(qiáng)的促血管生成能力，具體表現(xiàn)為內(nèi)皮細(xì)胞管狀結(jié)構(gòu)形成增多、VEGF分泌上升，并能通過(guò)上調(diào)ALP、BSP、OC等關(guān)鍵標(biāo)志物，促進(jìn)間充質(zhì)干細(xì)胞的成骨分化。
機(jī)制研究進(jìn)一步揭示，AstCSM2EVs主要通過(guò)miR-218-5p調(diào)控包括NOD樣受體和ECM-受體相互作用在內(nèi)的多條信號(hào)通路，從而協(xié)調(diào)上述再生過(guò)程。在兔股骨缺損模型中，局部施用AstCSM2EVs顯著促進(jìn)了骨再生，表現(xiàn)為骨體積分?jǐn)?shù)增加、小梁結(jié)構(gòu)改善，并有效抑制了局部炎癥反應(yīng)。
這些發(fā)現(xiàn)確立了AstCSM2EVs作為一種潛力的骨再生治療劑，凸顯了其在免疫調(diào)節(jié)、血管生成和成骨分化中的多重作用。本研究創(chuàng)新性地將細(xì)胞外囊泡與免疫調(diào)節(jié)性組織工程策略相結(jié)合，為骨缺損修復(fù)提供了新思路。
值得關(guān)注的是，該研究中關(guān)于M2型巨噬細(xì)胞來(lái)源EVs的表面標(biāo)志物分析，包括CD206、CD280、CD146、CD73、CD105和CD14，均在CytoFLEX nano納米流式細(xì)胞儀上完成。
研究結(jié)果顯示，CD73與CD146具有顯著的促血管生成作用；CD14陽(yáng)性細(xì)胞展現(xiàn)出成骨誘導(dǎo)潛力；而CD105則在再生內(nèi)皮細(xì)胞中高表達(dá)，通過(guò)調(diào)控TGF-β信號(hào)通路參與組織修復(fù)與血管生成過(guò)程。如圖F所示，與CSM2EV及未染色對(duì)照組相比，AstCSM2EVs中上述標(biāo)志物的陽(yáng)性群體比例顯著提升。正是憑借對(duì)納米級(jí)EVs進(jìn)行如此精細(xì)的表型分析，才得以將材料的生物學(xué)效應(yīng)、細(xì)胞的免疫狀態(tài)與外泌體的治療功能緊密關(guān)聯(lián)。
Fig. 4.(A) The schematic of AstCS extract promotes the polarization of naive M0 macrophages into M2 phenotype, subsequently facilitating the generation of M2- derived EVs. (B) TEM images and (C) size distribution analysis of M2EV, CSM2EV, and AstCSM2EV, (D) Western blot analysis of EV markers, (E) total protein quantification of different EVs, and (F) flow cytometry analysis showing the expression of surface markers (CD206/CD280, CD73/CD146, and CD14/CD105) on different EVs compared to unstained control. * indicates statistical significance (p<0.05). Data represent mean ±SD; n =6 for each group.
該研究是Ming-You Shie團(tuán)隊(duì)利用CytoFLEX nano納米流式分析儀自上市以來(lái)發(fā)表的第5篇研究。是什么支撐CytoFLEX nano在這類前沿研究中承擔(dān)重任？
傳統(tǒng)流式細(xì)胞術(shù)在外泌體檢測(cè)中面臨分辨率限制、背景噪聲干擾以及標(biāo)準(zhǔn)化方法缺失等挑戰(zhàn)。CytoFLEX nano作為專為納米顆粒檢測(cè)設(shè)計(jì)的新一代儀器，通過(guò)優(yōu)化流體動(dòng)力學(xué)、采用先進(jìn)光學(xué)組件并增強(qiáng)電子信號(hào)處理，實(shí)現(xiàn)了分辨率與靈敏度。儀器配備四激光器（405、488、561、638 nm），具備六熒光通道、五側(cè)向散射通道及一前向散射通道，支持單顆粒水平的多參數(shù)精準(zhǔn)檢測(cè)。
西安交通大學(xué)團(tuán)隊(duì)近期在《VIEW》（2025）上發(fā)表的系統(tǒng)性性能評(píng)估，為CytoFLEX nano的優(yōu)秀表現(xiàn)提供了客觀數(shù)據(jù)支撐。
熒光靈敏度評(píng)估
使用500 nm八峰熒光微球進(jìn)行測(cè)試，CytoFLEX nano在V447、B531、Y595通道中可清晰分辨八個(gè)熒光峰，在R670、R710、R792通道中也表現(xiàn)出熒光分辨能力與信號(hào)一致性。相較于傳統(tǒng)流式細(xì)胞儀，CytoFLEX nano在低熒光強(qiáng)度信號(hào)檢測(cè)中展現(xiàn)出更優(yōu)的信噪比與分離度，尤其適用于低表達(dá)表面抗原的外泌體檢測(cè)。
FIGURE 2 Fluorescence signal distribution histograms of 500 nm 8Peaks calibration beads measured using the CytoFLEX nano flow cytometer. (A) V447, (B) B531, (C) Y595, (D) R670, (E) R710, and (F) R792. The x-axis represents fluorescence intensity detected in each respective channel, and the y-axis indicates the number of events corresponding to each fluorescence intensity level.
粒徑檢測(cè)精度與分辨率
使用40–1000 nm系列標(biāo)準(zhǔn)微球進(jìn)行測(cè)試，CytoFLEX nano在VSSC1通道（40–200 nm）中表現(xiàn)出優(yōu)異的粒徑一致性，尤其在≥100 nm顆粒中測(cè)量值高度吻合標(biāo)準(zhǔn)值。在40–150 nm范圍內(nèi)表現(xiàn)突出，覆蓋典型外泌體粒徑范圍。
FIGURE 3 Particle size distribution of calibration microspheres measured using the CytoFLEX nano flow cytometer. The size distribution of standard microspheres (ranging from 40 to 150 nm in diameter) was analyzed using the VSSC1 detection channel. Particles/mL means the number of particles found in 1 mL of the sample.
濃度定量準(zhǔn)確性
使用NIST可追溯的100 nm聚苯乙烯微球進(jìn)行濃度梯度稀釋實(shí)驗(yàn)，CytoFLEX nano測(cè)得濃度與理論值高度一致（R2 = 0.9992），顯示出優(yōu)異的線性響應(yīng)與定量準(zhǔn)確性。其內(nèi)置的自動(dòng)清洗與基線監(jiān)測(cè)系統(tǒng)有效降低了交叉污染與背景干擾，確保在寬濃度范圍內(nèi)保持穩(wěn)定性能。
FIGURE 4 Particle size distribution histograms using the NIST-traceable 100 nm PS standard microsphere. (A) Measurement of particle size distribution and concentration of NIST-traceable 100 nm PS microspheres using the CytoFLEX nano flow cytometer. The microsphere stock concentration was 1.809 × 1013 particles/mL, and measurements were performed after a 1000-fold dilution. (B) Linear correlation between theoretical and measured concentrations of microspheres across dilution gradients. The x-axis represents the dilution factor, and the y-axis indicates the measured particle concentration (particles/mL) after dilution. Data were collected using both the CytoFLEX nano and CytoFLEX flow cytometers.
外泌體表面標(biāo)記檢測(cè)效率
在三種不同來(lái)源的外泌體（iPS-hTERT、MHCC 97L、血清）中，CytoFLEX nano對(duì)CD9、CD63、CD81的標(biāo)記效率顯著高于傳統(tǒng)儀器。尤其在40–80 nm顆粒區(qū)間，CytoFLEX nano能夠檢測(cè)到傳統(tǒng)儀器無(wú)法識(shí)別的外泌體亞群，進(jìn)一步揭示了外泌體群體的異質(zhì)性。
FIGURE 6 Evaluation of exosome labeling efficiency using fluorescence-conjugated antibodies. Exosome samples isolated from iPS-hTERT-exo, MHCC 97L-exo, and Serum-exo samples were labeled with FITC-conjugated CD9, PE-conjugated CD81, and APC-conjugated CD63 antibodies. Fluorescence intensity was measured by CytoFLEX nano flow cytometry to assess antibody binding efficiency.
不同分離方法對(duì)外泌體表征的影響
比較超速離心、Huize-kit和EXODUS三種方法，CytoFLEX nano清晰揭示了不同方法在得率與標(biāo)記效率上的差異。EXODUS方法顆粒富集效率高，但其表面標(biāo)記檢測(cè)效率較低；而超速離心雖得率較低，但標(biāo)記效率高，提示其在生物學(xué)表征中的可靠性，仍是外泌體分離純化的金標(biāo)準(zhǔn)。
FIGURE 7 Comparison of exosome yield obtained by different isolation methods. Exosomes were isolated from the same sample source using three methods: ultracentrifugation, Huize-kit, and EXODUS. The yield of exosomes was measured by particle concentration (particles/mL) using the CytoFLEX nano flow cytometer.
FIGURE 8 Comparison of exosome marker labeling efficiency using different isolation methods. Exosomes isolated by Huize-kit and EXODUS methods were labeled with specific surface marker antibodies.
與傳統(tǒng)儀器的對(duì)比分析
在相同樣本條件下，CytoFLEX nano的檢測(cè)下限低至40 nm，而傳統(tǒng)CytoFLEX僅為80 nm。在40–80 nm區(qū)間，CytoFLEX nano額外檢測(cè)到約20–30%的顆粒，這些多為傳統(tǒng)儀器遺漏的外泌體亞群。此外，CytoFLEX nano在多重?zé)晒鈽?biāo)記中的陽(yáng)性率更高，尤其在CD81等低表達(dá)標(biāo)記中優(yōu)勢(shì)明顯。
FIGURE 9 Comparison of exosome detection using CytoFLEX and CytoFLEX nano flow cytometer on identical samples. (A) Fluorescence detection of MHCC 97L-exo using CytoFLEX with the ultracentrifugation method. (B) Particle size distribution analysis of exosomes measured by both CytoFLEX and CytoFLEX nano flow cytometers. (C) Statistical analysis of particles measured by both CytoFLEX and CytoFLEX nano flow cytometers. (D) Comparison of fluorescence labeling efficiency across different particle size ranges detected by the two instruments.
CytoFLEX nano流式細(xì)胞儀憑借其在粒徑分辨率、熒光靈敏度、濃度定量及多參數(shù)分析方面的性能，已成為解析外泌體異質(zhì)性的理想平臺(tái)。這項(xiàng)系統(tǒng)性性能驗(yàn)證從技術(shù)底層證實(shí)：CytoFLEX nano具備的高靈敏度、高分辨率和定量準(zhǔn)確性，足以應(yīng)對(duì)真實(shí)研究中樣本來(lái)源多樣、前處理方法不一所帶來(lái)的挑戰(zhàn)。
選擇CytoFLEX nano納米流式分析儀，不僅是選擇一臺(tái)儀器，更是選擇了一個(gè)值得信賴的科研伙伴，助您在納米世界的探索中，見(jiàn)微知著，洞見(jiàn)未來(lái)。
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參考文獻(xiàn)：
· Cheng-Yu Chen, Jian-Jr Lee, Yen-Hong Lin, Ting-You Kuo, Der-Yang Cho, Ming-You Shie, Osteoimmunomodulation of astragalus-calcium silicate scaffolds-activated M2 macrophage-derived miR-218-rich exosome for enhanced bone regeneration, Materials Today Bio, Volume 35, 2025, 102286, ISSN 2590-0064,
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