內皮祖細胞（endothelialprogenitor cells, EPCs）旁分泌途徑在成血管及組織再生過程中發揮著積極作用，其功能也受外界複雜力學環境的影響。隨著「空間醫學」概念的產生，微重力環境對人體機能的調控逐漸成為研究熱點。前人研究已證實，在特定條件下微重力環境可激發血管相關幹/祖細胞促組織再生潛能，但相關機制尚不明確。在國家自然科學基金項目（批准號：82072421, 81772322和81572121）等的資助下，2021年1月，香港中文大學醫學院矯形外科及創傷學系的李剛教授團隊在Stem Cell Research & Therapy（影響因子5.116）雜誌上發表了題為Conditioned media from endothelial progenitorcells cultured in simulated microgravity promote angiogenesis and bone fracturehealing的研究論文。該研究闡述了微重力環境對EPCs成血管基因表達及旁分泌途徑的影響，並進一步探討了微重力EPCs條件培養基促進骨折後血管再生及骨癒合的作用及機制。
首先，作者將人外周血內皮祖細胞於微重力環境下培養，發現微重力環境抑制EPCs增殖，但激活了細胞內HIF-1α/eNOS/NO通路，通過過量釋放eNOS來源一氧化氮（NO）改善了EPCs促成血管功能，且NO介導的FAK/Erk1/2-MAPK通路激活是重要機制之一（如下圖）。
Fig. 1 MG suppressedEPCs proliferation but activated HIF-1α/eNOS/NO axis. A MTT assay wasperformed to detect EPCs proliferation under MG or NG exposure. BImmunofluorescence staining showed the expression levels of Ki67 under NG(left) or MG (right) exposure. DAPI was for nuclear counterstain. C Acluster of angiogenic genes expression was examined by qRT-PCR after 12-, 24-and 48-hour exposures of NG and MG, and GAPDH was used as the internalreference. D Western blot was employed to identify the expression ofHIF-1α, eNOS and iNOS at protein levels. E Griess assay was performed todetect the NO contents in MG-CM and NG-CM after 12-, 24- and 48-hour exposures,and the concentrations were normalized to cell numbers. This experiment wasrepeated independently three times. Data were presented as the mean ± standarddeviation. * P < 0.05, ** P < 0.01 and *** P <0.001.
Fig. 2 MG-CM promotedHUVECs proliferation, migration and angiogenesis in vitro partially throughincreased NO production. A The effects of DMEM, NG-CM, MG-CM and NOreduced MG-CM on HUVECs proliferation were analyzed by MTT assay. B TheKi67 expression in HUVECs was detected by immunofluorescence staining. DAPI wasfor nuclear counterstain. C-F Transwell assay and wound healing assaywere employed to detect the migration capacity changes of HUVECs aftertreatment followed by quantitative analyses. G The CD31 expressionlevels of HUVECs in each group were detected by immunofluorescence staining. H,I Tube formation assay was used to detect the angiogenic abilities ofHUVECs followed by quantitative analysis. J qRT-PCR results showed therelative expression levels of VEGF and MMP-9, with GAPDH as the internalreference. This experiment was repeated independently three times. Data werepresented as the mean ± standard deviation. * P < 0.05, ** P< 0.01 and *** P < 0.001.
Fig. 3 NO-inducedactivation of FAK/Erk1/2-MAPK signaling pathway contributed to enhancedpro-angiogenic properties of MG-CM. A Western blot was employed todetect the phosphorylation levels of FAK and Erk1/2. B Thephosphorylation level of Erk1/2 was examined after Erk1/2-MAPK-selectiveinhibitor PD98059 application for 4 hours. C, D Tube formation assay wasperformed to detect the angiogenic abilities of HUVECs when PD98059 was addedor not, followed by quantitative analysis. E Immunofluorescence stainingresults showed the expression of Ki67 and CD31 in different groups. DAPI wasfor nuclear counterstain. F The mRNA expression level of VEGF wasdetected by qRT-PCR, and GAPDH was used as internal reference. G Theschematic illustration of MG-induced HIF-1α/eNOS/NO activation of EPCspromoting HUVECs proliferation, migration and angiogenesis. This experiment wasrepeated independently three times. Data were presented as the mean ± standarddeviation. * P < 0.05, ** P < 0.01 and *** P <0.001.
體外實驗可用上圖Fig.3 G總結為：微重力刺激EPCs胞內HIF-1α/eNOS/NO通路上調，釋放的過量NO激活內皮細胞FAK/Erk1/2-MAPK信號通路，從而有利於內皮增殖、遷移和血管形成。隨後作者建立SD大鼠脛骨骨折模型，將條件培養基局部注射至斷端部位，發現微重力EPCs條件培養基在體內可直接改善成血管活動，加速骨折癒合（如下圖）。
Fig. 4MG-CM accelerated callus growth and improved mechanical properties of fracturebone. A The workflow of animal experiments, including surgery,interventions and detection. B The representative X-ray images of theregenerates in DMEM, NG-CM and MG-CM groups at 4, 7, 10 and 14 days aftersurgery. C The callus growth of rats was quantified by radiologicalscores. D Mechanical properties of fracture bone including ultimateload, stiffness and energy to failure were evaluated at post-operative 3 weeks.Data were presented as the mean ± standard deviation. * P < 0.05, ** P< 0.01 and *** P < 0.001.
Fig. 5MG-CM facilitated neovascularization of fracture areas in vivo. AMicrofil perfusion was performed to examine the newly formed vessels of theregenerated areas followed by micro-CT scan. B Total vessel volume wascalculated and analyzed based on reconstructive vessel images. C-HImmunohistochemistry staining was employed to detect the expression of CD31,VEGF-A and MMP-9 in regenerative areas followed by quantitative analyses. Datawere presented as the mean ± standard deviation. * P < 0.05, ** P< 0.01 and *** P < 0.001.
Fig. 6MG-CM promoted bone fracture repair in vivo. A The regenerative bone infracture areas was detected by micro-CT at post-operative 2 weeks. BQuantitative analysis of BV/TV of regenerative tissues in fracture areas. CH&E staining and Masson’s trichrome staining were performed to observe thehistological structures of regenerative tissues. D, EImmunohistochemistry staining was employed to detect the expression of OCN inthe newly formed bone followed by quantitative analysis. Data were presented asthe mean ± standard deviation. * P < 0.05, ** P < 0.01 and*** P < 0.001.
該研究預示著微重力設備有潛力作為新型生物反應器改善細胞功能，從而為骨癒合與骨再生生物學治療策略的研發和革新提供全新思路。該論文通訊作者為香港中文大學李剛教授和上海交通大學附屬第六人民醫院康慶林教授，上海交通大學醫學院孔令馳（兼香港中文大學訪問學者）和香港中文大學李嘉誠健康科學研究所王燕博士為該文共同第一作者。
原文信息
Kong L, Wang Y, Wang H, Pan Q, Zuo R, Bai S, ZhangX, Lee W, Kang Q, Li G. Conditioned media fromendothelial progenitor cells cultured in simulated microgravity promoteangiogenesis and bone fracture healing. Stem Cell Res Ther. 2021; 12: 47.(IF: 5.116)
原文連結（DOI）
https://stemcellres.biomedcentral.com/articles/10.1186/s13287-020-02074-y