骨髓干细胞来源外泌体通过促进海马能量代谢和微血管新生改善小鼠抑郁样行为

BMSC-Exo alleviates depression-like behaviors in mice by inducing hippocampal energy metabolism and microangiogenesis

目的:观察骨髓间充质干细胞来源外泌体(BMSC-Exo)对抑郁症模型小鼠的海马微血管、能量代谢及其行为学的改善作用。方法:(1)分离培养小鼠骨髓间充质干细胞,提取BMSC-Exo。采用透射电镜观察BMSC-Exo的形态,采用Zetaview分析仪测定BMSC-Exo的粒径分布范围,采用Western blotting实验检测BMSC-Exo中CD9和CD63的表达。(2)取2只小鼠,采用慢性不可预见性温和应激(CUMS)建立抑郁症模型后于海马分别注射磷酸盐缓冲液(PBS)、DiR标记的BMSC-Exo,24 h后采用活体成像系统检测小鼠体内摄取BMSC-Exo情况。(3)将36只小鼠按随机数字表法分为对照组、模型组和BMSC-Exo组,每组12只。后2组小鼠采用CUMS建立抑郁症模型。造模后BMSC-Exo组小鼠海马注射1 μL BMSC-Exo,对照组小鼠注射等量PBS。采用强迫游泳实验(FST)、悬尾实验(TST)及开放旷场实验(OFT)检测小鼠的行为学。采用碱性磷酸酶染色检测小鼠海马微血管数量及长度。采用微正电子发射断层显像/X线计算机体层成像(mPET/CT)检测小鼠海马的能量代谢情况。采用Western blotting实验检测小鼠海马葡萄糖转运蛋白1(GLUT1)的表达。结果:(1)BMSC-Exo呈典型的盘状囊泡样结构,粒径为(100.5±1.4) nm。Western blotting实验结果证实BMSC-Exo表达CD9、CD63。(2)活体成像检测结果显示注射PBS后小鼠的脑和肝脏未见明显荧光,注射BMSC-Exo后小鼠的脑和肝脏呈现显著局部荧光。(3)与对照组比较,模型组和BMSC-Exo组小鼠FST、TST的静止时间较长,OFT的中央区域运动路程、中央区域运动时间较短,差异均有统计学意义( P<0.05);与模型组比较,BMSC-Exo组小鼠FST、TST的静止时间较短,OFT的中央区域运动路程、中央区域运动时间较长,差异均有统计学意义( P<0.05)。与对照组比较,模型组和BMSC-Exo组小鼠海马标准摄取值(SUV)、微血管长度及微血管数量、海马GLUT1的表达较低,差异均有统计学意义( P<0.05);与模型组比较,BMSC-Exo组小鼠海马SUV值、微血管长度及微血管数量、GLUT1的表达较高,差异均有统计学意义( P<0.05)。3组小鼠海马微血管长度与SUV值呈正相关关系( r=0.540, P<0.001),海马微血管数量与SUV值呈正相关关系( r=0.600, P<0.001)。 结论:BMSC-Exo可促进抑郁症模型小鼠海马微血管新生,提高海马葡萄糖代谢,从而改善抑郁样行为。

Objective To investigate the effect of bone mesenchymal stem cells derived exosomes(BMSC-Exo)on improving hippocampal microangiogenesis,energy metabolism,and behaviors in depression mouse models.Methods(1)Mouse bone marrow mesenchymal stem cells were isolated and cultured to extract BMSC-Exo;BMSC-Exo morphology was observed by transmission electron microscopy,BMSC-Exo particle diameter ranges were determined by Zetaview analyzer,and expressions of CD9 and CD63 in BMSC-Exo were detected by Western blotting.(2)Depression models were established in 2 mice by chronic unforeseeable mild stress(CUMS);24 h after stereotaxic injection of phosphate buffer solution(PBS)or DiR labeled BMSC-Exo,BMSC-Exo uptake was detected by in vivo imaging system.(3)Thirty-six mice were randomly divided into control group,model group and BMSC-Exo group(n=12);CUMS was used to establish depression models in the latter 2 groups;brain stereotaxic injection of 1μL BMSC-Exo was given to mice in the BMSC-Exo group after modeling,and same amount of PBS was given to the control group;behaviors were observed by forced swimming test(FST),tail suspension test(TST)and open field test(OFT);hippocampal microvascular length and number were detected by alkaline phosphatase staining;energy metabolism in the hippocampus was detected by micro positron emission tomography/computed tomography(mPET/CT);glucose transporter 1(GLUT1)expression in the hippocampus was detected by Western blotting.Results(1)BMSC-Exo had a typical disk-like vesicle-like structure with particle size of(100.5±1.4)nm;Western blotting confirmed that CD9 and CD63 expressed in BMSC-Exo.(2)In vivo imaging showed no fluorescence in the brain and liver after PBS injection,but obvious local fluorescence after BMSC-Exo injection.(3)Compared with the control group,the model group and BMSC-Exo group had significantly longer rest time in FST and TST and shorter movement distance and time in the central region of OFT(P<0.05);compared with the model group,BMSC-Exo group had significantly shorter rest time in FST and TST and longer movement distance and time in the central region of OFT(P<0.05).Compared with the control group,the model group and BMSC-Exo group had significantly decreased standard uptake value(SUV)of regions of interest,microvascular length and number,and GLUT1 expression in the hippocampus(P<0.05);compared with the model group,the BMSC-Exo group had significantly higher SUV,microvascular length and number,and GLUT1 expression in the hippocampus(P<0.05).Positive correlations were noted between hippocampal microvascular length and SUV and between microvascular number and SUV in the 3 groups(r=0.540,P<0.001;r=0.600,P<0.001).Conclusion BMSC-Exo could promote microangiogenesis energy metabolism in the hippocampus to improve depression-like behaviors in depression mouse models.