人脐带干细胞外泌体对抑郁模型小鼠海马小胶质细胞极化和神经元凋亡的影响

Effects of human umbilical cord mesenchymal stem cells exosomes on hippocampal microglia polarization and neuronal apoptosis in depressed model mice

目的探讨人脐带间充质干细胞外泌体(human umbilical cord mesenchymal stem cells exosomes,hucMSC-Exo)对慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)构建的抑郁模型小鼠的抑郁样行为及海马小胶质细胞极化动态转换的影响。方法分离、培养hucMSC,通过超速离心法提取第5代hucMSC-Exo并进行鉴定。采用Western blot法检测hucMSC-Exo的生物标志物CD9和CD63。选取12只雄性SPF级C57BL/6J小鼠,采用CUMS法建立抑郁模型,采用旷场实验和强迫游泳实验检测CUMS小鼠抑郁样行为的变化。造模完成后12只小鼠按随机数字表法分为外泌体治疗组(CUMS+Exo组)和模型组(CUMS组),每组6只,CUMS+Exo组小鼠海马区脑立体定位注射hucMSC-Exo。微正电子发射断层显像/X线计算机体层成像(micropositron emission tomography/computed tomography,micro PET/CT)扫描技术检测两组小鼠海马炎性反应信号的表达情况。组织免疫荧光检测小鼠海马区M2型小胶质细胞和M1型小胶质细胞的比例。以微管蛋白β3(β3-tubulin)作为标志物,细胞免疫荧光染色检测皮质酮(corticosterone,CORT)构建的抑郁细胞模型中神经元轴突的长度变化。EdU染色检测神经元增殖情况。使用TUNEL染色观察小鼠海马组织神经元凋亡情况。采用GraphPad 8.0软件进行统计分析,组间比较采用t检验。结果电镜下,hucMSC-Exo呈现典型的"双层杯状"小囊泡样改变,粒径约为100 nm,Western blot证实有CD9及CD63标志性蛋白表达。micro PET/CT扫描成像实验中,CUMS+Exo组小鼠[18F]DPA-714摄取较CUMS组减少,差异有统计学意义[(0.91±0.02)g/mL,(0.81±0.05)g/mL,t=4.54,P=0.0011]。旷场实验中,CUMS+Exo组小鼠中心路径长度百分比[(3.40±0.44)%,(5.17±0.90)%,t=4.33,P=0.0015]及中心路径花费时间[(7.04±0.60)s,(10.22±1.41)s,t=6.02,P=0.0001]均高于CUMS组,而两组间运动总距离差异无统计学意义(t=0.48,P>0.05)。在强迫游泳实验中,CUMS+Exo组小鼠游泳不动时间较CUMS组减少,差异有统计学意义[(152.33±7.28)s,(133.50±4.32)s,t=5.45,P=0.0003]。组织免疫荧光实验中,CUMS+Exo组海马区M2型小胶质细胞比例高于CUMS组[(0.33±0.04),(0.59±0.12),t=5.11,P=0.0005],M1型小胶质细胞比例低于CUMS组[(0.56±0.06),(0.41±0.03),t=5.15,P=0.0004]。细胞免疫荧光实验中,CORT+Exo组神经元轴突长度长于CORT组[(3.99±0.99)μm,(6.76±1.11)μm,t=6.10,P=0.0001]。细胞增殖实验中,CORT+Exo组细胞增殖率高于CORT组[(0.74±0.07),(0.64±0.03),t=3.32,P=0.0018]。TUNEL染色实验中,CUMS+Exo组小鼠海马区神经元凋亡率低于CUMS组[(0.24±0.04),(0.39±0.04),t=6.11,P=0.0001]。结论hucMSC-Exo可以改善CUMS小鼠抑郁样行为,促进其海马区M1型极化小胶质细胞向M2型转换,减少神经元凋亡。

Objective To investigate the effects of human umbilical cord mesenchymal stem cells exosomes(hucMSC-Exo)on depression-like behavior and polarization dynamic transition of hippocampal microglia in chronic unpredictable mild stress(CUMS)mice.Methods The hucMSC was isolated,cultured,and the 5th generation hucMSC-Exo was extracted by ultracentrifugation and identified.Biological markers of CD9 and CD63 of hucMSC-Exo were detected by Western blot.A total of 12 SPF grade male C57BL/6J mice were randomly selected to establish depression model by CUMS method at the same time,and relevant behavioral experiments,including opening field test(OFT)and forced swimming test(FST)were used to detect changes of depression-like behavior in CUMS mice.After modeling,12 mice were randomly divided into two groups,with 6 mice in each group.One group of mice was the exosome treatment group(CUMS+Exo group)after stereotactic brain injection of hucMSC-Exo in the hippocampus,and the other group was the CUMS group.The expression of inflammatory signals in the hippocampus of mice in both groups were detected by micro PET/CT scanning technique.The proportion of M2-type microglia and M1-type microglia in the hippocampus of mice was detected by tissue immunofluorescence.β3-tubulin immunofluorescence staining was used to detect the length changes of neuronal axons in the depressed cell model constructed with corticosterone(CORT).EdU staining was used to detect neuronal proliferation.TUNEL staining was used to observe the apoptosis of neurons in the hippocampus of mice.Statistical analysis was conducted by GraphPad 8.0 software,and t-test was used for inter group comparison.Results Under the electron microscope,hucMSC-Exo showed typical"double-layer cup-like"small vesicle-like changes and the particle diameter was about 100 nm.Western blot confirmed the expression of lconic proteins of CD9 and CD63.In the micro PET/CT scans,the uptake of[18F]DPA-714 in the CUMS+Exo group was lower than that of the CUMS group,and the difference was statistically significant((0.91±0.02)g/mL,(0.81±0.05)g/mL,t=4.54,P=0.0011).In the opening field test,the percentage of central path length((3.40±0.44)%,(5.17±0.90)%,t=4.33,P=0.0015)and the time spent on the central path((7.04±0.60)s,(10.22±1.41)s,t=6.02,P=0.0001)in CUMS+Exo group were higher than those in CUMS group,while the total distances of the two groups were not statistically significant(t=0.48,P>0.05).In the forced swimming test,the immobility time in the CUMS+Exo group was less than that in the CUMS group((152.33±7.28)s,(133.50±4.32)s,t=5.45,P=0.0003).In tissue immunofluorescence experiments,compared with the CUMS group,the proportion of M2-type microglia of hippocampus in the CUMS+Exo group((0.33±0.04),(0.59±0.12),t=5.11,P=0.0005)increased and the proportion of M1-type microglia((0.56±0.06),(0.41±0.03),t=5.15,P=0.0004)decreased in the CUMS+Exo group.β3-tubulin-labeled immunofluorescence results showed an increase of neuronal axon length in the CORT+Exo group compared with that in the CORT group((3.99±0.99)μm,(6.76±1.11)μm,t=6.10,P=0.0001).The results of cell proliferation test showed an increase of proliferation rate in the CORT+Exo group compared with that in the CORT group((0.74±0.07),(0.64±0.03),t=3.32,P=0.0018).In the TUNEL staining experiment,the apoptosis rate of neurons in the hippocampal region of mice in the CUMS+Exo group was lower than that of CUMS group((0.24±0.04),(0.39±0.04),t=6.11,P=0.0001).Conclusion hucMSC-Exo can promote the conversion of M1 polarized microglia to M2 type microglia to alleviate depression-like behavior and reduce neuronal apoptosis in CUMS mice.