人脂肪间充质干细胞来源类外泌体纳米囊泡与外泌体对人脐静脉内皮细胞生物学功能影响的比较研究

Comparative study of exosome-mimetic nanovesicles and exosomes derived from human adipose mesenchymal stem cells on the biological function of human umbilical vein endothelial cells

目的探讨不同浓度人脂肪间充质干细胞(hADMSCs)来源类外泌体纳米囊泡(NVs)和外泌体(EXOs)对人脐静脉内皮细胞(HUVECs)生物学功能的影响。方法 (1)通过水动力抽脂方式, 从2019年6月至2020年8月就诊于福建医科大学附属协和医院的10例女性大腿获取脂肪组织(年龄18~65岁), 通过酶解分离提取第4代hADMSCs, 对其进行成脂、成骨诱导分化, 流式细胞仪鉴定其表面蛋白标志物。(2)制备hADMSCs-NVs和hADMSCs-EXOs并电镜观察其形态, 以粒径分析仪分析其表面蛋白标志物, 通过纳米颗粒跟踪仪分析颗粒大小, 通过蛋白定量和纳米颗粒跟踪仪分别检测1×10;个hADMSCs产生的NVs和EXOs总蛋白含量和颗粒数。(3)设置对照组(加入等量基础培养基)、40、60、80 μg/ml NVs组和20、40、60 μg/ml EXOs组, 分别通过CCK-8增殖实验、细胞划痕试验、血管形成实验检测各组HUVECs增殖、迁移、血管再生能力。采用Graphpad Prism 7.0进行统计分析, 计量资料以±s表示, 多组间比较应用重复测量方差分析, 组间两两比较应用Tukey检验, P<0.05表示差异有统计学意义。结果 (1)光学显微镜下观察到第4代hADMSCs形态呈细长纺锤状;成脂诱导21 d后, 油红O染色可见细胞内透明的脂滴被染成红色;成骨诱导14 d后, 碱性磷酸酶钙钴法染色可见大量棕黑色着色区;hADMSCs表面蛋白标志物CD90、CD29为阳性。(2)透射电镜下可见hADMSCs-NVs和EXOs结构相似, 都呈盘状囊泡;NVs和EXOs表面蛋白标志物CD9、CD81、IgG表达量相似;NVs和EXOs两者的粒径大小大致相同, 分别为(72.00±21.51)nm和(81.27±22.37)nm;1×106个hADMSCs产生的NVs总蛋白含量为(140.7±5.1) μg, 是EXOs的[(1.3±0.3) μg] 100倍左右;NVs颗粒数[(644.5±17.1)×108个/ml]是EXOs[(7.1±0.1)×108个/ml]的90倍左右。(3)CCK-8增殖实验中, 培养12、24、48 h各组HUVECs生长趋势较为一致, 吸光度值比较差异有统计学意义(P<0.01);培养48 h, 60 μg/ml NVs、40 μg/ml EXOs组吸光度值均大于对照组(均P<0.01), 而两实验组之间差异无统计学意义(P>0.05)。细胞划痕后8、24 h, 各组划痕宽度改变不完全相同, 差异有统计学意义(P<0.01);划痕后24 h, 60 μg/ml NVs、40 μg/ml EXOs组划痕宽度改变均大于对照组(均P<0.01), 而两实验组之间差异无统计学意义(P>0.05)。血管形成实验中, 各组血管分支点数量与血管长度不完全相同, 差异有统计学意义(P<0.01);60 μg/ml NVs、40 μg/ml EXOs组HUVECs形成的毛细血管分支点数量均多于对照组(均P<0.01), 而两实验组之间差异无统计学意义(P>0.05);60 μg/ml NVs、40μg/ml EXOs组毛细血管长度均长于对照组(均P<0.01), 而两实验组之间差异无统计学意义(P>0.05)。结论 hADMSCs-NVs的形态、大小与EXOs相似, 而总蛋白量却是EXOs的100倍左右;hADMSCs-NVs和EXOs对HUVECs生物学功能的作用相近, 两者最适宜的浓度分别为60 μg/ml和40 μg/ml。

Objective To investigate the effect of different concentrations of human adipose-derived mesenchymal stromal cells(hADMSCs)derived exosome-mimetic nanovesicles(NVs)and exosomes(EXOs)on the biological function of human umbilical vein endothelial cells(HUVECs).Methods(1)Through hydrodynamic liposuction,adipose tissue was obtained from the thighs of 10 women(aged 18-65 years)in Fujian Medical University Union Hospital from June 2019 to August 2020.The hADMSCs were isolated by enzymatic hydrolysis,cultured to passage 4 and induced into adipocytes and osteocytes.The surface protein markers were identified by flow cytometry.(2)hADMSCs-NVs and hADMSCs-EXOs were prepared and observed under an electron microscope.Their surface protein markers were analyzed with particle size analyzer,particle size was analyzed with nanoparticle tracker.Protein quantitative analysis and nanoparticle tracking were used to detect the total protein and particle number of NVs and EXOs produced by 1×106 hADMSCs.(3)The control group(DMEM basic medium),40,60,80μg/ml NVs groups and 20,40,60μg/ml EXOs groups were set to compare the proliferation,migration and angiogenesis of HUVECs through CCK-8 proliferation test,cell scratch test and angiogenesis test respectively.Graphpad Prism 7.0 was used for statistical analysis,and the measurement data were expressed as Mean±SD.Repeated measurement analysis of variance was applied to the comparison between multiple groups,and Tukey test was applied to pairwise comparison.P<0.05 represented statistical significance.Results(1)The fourth generation of hADMSCs were slender spindle-shaped cells under optical microscope.After 21 days of adipogenesis induction,the transparent lipid droplets inside the cells were stained red by oil red O staining.After 14 days of osteogenesis induction,a large proportion of brown black staining area was observed by alkaline phosphatase calcium cobalt staining.The surface protein markers CD90 and CD29 of hADMSCs were positive.(2)Under transmission electron microscope,the structures of hADMSCs-NVs and EXOs were similar,both were discoid vesicles.The expression levels of CD9,CD81 and IgG were similar between NVS and EXOs.The particle sizes of NVs and EXOs were about the same,which were(72.0±21.51)nm and(81.27±22.37)nm.The total protein content of NVs produced by 1×106 hADMSCs was(140.7±5.1)μg,about 100 times that of EXOs,which was(1.3±0.3)μg.The number of NVs[(644.5±17.1)×108/ml]particles was about 90 times that of EXOs[(7.1±0.1)×108/ml].(3)In CCK-8 proliferation assay,at 12,24 and 48 hours after culture,the growth trend of HUVECs in the groups were generally consistent,and the difference in absorbance value was statistically significant(P<0.01);at 48 hours after culture,the absorbance values of 60μg/ml NVs and 40μg/ml EXOs were higher than those in the control group(all P<0.01),but there was no significant difference between the two experimental groups(P>0.05).At 8 and 24 hours after cell scratch assay,the changes of scratch width in each group were different,and the difference was statistically significant(P<0.01);at 24 hours after scratch,the change of scratch width in 60μg/ml NVs and 40μg/ml EXOs groups were greater than that in the control group(all P<0.01),but there was no significant difference between the two experimental groups(P>0.05).In the angiogenesis assay,the number of branch points and the length of blood vessels in each group were different,and the difference was statistically significant(P<0.01).The number of capillary branches formed by HUVECs in 60μg/ml NVs and 40μg/ml EXOs groups were higher than that in the control group(all P<0.01),but there was no significant difference between the two experimental groups(all P>0.05).The capillary length of 60μg/ml NVs and 40μg/ml EXOs groups were longer than that of the control group(all P<0.01),but there was no significant difference between the two experimental groups(P>0.05).Conclusions The shape and size of NVs were similar to EXOs,while the total protein content of NVs was about 100 times that of EXOs.The effects of hADMSCs-NVs and EXOs on the biological functions of HUVECs are similar and the optimum concentrations of NVs and EXOs are 60μg/ml and 40μg/ml,respectively.