不同激活条件下PRR源性外泌体特征及其对HMEC-1和BJ增殖能力差异的比较研究  

Characteristics of PRR-derived exosomes and the proliferation abilities of HMEC-1 and BJ under different activation conditions:a comparative study

作　　者：高立兰[1] 吕孟兴[1] 刘建香[1] 胡美坤 金晓红[1] 屈柯暄 GAO Lilan;LYU Mengxing;LIU Jianxiang;HU Meikun;JIN Xiaohong;QU Kexuan(Department of Blood Transfusion,Kunming Children′s Hospital,Kunming 650228,China)

机构地区：[1]昆明市儿童医院输血科,云南昆明650228

出　　处：《中国输血杂志》2025年第3期343-351,374,共10页Chinese Journal of Blood Transfusion

基　　金：儿童富血小板血浆制备的影响因素及质量控制的研究(2023-03-236-K01)。

摘　　要：目的 比较不同激活条件下富血小板血浆源性外泌体(platelet-rich plasma derived exosomes, PRP-Exos)的特征及其人微血管内皮细胞(HMEC-1)和人皮肤成纤维细胞(BJ)增殖能力的差异。方法 招募10名健康志愿者,采集EDTA-K_2抗凝静脉血10 mL,应用二次离心法制备提取PRP-Exos。通过透射电镜(TEM)、纳米颗粒追踪分析(NTA)和免疫印迹法(WB)检测技术,比较PRP-Exos的形态特征、粒径分布和浓度及表面标志蛋白的表达;通过体外细胞培养,比较不同激活剂组PRP-Exos对人微血管内皮细胞(HMCE-1)和人皮肤成纤维细胞(BJ)形态变化和增殖能力的差异。结果 1)TEM观察显示:不同激活剂组PRP-Exos形态不同。2)NTA分析显示:与生理盐水组粒径(109.60±2.71)nm相比,凝血酶组粒径(104.93±1.55)nm和混合剂组粒径(103.83±1.58)nm相对较小,葡萄糖酸钙组粒径(113.57±4.70)nm较大且粒径分布尺寸较宽,不同组间比较差异有统计学意义(F=7.019,P<0.05);凝血酶和葡萄糖酸钙混合剂组获得的外泌体浓度最高[(4.87±0.63)×10^(6 )particles/mL,P<0.001],凝血酶组[(2.75±0.13)×10^(6 )particles/mL,P<0.01]和葡萄糖酸钙组[(3.82±0.06)×10^(6 )particles/mL,P<0.001]与生理盐水组相比均获得更高浓度的外泌体;葡萄糖酸钙组略高于凝血酶组,两组之间比较存在明显差异性(P<0.05)。3)WB分析显示:PRP-Exos表面表达CD41、CD81及TSG101,其中CD41、TSG101在凝血酶和葡萄糖酸钙混合剂组中的蛋白信号强度最强(P<0.001)。4)体外细胞培养结果显示:凝血酶和葡萄糖酸钙混合剂组PRP-Exos可显著促HMEC-1和BJ的增殖(P<0.05)。结论 凝血酶和葡萄糖酸钙混合剂组PRP可获得最高浓度的外泌体;在体外培养条件下,凝血酶和葡萄糖酸钙混合剂组PRP-Exos可显著促进HMEC-1和BJ的增殖。Objective To compare the characteristics of platelet-rich plasma derived exosomes(PRP-Exos)under dif-ferent activation conditions and their differential effects on the proliferation capacit of human microvascular endothelial cells(HMEC-1)and human skin fibroblasts(BJ).Methods Ten healthy volunteers were recruited,and 10 mL of venous blood anticoagulated with EDTA-K2 was collected.PRP-Exos were prepared and extracted by the secondary centrifugation method.Transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and Western blotting(WB)de-tection techniques were used to compare the morphological characteristics,particle size distribution,concentration,and the expression of surface marker proteins of PRP-Exos.Through in vitro cell culture,the differences in morphological changes and proliferation abilities of HMEC-1 and BJ cells induced by PRP-Exos in different activator groups were compared.Re-sults 1)TEM observation showed that the morphologies of PRP-Exos in different activator groups were different.2)NTA analysis showed that compared with the particle size of(109.60±2.71)nm in the normal saline group,the particle sizes of the thrombin group[(104.93±1.55)nm]and the mixed agent group[(103.83±1.58)nm]were relatively smaller,while the particle size of the calcium gluconate group[(113.57±4.70)nm]was larger and its particle size distribution range was wider.There were statistically significant differences among different groups(F=7.019,P<0.05).The concen-tration of exosomes obtained in the group with the mixture of calcium gluconate and thrombin was the highest[(4.87±0.63)×10^(6) particles/mL,P<0.001].Both the thrombin group[(2.75±0.13)×10^(6) particles/mL,P<0.01]and the calcium gluconate group[(3.82±0.06)×10^(6) particles/mL,P<0.001]obtained higher concentrations of exosomes com-pared with the normal saline group.The concentration in the calcium gluconate group was slightly higher than that in the thrombin group,and there was a significant difference between the two groups(P<0.05).3)WB anal

关 键 词：PRP 外泌体 凝血酶 葡萄糖酸钙 人微血管内皮细胞 人皮肤成纤维细胞 细胞增殖 

分 类 号：R457.1[医药卫生—治疗学]