Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism
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摘要:
目的 建立人真皮成纤维细胞（HDF）高糖老化模型，探讨人蜕膜间充质干细胞（dMSC）来源外泌体对高糖老化HDF增殖、迁移、凋亡的影响及其可能机制。 方法 采用实验研究方法。收集2021年1—3月解放军总医院第四医学中心收治的4例男性包茎患者（18~22岁）环切术后废弃包皮组织，分离培养获取原代HDF。取第6代HDF，按照随机数字表法分为低糖组和高糖组，分别采用低糖完全培养基和高糖完全培养基进行每72小时换液、不传代培养，10 d后取细胞，于接种后24 h，采用β-半乳糖苷酶试剂盒检测细胞衰老情况；于接种后48 h，采用蛋白质印迹法检测细胞衰老相关蛋白p16、p53表达情况；于接种后24、48、72 h，采用细胞计数试剂盒8（CCK-8）法检测细胞增殖情况；于接种后48 h，采用脱氧尿嘧啶核苷（EdU）染色法检测细胞增殖情况，采用流式细胞术检测细胞周期及凋亡情况；于接种后24 h，采用Transwell实验测定细胞迁移能力。取人dMSC培养48~72 h，采用差速高速离心法获取其外泌体，采用透射电子显微镜观察dMSC外泌体形态，采用纳米颗粒追踪分析法检测dMSC外泌体的粒径分布，采用蛋白质印迹法检测dMSC外泌体标志蛋白CD9、肿瘤易感基因101（TSG101）的表达。取dMSC外泌体及前述高糖完全培养基诱导老化的HDF共孵育24 h，采用PKH67试剂盒检测细胞摄取外泌体的情况。取前述高糖完全培养基诱导老化的HDF，同前分为单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组，分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度为50 μg/mL dMSC外泌体、终质量浓度为100 μg/mL dMSC外泌体进行常规细胞培养。分组后同前于对应时间点采用CCK-8法和EdU染色法、流式细胞术、Transwell实验分别检测细胞增殖、细胞周期和凋亡及细胞迁移情况。根据前述结果，另取经高糖完全培养基诱导老化的HDF，分为单纯高糖组、高糖+高浓度外泌体组并同前处理。分组培养48 h，采用实时荧光定量反转录PCR法检测单纯高糖组和高糖+高浓度外泌体组细胞衰老相关的微小RNA-145-5p（miR-145-5p）、miR-498、miR-503-5p及其靶基因钙/钙调素依赖性蛋白激酶1D（CAMK1D）、人第10号染色体缺失的磷酸酶及张力蛋白同源的基因（PTEN基因）和细胞周期蛋白D1的mRNA表达情况。对数据行析因设计方差分析、单因素方差分析、LSD-t检验和独立样本t检验。 结果 接种后24 h，高糖组HDF β-半乳糖苷酶阳性染色率为（38.4±4.2）%，明显高于低糖组的（16.5±2.2）%（t=4.65，P<0.01）。接种后48 h，高糖组HDF的衰老相关蛋白p16和p53的表达量均明显高于低糖组（t值分别为11.85、3.02，P<0.05或P<0.01）。接种后24、48、72 h，高糖组HDF的增殖活性均明显低于低糖组（t值分别为4.13、9.90、15.12，P<0.01）。接种后48 h，高糖组HDF的EdU阳性染色率明显低于低糖组（t=3.83， P<0.05）。接种后48 h，高糖组HDF周期的G2/M+S亚群在3个亚群（G0/G1、S和G2/M）的占比明显低于低糖组（t=8.74，P<0.01）。接种后24 h，高糖组HDF穿过Transwell滤膜到达下室的细胞数量为（37±6）个，明显少于低糖组的（74±7）个（t=8.42，P<0.01）。接种后48 h，高糖组HDF凋亡率明显高于低糖组（t=8.48，P<0.01）。dMSC外泌体为边缘清晰、大小分布均匀的杯状或者圆形囊泡，粒径基本处于80~200 nm。dMSC外泌体标志性蛋白CD9、TSG101表达均呈阳性。共孵育24 h，外泌体被HDF摄入胞内，主要分布于细胞核周围。分组培养24、48、72 h，高糖+低浓度外泌体组和高糖+高浓度外泌体组HDF增殖活性均明显高于单纯高糖组（t值分别为6.36、6.10、7.76，8.92、12.17、10.74，P<0.01），高糖+高浓度外泌体组HDF增殖活性均明显高于高糖+低浓度外泌体组（t值分别为7.92、4.82、4.72，P<0.01）。分组培养48 h，与单纯高糖组比较，高糖+低浓度外泌体组和高糖+高浓度外泌体组HDF EdU阳性染色率均明显升高（t值分别为5.32、9.88，P<0.01）；与高糖+低浓度外泌体组比较，高糖+高浓度外泌体组HDF EdU阳性染色率显著升高（t=5.27，P<0.01）。分组培养48 h，与单纯高糖组比较，高糖+低浓度外泌体组和高糖+高浓度外泌体组HDF中的G0/G1期亚群占比均显著降低（t值分别为3.81、4.31，P<0.05），G2/M+S亚群占比均明显升高（t值分别为3.81、4.31，P<0.05）。分组培养24 h，与单纯高糖组相比，高糖+低浓度外泌体组和高糖+高浓度外泌体组HDF穿过滤膜的数量均明显增多（t值分别为10.14、13.39，P<0.01）；与高糖+低浓度外泌体组相比，高糖+高浓度外泌体组HDF穿过滤膜的数量明显增多（t=6.27，P<0.01）。分组培养48 h，与单纯高糖组比较，高糖+低浓度外泌体组和高糖+高浓度外泌体组HDF凋亡率均明显降低（t值分别为3.72、5.53，P<0.05或P<0.01）。分组培养48 h，与单纯高糖组相比，高糖+高浓度外泌体组HDF的miR-145-5p、miR-498 mRNA表达量均明显上升（t值分别为13.03、8.90，P<0.01），miR-503-5p mRNA表达量明显下降（t=3.85，P<0.05）；高糖+高浓度外泌体组中HDF的CAMK1D、PTEN基因mRNA表达量均明显低于单纯高糖组（t值分别为8.83、5.97，P<0.01），细胞周期蛋白D1 mRNA表达量明显高于单纯高糖组（t=4.03，P<0.05）。 结论 人dMSC来源外泌体可显著提高高糖老化HDF的增殖和迁移能力，抑制其凋亡。这可能与HDF内miR-145-5p和miR-498表达增高抑制了CAMK1D和PTEN基因的表达及miR-503-5p表达下降促进了细胞周期蛋白D1的表达有关。 Abstract:Objective To establish a high glucose senescent model of human dermal fibroblasts (HDFs), and to investigate the effects of exosomes derived from human decidua mesenchymal stem cells (dMSCs) on the proliferation, migration, and apoptosis of senescent HDFs and possible mechanism. Methods The experimental research method was used. From January to March 2021, discarded foreskin tissue was collected for isolation and culture of primary HDFs from 4 male phimosis patients (aged 18-22 years) admitted for circumcision in the Fourth Medical Center of the PLA General Hospital. The 6th passage of HDFs were taken and divided into low glucose group and high glucose group according to the random number table, and subsequently cultured in low-glucose complete medium and high-glucose complete medium, respectively, with medium changed every 72 h without subculturing. After 10 days of culture, the cells were taken and measured for cellular senescence using the β-galactosidase kit at 24 h after seeding; the expression of senescence-related proteins p16 and p53 was assessed by Western blotting at 48 h after seeding; cell proliferation was detected at 24, 48, and 72 h after seeding using the cell counting kit 8 (CCK-8) method; the cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) staining method, cell cycle and apoptosis were measured by flow cytometry after 48 h of seeding; Transwell experiment was used for the calculation of cell migration rate at 24 h after seeding. The human dMSCs were taken and cultured for 48-72 h from which the exosomes were extracted by differential high speed centrifugal method. The morphology of dMSC exosomes was observed by transmission electron microscopy, the particle size distribution of dMSC exosomes was measured by nanoparticle tracking analysis, and the expression of dMSC-exosomes marker proteins CD9 and tumor susceptibility gene101 (TSG101) were detected by Western blotting. The dMSC exosomes and high-glucose complete medium-induced senescent HDFs were co-cultured for 24 hours, then PKH67 kit was used to detect the uptake of exosomes by HDFs. High-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group, high glucose+low concentration of exosomes group, and high glucose+high concentration of exosomes group according to the same method above. The high-glucose complete medium with equal volume of phosphate buffered saline, dMSC exosomes with final concentration of 50 μg/mL, and dMSC exosomes with final concentration of 100 μg/mL were added to the corresponding groups for conventional cell culture, respectively. After grouped, the cell proliferation, cell cycle and apoptosis as well as cell migration were detected by CCK-8 method and EdU staining method, flow cytometry, and Transwell experiment at the corresponding time points as before, respectively. Based on the previous results, high-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group and high glucose+high concentration of exosomes group for the same treatment. After being grouped and cultured for 48 h, real-time fluorescent quantitative polymerase chain reaction was used to evaluate the mRNA expression of senescent-related microRNA (miR)-145-5p, miR-498, miR-503-5p, calcium/calmodulin dependent protein kinase 1D (CAMK1D), phosphates and tensin homologue deleted on chromosome ten (PTEN) gene, and Cyclin D1 in high glucose alone group and high glucose+high concentration of exosomes group. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, and independent sample t test. Results At 24 h after seeding, the rate of β-galactosidase-positive staining of HDF in high glucose group was (38.4±4.2)%, which was significantly higher than (16.5±2.2)% of low glucose group (t=4.65, P<0.01). At 48 h after seeding, the expression levels of senescence-related proteins p16 and p53 both were significantly higher in HDFs of high glucose group than those in low glucose group (with t values of 11.85 and 3.02, respectively, P<0.05 or P<0.01). At 0, 24, 48, and 72 h after seeding, the cell proliferation viability of HDFs in high glucose group was all significantly lower than in low glucose group (with t values of 4.13, 9.90, and 15.12, respectively, P<0.01). At 48 h after seeding, the rate of EdU-positive staining of HDFs in high glucose group was obviously lower than that of low glucose group (t=3.83, P<0.05). At 48 h after seeding, the percentage of G2/M+S subpopulations in three subpopulations (G0/G1, S, and G2/M) of HDF cycle was significantly lower in high glucose group than that in low glucose group (t=8.74, P<0.01). At 24 h after seeding, the number of HDFs migrated through the filter membrane to the lower chamber was 37±6 in high glucose group, which was significantly less than 74±7 in low glucose group (t=8.42, P<0.01). At 48 h after seeding, the HDF apoptosis rate was significantly higher in high glucose group than in low glucose group (t=8.48, P<0.01). The dMSC exosomes were cup-shaped or round vesicles with well-defined edges and uniform size distribution. The size of dMSC exosomes was basically in the range of 80-200 nm. Exosomal markers including CD9 and TSG101 were positively presented on the dMSC exosomes. After being co-cultured for 24 hours, the dMSC exosomes were taken up intracellularly by HDFs and mainly distributed around the nucleus of HDFs. After being grouped and cultured for 24, 48, and 72 h, the HDF proliferation viabilities in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 6.36, 6.10, 7.76, 8.92, 12.17, and 10.74, respectively, P<0.01), the HDF proliferation viability in high glucose+high concentration of exosomes group was significantly higher than in high glucose+low concentration of exosomes group (with t values of 7.92, 4.82, and 4.72, respectively, P<0.01). After being grouped and cultured for 48 h, the percentages of EdU-positive HDFs in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 5.32 and 9.88, respectively, P<0.01), the percentage of EdU-positive HDFs in high glucose+high concentration of exosomes group was notably higher than in high glucose+low concentration of exosomes group (t=5.27, P<0.01). After being grouped and cultured for 48 h, the proportion of G0/G1 subpopulation in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was distinctly lower (with t values of 3.81 and 4.31, respectively, P<0.05), while the proportion of G2/M+S subpopulation was markedly higher (with t values of 3.81, 4.31, respectively, P<0.05) than in high glucose alone group. After being grouped and cultured for 24 h, the number of HDFs migrated through the filter membrane in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was significantly higher than in high glucose alone group (with t values of 10.14 and 13.39, respectively, P<0.01), the number of HDFs migrated through the filter membrane in high glucose+high concentration of exosomes group was significantly increased than in high glucose+low concentration of exosomes group (t=6.27, P<0.01). After being grouped and cultured for 48 h, the HDF apoptosis rates in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly lower than in high glucose alone group (with t values of 3.72 and 5.53, respectively, P<0.05 or P<0.01). After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of miR-145-5p and miR-498 were both obviously higher (with t values of 13.03 and 8.90, respectively, P<0.01), while the mRNA expression level of miR-503-5p was significantly lower (t=3.85, P<0.05) in high glucose+high concentration of exosomes group. After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of CAMK1D and PTEN gene were both significantly lower (with t values of 8.83 and 5.97, respectively, P<0.01), while the mRNA expression level of Cyclin D1 was significantly higher in high glucose+high concentration of exosomes group (t=4.03, P<0.05). Conclusions The dMSC exosomes are capable of improving cell proliferation and migration, and inhibiting cell apoptosis of high-glucose senescent HDFs. This may be related to the mechanism by which the increased expressions of intracellular miR-145-5p and miR-498 inhibit the expression of CAMK1D and PTEN gene, and the decreased expression of miR-503-5p promote the expression of Cyclin D1. -
Key words:
- Mesenchymal stem cells /
- Exosomes /
- Cell aging /
- Glucose solution, hypertonic /
- Fibroblasts
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参考文献
(40) [1] LiB, BianX, HuW, et al. Regenerative and protective effects of calcium silicate on senescent fibroblasts induced by high glucose[J]. Wound Repair Regen,2020,28(3):315-325. DOI: 10.1111/wrr.12794. [2] LiM,ZhaoY,HaoH,et al.Umbilical cord-derived mesenchymal stromal cell-conditioned medium exerts in vitro antiaging effects in human fibroblasts[J].Cytotherapy,2017,19(3):371-383.DOI: 10.1016/j.jcyt.2016.12.001. [3] RaniS,RyanAE,GriffinMD,et al.Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications[J].Mol Ther,2015,23(5):812-823.DOI: 10.1038/mt.2015.44. [4] NikfarjamS,RezaieJ,ZolbaninNM,et al.Mesenchymal stem cell derived-exo somes: a modern approach in translational medicine[J].J Transl Med,2020,18(1):449.DOI: 10.1186/s12967-020-02622-3. [5] KomakiM,NumataY,MoriokaC,et al.Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis[J].Stem Cell Res Ther,2017,8(1):219.DOI: 10.1186/s13287-017-0660-9. [6] ZhaoB,ZhangY,HanS,et al.Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation[J].J Mol Histol,2017,48(2):121-132.DOI: 10.1007/s10735-017-9711-x. [7] DashBC,XuZ,LinL,et al.Stem cells and engineered scaffolds for regenerative wound healing[J].Bioengineering (Basel),2018, 5(1):23. DOI: 10.3390/bioengineering5010023. [8] LermanOZ,GalianoRD,ArmourM,et al.Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia[J].Am J Pathol,2003,162(1):303-312.DOI: 10.1016/S0002-9440(10)63821-7. [9] ThomasK,KiwitM,KernerW.Glucose concentration in human subcutaneous adipose tissue: comparison between forearm and abdomen[J].Exp Clin Endocrinol Diabetes,1998,106(6):465-469.DOI: 10.1055/s-0029-1212017. [10] WeiQ,WangY,MaK,et al.Extracellular vesicles from human umbilical cord mesenchymal stem cells facilitate diabetic wound healing through miR-17-5p-mediated enhancement of angiogenesis[J/OL].Stem Cell Rev Rep,2021(2021-05-04)[2021-10-13]. https://pubmed.ncbi.nlm.nih.gov/33942217/. DOI:10.1007/s12015-021-10176-0. [published online ahead of print]. [11] ChengL,ZhangK,WuS,et al.Focus on mesenchymal stem cell-derived exosomes: opportunities and challenges in cell-free therapy[J].Stem Cells Int,2017,2017:6305295.DOI: 10.1155/2017/6305295. [12] LuK,LiHY,YangK,et al.Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells[J].Stem Cell Res Ther,2017,8(1):108.DOI: 10.1186/s13287-017-0563-9. [13] Casado-DíazA,Quesada-GómezJM,DoradoG.Extracellular vesicles derived from mesenchymal stem cells (MSC) in regenerative medicine: applications in skin wound healing[J].Front Bioeng Biotechnol,2020,8:146.DOI: 10.3389/fbioe.2020.00146. [14] HuY,RaoSS,WangZX,et al.Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function[J].Theranostics,2018,8(1):169-184.DOI: 10.7150/thno.21234. [15] PomattoM,GaiC,NegroF,et al.Differential therapeutic effect of extracellular vesicles derived by bone marrow and adipose mesenchymal stem cells on wound healing of diabetic ulcers and correlation to their cargoes[J].Int J Mol Sci,2021, 22(8):3851. DOI: 10.3390/ijms22083851. [16] WangC,WangM,XuT,et al.Engineering bioactive self-healing antibacterial exosomes hydrogel for promoting chronic diabetic wound healing and complete skin regeneration[J].Theranostics,2019,9(1):65-76.DOI: 10.7150/thno.29766. [17] McBrideJD,Rodriguez-MenocalL,GuzmanW,et al.Bone marrow mesenchymal stem cell-derived CD63+ exosomes transport Wnt3a exteriorly and enhance dermal fibroblast proliferation, migration, and angiogenesis in vitro[J].Stem Cells Dev,2017,26(19):1384-1398.DOI: 10.1089/scd.2017.0087. [18] YangJ,ChenZ,PanD,et al.Umbilical cord-derived mesenchymal stem cell-derived exosomes combined pluronic F127 hydrogel promote chronic diabetic wound healing and complete skin regeneration[J].Int J Nanomedicine,2020,15:5911-5926.DOI: 10.2147/IJN.S249129. [19] ZhangW,BaiX,ZhaoB,et al.Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K/Akt signaling pathway[J].Exp Cell Res,2018,370(2):333-342.DOI: 10.1016/j.yexcr.2018.06.035. [20] FangS,XuC,ZhangY,et al.Umbilical cord-derived mesenchymal stem cell-derived exosomal microRNAs suppress myofibroblast differentiation by inhibiting the transforming growth factor-β/SMAD2 pathway during wound healing[J].Stem Cells Transl Med,2016,5(10):1425-1439.DOI: 10.5966/sctm.2015-0367. [21] HuL,WangJ,ZhouX,et al.Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts[J].Sci Rep,2016,6:32993.DOI: 10.1038/srep32993. [22] WiklanderOP, NordinJZ, O'LoughlinA,et al.Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting[J].J Extracell Vesicles,2015,4:26316.DOI: 10.3402/jev.v4.26316. [23] YangD,ZhangW,ZhangH,et al.Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics[J].Theranostics,2020,10(8):3684-3707.DOI: 10.7150/thno.41580. [24] DingM,WangC,LuX,et al.Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling[J].Anal Bioanal Chem,2018,410(16):3805-3814.DOI: 10.1007/s00216-018-1052-4. [25] KusumaGD,CarthewJ,LimR,et al.Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect[J].Stem Cells Dev,2017,26(9):617-631.DOI: 10.1089/scd.2016.0349. [26] WuD,KangL,TianJ,et al.Exosomes derived from bone mesenchymal stem cells with the stimulation of Fe3O4 nanoparticles and static magnetic field enhance wound healing through upregulated miR-21-5p[J].Int J Nanomedicine,2020,15:7979-7993.DOI: 10.2147/IJN.S275650. [27] YangH,LinJ,JiangJ,et al.miR-20b-5p functions as tumor suppressor microRNA by targeting cyclinD1 in colon cancer[J].Cell Cycle,2020,19(21):2939-2954.DOI: 10.1080/15384101.2020.1829824. [28] DimitrovaN,GochevaV,BhutkarA,et al.Stromal expression of miR-143/145 promotes neoangiogenesis in lung cancer development[J].Cancer Discov,2016,6(2):188-201.DOI: 10.1158/2159-8290.CD-15-0854. [29] DimitrovaN,GochevaV,BhutkarA,et al.Stromal expression of miR-143/145 promotes neoangiogenesis in lung cancer development[J].Cancer Discov,2016,6(2):188-201.DOI: 10.1158/2159-8290.CD-15-0854. [30] LawsonJ,DickmanC,MacLellanS,et al.Selective secretion of microRNAs from lung cancer cells via extracellular vesicles promotes CAMK1D-mediated tube formation in endothelial cells[J].Oncotarget,2017,8(48):83913-83924.DOI: 10.18632/oncotarget.19996. [31] GrasC,RatusznyD,HadamitzkyC,et al.miR-145 contributes to hypertrophic scarring of the skin by inducing myofibroblast activity[J].Mol Med,2015,21(1):296-304.DOI: 10.2119/molmed.2014.00172. [32] ChaiC,WuH,WangB,et al.MicroRNA-498 promotes proliferation and migration by targeting the tumor suppressor PTEN in breast cancer cells[J].Carcinogenesis,2018,39(9):1185-1196.DOI: 10.1093/carcin/bgy092. [33] DuanXM,LiuXN,LiYX,et al.MicroRNA-498 promotes proliferation, migration, and invasion of prostate cancer cells and decreases radiation sensitivity by targeting PTEN[J].Kaohsiung J Med Sci,2019,35(11):659-671.DOI: 10.1002/kjm2.12108. [34] LiJ,ZhangF,LiH,et al.Circ_0010220-mediated miR-503-5p/CDCA4 axis contributes to osteosarcoma progression tumorigenesis[J].Gene,2020,763:145068.DOI: 10.1016/j.gene.2020.145068. [35] MarkopoulosGS,RoupakiaE,TokamaniM,et al.Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts[J].Exp Gerontol,2017,96:110-122.DOI: 10.1016/j.exger.2017.06.017. [36] JiangL,ZhaoZ,ZhengL,et al.Downregulation of miR-503 promotes ESCC cell proliferation, migration, and invasion by targeting cyclin D1[J].Genomics Proteomics Bioinformatics,2017,15(3):208-217.DOI: 10.1016/j.gpb.2017.04.003. [37] BovyN,BlommeB,FrèresP,et al.Endothelial exosomes contribute to the antitumor response during breast cancer neoadjuvant chemotherapy via microRNA transfer[J].Oncotarget,2015,6(12):10253-10266.DOI: 10.18632/oncotarget.3520. [38] HouSQ,OuyangM,BrandmaierA,et al.PTEN in the maintenance of genome integrity: From DNA replication to chromosome segregation[J].Bioessays,2017,39(10):10. DOI: 10.1002/bies.201700082. [39] LiB,LuanS,ChenJ,et al.The MSC-derived exosomal incRNA H19 promotes wound healing in diabetic foot ulcers by upregulating PTEN via microRNA-152-3p[J].Mol Ther Nucleic Acids,2020,19:814-826.DOI: 10.1016/j.omtn.2019.11.034. [40] LongJ,OuC,XiaH,et al.MiR-503 inhibited cell proliferation of human breast cancer cells by suppressing CCND1 expression[J].Tumour Biol,2015,36(11):8697-8702.DOI: 10.1007/s13277-015-3623-8. -
1 2组人真皮成纤维细胞中接种后24 h β-半乳糖苷酶表达情况 光学显微镜×100，图中标尺为200 μm。1A.低糖组β-半乳糖苷酶染色；1B.高糖组β-半乳糖苷酶阳性染色率较图1A高
注：低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L；图中蓝绿色指示细胞β-半乳糖苷酶阳性染色
2 蛋白质印迹法检测2组人真皮成纤维细胞接种后48 h衰老相关蛋白表达
注：1为低糖组，2为高糖组；低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L
3 细胞计数试剂盒8法检测2组人真皮成纤维细胞接种各时间点的增殖活性（样本数为3，
注：低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L；处理因素主效应，F=244.90，P<0.001；时间因素主效应，F=892.30，P<0.001；两者交互作用，F=56.23，P<0.001；与低糖组比较，aP<0.01
4 EdU染色法检测2组人真皮成纤维细胞培养48 h增殖活力 EdU-hochst×200，图中标尺为100 μm。4A、4B、4C.分别为低糖组细胞EdU染色、细胞核染色、细胞EdU与细胞核共染色情况；4D、4E、4F.分别为高糖组细胞EdU染色、细胞核染色、细胞EdU与细胞核共染色情况，细胞核完整，图4D EdU阳性细胞较图4A明显减少
注：低糖组、高糖组细胞培养基中的葡萄糖浓度分别为5.5、35.0 mmol/L；以细胞脱氧尿嘧啶核苷（EdU）阳性染色为绿色，细胞核阳性染色为蓝色，绿色+蓝色双荧光染色为增殖细胞
5 流式细胞术检测2组人真皮成纤维细胞培养48 h的细胞周期情况。5A、5B.分别为低糖组、高糖组
注：低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L；图5A、5B中横坐标a、b、c、d、e、f分别为0、500、1 000、1 600、2 000、2 500；图中第1个红色波峰为G1期，第2个红色波峰为G2期，蓝色斜线区为S期
6 Transwell实验观察2组人真皮成纤维细胞培养24 h迁移情况 结晶紫×200，图中标尺为100 μm。6A、6B.分别为低糖组、高糖组，图6B穿膜细胞数较图6A少
注：低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L；图中紫色指示穿膜细胞
7 流式细胞术检测2组人真皮成纤维细胞培养48 h凋亡情况。7A.低糖组绝大多数细胞处于左下象限，凋亡细胞少；7B.高糖组细胞右上、右下象限凋亡细胞数较图7A明显增多
注：低糖组、高糖组细胞培养基中的葡萄糖物质的量浓度分别为5.5、35.0 mmol/L；FITC为异硫氰酸荧光素；图中左下象限显示活细胞，右下象限显示早期凋亡细胞，右上象限显示晚期凋亡细胞和坏死细胞，左上象限显示细胞收集过程中出现的损伤细胞
11 人真皮成纤维细胞（HDF）与人dMSC外泌体共培养24 h后，外泌体被HDF内吞入细胞质，并聚集于细胞核周围培养 4',6-二脒基-2-苯基吲哚-罗丹明标记的鬼笔环肽-PKH67×400，图中标尺为25 μm
注：紫色指示细胞核，酱红色指示细胞骨架，绿色为人蜕膜间充质干细胞（dMSC）来源外泌体
12 细胞计数试剂盒8法检测3组人真皮成纤维细胞培养各时间点活力（样本数为3，
注：单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度50 μg/mL人蜕膜间充质干细胞外泌体、终质量浓度100 μg/mL 人蜕膜间充质干细胞外泌体进行常规细胞培养；处理因素主效应，F=107.80，P<0.001；时间因素主效应，F=2 992.00，P<0.001；两者交互作用，F=24.99，P<0.001；与单纯高糖组比较，aP<0.01；与高糖+低浓度外泌体组比较，bP<0.01
13 EdU染色法检测3组HDF培养48 h的增殖活力 EdU-hoechst×200，图中标尺为100 μm。13A、13B、13C.分别为单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组细胞EdU与细胞核共染色情况，细胞核完整，图13B EdU阳性细胞较图13A明显增多，且图13C EdU阳性染色细胞数最多
注：单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度50 μg/mL人蜕膜间充质干细胞外泌体、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；细胞脱氧尿嘧啶核苷（EdU）阳性染色为绿色，细胞核阳性染色为蓝色，绿色+蓝色双荧光染色为增殖的人真皮成纤维细胞（HDF）
14 流式细胞术检测3组人真皮成纤维细胞培养48 h的细胞周期情况。14A、14B、14C.分别为单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组
注：单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度50 μg/mL人蜕膜间充质干细胞外泌体、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；图中第1个红色波峰为G1期，第2个红色波峰为G2期,蓝色斜线区为S期
15 Transwell实验观察3组人真皮成纤维细胞培养24 h迁移能力 结晶紫×200，图中标尺为100 μm。15A、15B、15C.分别为单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组穿膜细胞生长情况，图15B、15C穿膜细胞数均较图15A多，且图15C穿膜细胞数最多
注：单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度50 μg/mL人蜕膜间充质干细胞外泌体、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；图中紫色指示穿膜细胞
16 流式细胞术检测3组人真皮成纤维细胞（HDF）培养48 h凋亡情况。16A.单纯高糖组HDF绝大多数细胞处于左下象限，凋亡细胞较多；16B.高糖+低浓度外泌体组凋亡HDF较单纯高糖组减少；16C.高糖+高浓度外泌体组右上/下象限凋亡HDF较图16A明显减少，且凋亡细胞数低于图16B
注：单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度50 μg/mL人蜕膜间充质干细胞外泌体、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；FITC为异硫氰酸荧光素；图中左下象限显示活细胞，右下象限显示早期凋亡细胞，右上象限显示晚期凋亡细胞和坏死细胞，左上象限显示细胞收集过程中出现的损伤细胞
表1 2组高糖诱导老化人真皮成纤维细胞培养48 h衰老相关微小RNA表达情况比较（
组别 样本数 miR-145-5p miR-498 miR-503-5p 单纯高糖组 3 1.01±0.14 1.06±0.21 1.05±0.37 高糖+高浓度外泌体组 3 12.15±1.47 2.89±0.29 0.22±0.03 t值 13.03 8.90 3.85 P值 <0.001 <0.001 0.018 注：单纯高糖组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；miR为微小RNA 
下载: 导出CSV
表2 2组高糖诱导老化人真皮成纤维细胞中衰老相关基因的mRNA表达情况比较（
组别 样本数 CAMK1D PTEN基因 细胞周期蛋白D1 单纯高糖组 3 1.00±0.11 1.00±0.04 1.14±0.18 高糖+高浓度外泌体组 3 0.37±0.06 0.52±0.13 2.85±0.70 t值 8.83 5.97 4.03 P值 <0.001 0.003 <0.015 注：单纯高糖组、高糖+高浓度外泌体组分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度100 μg/mL人蜕膜间充质干细胞外泌体进行常规细胞培养；CAMK1D为钙/钙调素依赖性蛋白激酶1D，PTEN基因为人第10号染色体缺失的磷酸酶及张力蛋白同源的基因
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