胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响

边晓玮; 张翠萍; 李炳旻; 马奎; 杨劼; 付小兵

doi:10.16016/j.1000-5404.202001190

您当前的位置：

首页 >

文章列表页 >

胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响

基础医学 | 更新时间：2026-01-09

- 胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响
- Effects of exosomes derived from placental mesenchymal stem cells on high glucose-induced senescence in human dermal fibroblasts
- 第三军医大学学报 2020年42卷第12期页码：1163-1173
- 作者机构：
  
  1.300070 天津，天津医科大学研究生院
  2.100048 北京，中国人民解放军总医院创新医学研究部组织修复与再生研究中心，皮肤损伤修复与再生北京市重点实验室
- 作者简介：
  
  [ 付小兵，E-mail：fuxiaobing@vip.163.com FU Xiaobing, E-mail:fuxiaobing@vip.163.com
- 基金信息：
  
  国家自然科学基金重点项目(81571905);国家重点研发计划(2017YFC1103300)
- DOI：10.16016/j.1000-5404.202001190
  中图分类号： R322.99; R329.28; R587.102
- 收稿：2020-01-22，
  
  修回：2020-03-08，
  
  纸质出版：2020-06-30
- 稿件说明：
移动端阅览
边晓玮, 张翠萍, 李炳旻, 等. 胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响[J]. 第三军医大学学报, 2020,42(12):1163-1173.

Xiaowei BIAN, Cuiping ZHANG, Bingmin LI, et al. Effects of exosomes derived from placental mesenchymal stem cells on high glucose-induced senescence in human dermal fibroblasts[J]. Journal of Third Military Medical University, 2020, 42(12): 1163-1173.
边晓玮, 张翠萍, 李炳旻, 等. 胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响[J]. 第三军医大学学报, 2020,42(12):1163-1173. DOI： 10.16016/j.1000-5404.202001190.

Xiaowei BIAN, Cuiping ZHANG, Bingmin LI, et al. Effects of exosomes derived from placental mesenchymal stem cells on high glucose-induced senescence in human dermal fibroblasts[J]. Journal of Third Military Medical University, 2020, 42(12): 1163-1173. DOI： 10.16016/j.1000-5404.202001190.

摘要

目的

探索高糖(high glucose

HG)促进体外培养的人皮肤成纤维细胞(human dermal fibroblasts

HDFs)的衰老条件，建立高糖老化模型；观察胎盘间充质干细胞来源的外泌体(exosomes

Exos)对高糖培养的成纤维细胞增殖、迁移及衰老的影响。

方法

分离人包皮组织的HDFs。实验分为3组：正常对照组(5.5 mmol/L葡萄糖)、HG1组(26 mmol/L葡萄糖)、HG2组(35 mmol/L葡萄糖)。CCK-8试剂盒检测各组细胞在1、3、5、7 d的增殖情况；各组培养7 d后进行划痕实验，观察各组HDFs在24 h内的迁移率；2′，7′二氯荧光素二醋酸(DCFH)法检测各组培养7 d后细胞内活性氧簇(ROS)水平；细胞衰老β-半乳糖苷酶染色试剂盒检测各组培养7 d后β-半乳糖苷酶活性。体外培养胎盘间充质干细胞(placenta mesenchymal stem cells

PMSCs)，差速高速离心法分离Exos。将上述培养的HDFs分为3组：HG2组(35 mmol/L葡萄糖)、HG2+低浓度Exos(5 μg/mL)组、HG2+高浓度Exos(50 μg/mL)组，培养3 d后，检测细胞增殖情况、迁移率、ROS水平和β-半乳糖苷酶活性；流式细胞术检测细胞周期；Western blot分析衰老相关蛋白p53和p21表达情况。

结果

与对照组相比，HG1和HG2组培养的HDFs增殖、迁移能力明显下降(

0.01)，ROS水平显著升高(

0.01)，β-半乳糖苷酶活性显著增加(

0.01)，而且各指标尤其以HG2组变化最为明显；Exos实验结果显示：高浓度Exos明显促进高糖培养HDFs的增殖和迁移(

0.01)，显著降低细胞内的ROS水平和β-半乳糖苷酶活性(

0.01)，同时使细胞内p53和p21蛋白的表达水平显著下降(

0.01)，而低浓度Exos无明显效果。

结论

胎盘间充质干细胞来源的Exos促进高糖培养的HDFs增殖、迁移，缓解细胞的氧化应激损伤和衰老状态，其机制可能与衰老相关蛋白p53和p21表达下调有关。

Abstract

Objective

To explore the optimal conditions for inducing senescence in human dermal fibroblasts (HDFs) cultured in high glucose and observe the effect of exosomes derived from human placental mesenchymal stem cells (PMSCs) on the proliferation

migration and senescence of high glucose-induced fibroblasts.

Methods

HDFs were isolated from human foreskin tissue and cultured in the presence of 5.5 mmol/L glucose (normal control group)

26 mmol/L glucose (HG1 group) and 35 mmol/L glucose (HG2 group). CCK-8 assay was used to detect cell proliferation in each group on days 1

5 and 7 of cell culture. The cells cultured for 7 d were tested for migration within 24 h using cell scratch assay; reactive oxygen species (ROS) production in the cells was detected using 2′

7′-dichlorofluorescein diacetic acid (DCFH) method

and β-galactosidase activity was analyzed using a β-galactosidase staining kit. The HDFs cultured in 35 mmol/L glucose for 7 d were exposed for 3 days to 5 μg/mL and 50 μg/mL exosomes

which were isolated from human PMSCs by differential ultracentrifugation

and the cell proliferation

migration

ROS levels and β-galactoside activity were assessed. Flow cytometry was used to analyze the cell cycle changes and Western blotting was performed to detect the expression of aging-related proteins p53 and p21 in the cells following treatment with the exosomes.

Results

Compared with the control cells

the HDFs in HG1 and HG2 groups exhibited significantly decreased proliferation and migration ability (

0.01) and increased ROS production (

0.01) and β-galactoside activity (

0.01). Treatment with the exosomes obviously promoted the proliferation and migration of HDFs cultured in high glucose (

0.01). At the concentration of 50 μg/mL

PMSC-derived exosomes significantly decreased ROS production and β-galactoside activity (

0.01) and obviously down-regulated the expression levels of p53 and p21 (

0.01) in the HDFs cultured in high glucose

whereas a low concentration (5 μg/mL) of the exosomes did not produce such effects.

Conclusion

Exosomes derived from PMSCs promote the proliferation and migration and alleviate oxidative stress-induced damage and cell senescence in high glucose-induced HDFs

the mechanism of which may involve the down-regulation of the aging-related proteins p53 and p21.

关键词

Keywords

references

LEFRANCOIS T, MEHTA K, SULLIVAN V, et al . Evidence based review of literature on detriments to healing of diabetic foot ulcers[J ] . Foot Ankle Surg, 2017, 23(4): 215-224. DOI: 10.1016/j.fas.2016.04.002.

JOHNSTONE R M, ADAM M, HAMMOND J R, et al . Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)[J ] . J Biol Chem, 1987, 262(19): 9412-9420.

RANI S, RYAN A E, GRIFFIN M D, 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.

COOPER D R, WANG C Y, PATEL R, et al . Human adipose-derived stem cell conditioned media and exosomes containing MALAT1 promote human dermal fibroblast migration and ischemic wound healing[J ] . Adv Wound Care (New Rochelle), 2018, 7(9): 299-308. DOI: 10.1089/wound.2017.0775.

TAKEUCHI R, KATAGIRI W, ENDO S, et al . Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis[J ] . PLoS ONE, 2019, 14(11): e0225472. DOI: 10.1371/journal.pone.0225472.

LI X C, CHEN C Y, WEI L M, et al . Exosomes derived from endothelial progenitor cells attenuate vascular repair and accelerate reendothelialization by enhancing endothelial function[J ] . Cytotherapy, 2016, 18(2): 253-262. DOI: 10.1016/j.jcyt.2015.11.009.

SHABBIR A, COX A, RODRIGUEZ-MENOCAL L, et al . Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts, and enhance angiogenesis in vitro[J ] . Stem Cells Dev, 2015, 24(14): 1635-1647. DOI: 10.1089/scd.2014.0316.

ZHANG J Y, GUAN J J, NIU X, et al . Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesisand angiogenesis[J ] . J Transl Med, 2015, 13: 49. DOI: 10.1186/s12967-015-0417-0.

KOMAKI M, NUMATA Y, MORIOKA C, 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.

ZHAO B, ZHANG Y J, HAN S C, et al . Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation[J ] . J Mol Hist, 2017, 48(2): 121-132. DOI: 10.1007/s10735-017-9711-x.

FALANGA V. Wound healing and its impairment in the diabetic foot[J]. Lancet, 2005, 366(9498): 1736-1743. DOI: 10.1016/S0140-6736(05)67700-8.

DESJARDINS-PARKH E, FOSTER D S, LONGAKER M T. Fibroblasts and wound healing: an update[J]. Regen Med, 2018, 13(5): 491-495. DOI: 10.2217/rme-2018-0073.

ROGNONI E, WATT F M. Skin cell heterogeneity in development, wound healing, and cancer[J]. Trends Cell Biol, 2018, 28(9): 709-722. DOI: 10.1016/j.tcb.2018.05.002.

SUNDARAM G M,COMMON J E, GOPAL F E, et al . 'See-saw' expression of microRNA-198 and FSTL1 from a single transcript in wound healing[J ] . Nature, 2013, 495(7439): 103-106. DOI: 10.1038/nature11890.

MAEDA M, HAYASHI T, MIZUNO N, et al . Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase[J ] . PLoS ONE, 2015, 10(4): e0123169. DOI: 10.1371/journal.pone.0123169.

THOMAS K, KIWIT M, KERNER W. 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.

LEE J, SHIN Y K, SONG J Y, et al . Protective mechanism of morin against ultraviolet B-induced cellular senescence in human keratinocyte stem cells[J ] . Int J Radiat Biol, 2014, 90(1): 20-28.DOI: 10.3109/09553002.2013.835502.

HE T, GUAN X, WANG S, et al . Resveratrol prevents high glucose-induced epithelial-mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway[J ] . Mol Cell Endocrinol, 2015, 402: 13-20. DOI: 10.1016/j.mce.2014.12.010.

KANIKARLA-MARIE P, JAIN S K. L-Cysteine supplementation reduces high-glucose and ketone-induced adhesion of monocytes to endothelial cells by inhibiting ROS[J]. Mol Cell Biochem, 2014, 391(1/2): 251-256. DOI: 10.1007/s11010-014-2009-3.

LAMERS M L , ALMEIDA M E, VICENTE-MANZANARES M, et al . High glucose-mediated oxidative stress impairs cell migration[J ] . PLoS ONE, 2011, 6(8): e22865. DOI: 10.1371/journal.pone.0022865.

LOBO S E, LEONEL L C, MIRANDA C M, et al . The placenta as an organ and a source of stem cells and extracellular matrix: a review[J ] . Cells Tissues Organs (Print), 2016, 201(4): 239-252. DOI: 10.1159/000443636.

ZHANG K Y, ZHAO X N, CHEN X N, et al . Enhanced therapeutic effects of mesenchymal stem cell-derived exosomes with an injectable hydrogel for hindlimb ischemia treatment[J ] . ACS Appl Mater Interfaces, 2018, 10(36): 30081-30091. DOI: 10.1021/acsami.8b08449.

BIER A, BERENSTEIN P, KRONFELD N, et al . Placenta-derived mesenchymal stromal cells and their exosomes exert therapeutic effects in Duchenne muscular dystrophy[J ] . Biomaterials, 2018, 174: 67-78. DOI: 10.1016/j.biomaterials.2018.04.055.

TAO S C, GUO S C, LI M, et al . Chitosan wound dressings incorporating exosomes derived from microRNA-126-overexpressing synovium mesenchymal stem cells provide sustained release of exosomes and heal full-thickness skin defects in a diabetic rat model[J ] . Stem Cells Transl Med, 2017, 6(3): 736-747. DOI: 10.5966/sctm.2016-0275.

KIM S, LEE S K, KIM H, et al . Exosomes secreted from induced pluripotent stem cell-derived mesenchymal stem cells accelerate skin cell proliferation[J ] . Int J Mol Sci, 2018, 19(10): E3119. DOI: 10.3390/ijms19103119.

LEE H, CHA H, PARK J H. Derivation of cell-engineered nanovesicles from human induced pluripotent stem cells and their protective effect on the senescence of dermal fibroblasts[J]. Int J Mol Sci, 2020, 21(1): E343. DOI: 10.3390/ijms21010343.

OH M, LEE J, KIM Y J, et al . Exosomes derived from human induced pluripotent stem cells ameliorate the aging of skin fibroblasts[J ] . Int J Mol Sci, 2018, 19(6): E1715. DOI: 10.3390/ijms19061715.

BAE Y U, SON Y, KIM C H, et al . Embryonic stem cell-derived mmu-miR-291a-3p inhibits cellular senescence in human dermal fibroblasts through the TGF-β receptor 2 pathway[J ] . J Gerontol A Biol Sci Med Sci, 2019, 74(9): 1359-1367. DOI: 10.1093/gerona/gly208.

LI X, XIE X Y, LIAN W S, et al . Exosomes from adipose-derived stem cells overexpressing Nrf2 accelerate cutaneous wound healing by promoting vascularization in a diabetic f oot ulcer rat model[J ] . Exp Mol Med, 2018, 50(4): 29. DOI: 10.1038/s12276-018-0058-5.

LIU S Q, MAHAIRAKI V, BAI H, et al . Highly purified human extracellular vesicles produced by stem cells alleviate aging cellular phenotypes of senescent human cells[J ] . Stem Cells, 2019, 37(6): 779-790. DOI: 10.1002/stem.2996.

KONG J G, PARK J B, LEE D, et al . Effect of high glucose on stress-induced senescence of nucleus pulposus cells of adult rats[J ] . AsianSpine J, 2015, 9(2): 155-161. DOI: 10.4184/asj.2015.9.2.155.

CHOI O R, RYU M S, LIM I K. Shifting p53-induced senescence to cell death by TIS21(/BTG2/Pc3) gene through posttranslational modification of p53 protein[J]. Cell Signal, 2016, 28(9): 1172-1185. DOI: 10.1016/j.cellsig.2016.05.014.

BEAUSÉJOUR C M, KRTOLICA A, GALIMI F, et al . Reversal of human cellular senescence: roles of the p53 and p16 pathways[J ] . EMBO J, 2003, 22(16): 4212-4222. DOI: 10.1093/emboj/cdg417.

JIN H, LIAN N, ZHANG F, et al . Activation of PPARγ/P53 signaling is required for curcumin to induce hepatic stellate cell senescence[J ] . Cell D eath Dis, 2016, 7: e2189. DOI: 10.1038/cddis.2016.92.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

谷胱甘肽过氧化物酶7通过PI3K-AKT通路调控肝癌细胞增殖和衰老

二甲双胍通过PP2A/AKT通路诱导结直肠癌细胞衰老

骨髓间充质干细胞外泌体调控肾癌细胞胆固醇代谢及其增殖、侵袭和迁移

高糖环境下抑瘤素对人皮肤成纤维细胞增殖和细胞外基质分泌的影响

miR-186-5p负性调控CDCA3对高脂高糖诱导的胰岛β细胞具有保护作用