脂肪间充质干细胞在功能化自组装纳米多肽水凝胶三维培养下旁分泌的研究  

Paracrine of human adipose - derived mesenchymal stem cells in three - dimensional culture containing functionized self assembling peptide nanofiber hydrogel

作　　者：凌建民[1] 田爱玲[1] 范鲁峰[1] 邵闻冲 武涵 焦阳[1] 胡三元[1] 孙念峰[1] 

机构地区：[1]山东大学齐鲁医院普外科,济南250012

出　　处：《中华实验外科杂志》2018年第2期250-252,共3页Chinese Journal of Experimental Surgery

基　　金：山东省重点研发计划项目（2016GSF201219）;山东省自然科学基金（ZR2017MH072）;山东大学齐鲁医院（青岛）科研基金（QDKY2015ZD05）

摘　　要：目的探讨脂肪间充质干细胞（ADMSCs）在自组装纳米多肽水凝胶介导的三维培养条件下旁分泌促血管生成激素及其影响因素。方法分离、培养人ADMSCs，并合成RADAI-16、RGD、KLT 3种多肽且按体积比2∶1∶1制备RADAI-16∶KLT∶RGD多肽混合溶液，原子力显微镜（AFM）下观察多肽水凝胶形态（n＝4）。使用水凝胶对ADMSCs进行三维培养，并设置ADMSCs普通培养组（37 ℃、5%CO2细胞培养箱）和缺氧培养组（37 ℃、10%CO2、1%O2乏氧培养箱）作为对照。酶联免疫吸附试验（ELISA）法检测各组细胞培养上清内血管内皮生长因子（VEGF）（n＝3）、肝细胞生长因子（HGF）（n＝3）浓度；Western blot检测细胞内血红素氧合酶-1（HO-1）（n＝3）的表达。结果分离培养的ADMSCs为长梭形，呈＂漩涡式＂生长。RADA16-I、RGD、KLT及自组装多肽溶液在AFM下均为纳米纤维状，直径10～20 nm，长度100～500 nm。ELISA法测得普通培养1 d、缺氧培养1 d及三维培养1 d后细胞培养上清中HGF浓度分别为（47.31±6.75）、（247.86±17.59）、（297.25±17.95） ng/L；VEGF浓度分别为（218.30±3.03）、（267.13±4.27）、（289.14±3.11） ng/L，缺氧及三维培养条件下细胞旁分泌HGF、VEGF均增高，但三维培养条件下增高更明显（P＝0.000）。Western blot结果显示三维培养组细胞内HO-1的表达量约为普通培养组2倍（P＝0.000），为缺氧培养组1.2倍（P＝0.033）。结论功能性自组装纳米多肽水凝胶介导ADMSCs的三维培养能明显促进其旁分泌作用，且缺氧是其重要影响因素之一。Objective To study the paracrine of human adipose-derived mesenchymal stem cells （ADMSCs） in three-dimensional culture containing functionized self assembling peptide nanofiber hydrogel and the influencing factors.Methods ADMSCs were isolated and extracted. Three kinds of peptides （RADAI-16, RGD, KLT） were prepared and functionized self assembling peptide nanofiber hydrogel was made by mixing RADAI-16, RGD and KLT with a volume ratio of 2∶1∶1. Under AFM, the morphology of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was observed （n=4）. ADMSCs were cultured under the three-dimensional condition containing peptide nanofiber hydrogel. ADMSCs were subjected to in situ three-dimensional cultivation for 1 day, then the concentrations of hepatocyte growth factor （HGF, n=3） and vascular endothelial growth factor （VEGF, n=3） were determined in the supernatatn by enzyme linked immunosorbent assay （ELISA）. The cells were extracted in peptide nanofiber hydrogel and the expression of heme oxygenase-1 （HO-1, n=3） was detected by Western blotting. The cultured ADMSCs in hypoxic condition and normal circumstance served as controls.Results ADMSCs showed long spindle shape and grew in a whirlpool shape. The diameter of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was （17.34±1.82）, （15.50±1.41）, （13.77±1.18） and （20.26±1.25） nm, respectively. The concentration of HGF under three conditions was （47.31±6.75）, （247.86±17.59）, and （297.25±17.95） ng/L, respectively, and that of VEGF was （218.30±3.03）, （267.13±4.27） and （289.14±3.11） ng/L, respectively. Both HGF and VEGF were paracrined more in condition of functionized self assembling peptide nanofiber hydrogel （P=0.000）. Western blotting results showed that the expression of HO-1 in the three-dimensional culture group was about 2 times （P=0.000） higher than that in the ordinary culture group, and was 1.2 times higher than that in

关 键 词：脂肪间充质干细胞 三维培养 功能性自组装纳米多肽 旁分泌 缺氧 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]