缓释的碱性成纤维细胞生长因子、血管内皮细胞生长因子对于水凝胶中内皮祖细胞集落形成单位的影响  

Effect of basic fibroblast growth factor and vascular endothelial growth factor on endothelial progenitorceils colony proliferation, differentiation and maturation in hydrogel and vascularization

作　　者：韩艳久[1] 刘国辉[1] 欧阳柳[1] 王小红[1] 

机构地区：[1]华中科技大学 同济医学院附属协和医院骨科,湖北武汉430022

出　　处：《生物医学工程与临床》2015年第1期1-6,共6页Biomedical Engineering and Clinical Medicine

基　　金：国家自然科学基金资助项目(81141077)

摘　　要：目的研究碱性成纤维细胞生长因子(b FGF)和血管内皮细胞生长因子(VEGF)对于水凝胶中内皮祖细胞集落形成单位(EPCs)增殖、分化、成熟,以及血管化的影响。方法制备分别含有b FGF/VEGF和不含b FGF/VEGF的精氨酸-甘氨酸-天冬氨酸(RGD)水凝胶,分为A、B 2组。同时包埋EPCs,流式细胞仪检测培养7 d后的细胞存活率,倒置显微镜观察EPCs的生长及长入水凝胶的情况并计数,分析b FGF、VEGF对EPCs存活率的影响。培养7 d后用定量实时聚合酶链反应(RT-PCR)检测总RNA量,并检测PECAM1、CD34、KDR、Angiopoietin-2、pcdh12基因的表达。然后用酶联免疫吸附分析(ELISA)法检测2组去水凝胶的培养上清液中基质金属蛋白酶(MMP)-2、MMP-9的表达,并用荧光法检测水凝胶的降解速度,b FGF、VEGF对EPCs释放MMP的影响。最后以鸡胚绒毛尿囊膜(CAM)技术检测含有b FGF、VEGF的水凝胶体外诱导血管新生的作用。结果 A组水凝胶,消化7 d后,流式细胞仪检测发现39.43%的EPCs尚存,B组4.14%;两组相比,差异有统计学意义(P<0.05)。水凝胶表面种植EPCs可观察到的细胞数A组为378.333 3,B组为302.333 3;两组相比,差异有统计学意义(P<0.05)。ELISA法检测2组中MMP-2、MMP-9的表达,发现水凝胶中MMP-2和MMP-9随时间推移在72 h内持续释放,A组较B组释放浓度显著增加,差异有统计学意义(P<0.05)。荧光法检测水凝胶的降解发现,从第1天开始,A组水凝胶降解百分比急速升高,之后增长速度减缓,而B组呈继续增长之势,差异有统计学意义。定量RT-PCR检测发现A组较B组基因Ct值高,且差异有统计学意义(P<0.05)。CAM技术检测发现A组(空白组)、B组(不含b FGF、VEGF组)、C组(含b FGF、VEGF组)的新生血管总数分别为9、25、36;两两比较,差异有统计学意义(P<0.05)。结论体外研究证实,缓释的b FGF、VEGF不仅能促进水凝胶中EPCs增殖、分化、成熟,以及血管化,还能刺激血管新生。Objective To study the impact of basic fibroblast growth factor（bFGF） and vascular endothelial growth factor（VEGF） on endothelial progenitorceils colony（EPCs） proliferation, differentiation and maturation in hydrogel and vascularization. Methods The arginine-glycine- aspartic acid（RGD） hydrogels with or without bFGF and VEGF were prepared, which were divided into group A and B. The EPCs were embedded at the same time to test cell survival rate with flow cytometer after 7 days, and inverted microscope was used to observe the growth of EPCs and evolved hydrogel, and the impact of bFGF and VEGF on survival rate of EPCs was analyzed. Quantitative real-time polymerase chain reaction（RT-PCR） was used to test the total RNA level after 7 days and the gene expression of PECAM1, CD34, KDR, Angiopoitin-2 and pcdhl2. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of matrix metalloproteinases（MMP）-2 and MMP-9 in de-hydrogel culture. The speed of hydrogel degradation and the impact of bFGF and VEGF on the release of MMP by EPCs were tested by fluorescent method. Finally, the impact of bFGF and VEGF on aquogelin in vitro induction of angiogenesis was tested with chick chorioallantoic membrane（CAM）. Results The 39.43 % of EPCs survives after 7 days tested with flow cytometry in group A hydrogel in comparison with 4.14 % in group B, and there was statistically significant difference between 2 groups（P 〈 0.05）. The observable cell number in group A was 378.333 3 while that in group B was 302.333 3, and there was statistically significant difference between 2 groups（P 〈 0.05）. The MMP-2 and MMP-9 expression in 2 groups tested by ELISA showed that MMP-2 and MMP-9 in hydrogel were released continuously within 72-hour, the release speed of group A was faster than that of group B, and the difference was statistically significant between 2 groups（P 〈0.05）. The degradation of hydrogel tested with fluorescent method revealed that the degradation rate of hydrogel

关 键 词：碱性成纤维细胞生长因子(bFGF) 血管内皮细胞生长因子(VEGF) 内皮祖细胞集落形成单位(EPCs) 基质金属蛋白酶(MMP) 鸡胚绒毛尿囊膜(CAM)技术 血管新生 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]