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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 investigate the effects of basic fibroblast growth factor (b FGF) and vascular endothelial growth factor (VEGF) on the proliferation, differentiation, maturation and vascularization of endothelial progenitor cells (EPCs) in hydrogels. Methods Arginine-glycine-aspartic acid (RGD) hydrogels containing b FGF / VEGF and b FGF / VEGF were prepared and divided into groups A and B 2. EPCs were also embedded. The viability of EPCs was detected by flow cytometry after 7 days. The growth of EPCs and the growth of hydrogel were observed under inverted microscope. The effects of bFGF and VEGF on the survival rate of EPCs were analyzed. After cultured for 7 days, the amount of total RNA was detected by quantitative real-time polymerase chain reaction (RT-PCR) and the expression of PECAM1, CD34, KDR, Angiopoietin-2 and pcdh12 genes were detected. Then the enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of matrix metalloproteinase (MMP) -2 and MMP-9 in the culture supernatant of the two groups of dehydrogel and the degradation rate of the hydrogel was detected by fluorescence method. Effect of FGF and VEGF on the release of MMPs from EPCs. Finally, chick embryo chorioallantoic membrane (CAM) technique was used to detect the in vitro angiogenesis induced by hydrogel containing b FGF and VEGF. Results A group of hydrogels, digestion after 7 days, the flow cytometry found that 39.43% of EPCs survived, B group 4.14%; two groups, the difference was statistically significant (P <0.05). The number of observed cells in EPCs grown on hydrogel surface was 378.333 3 in group A and 302.333 3 in group B, respectively. There was significant difference between the two groups (P <0.05). The expression of MMP-2 and MMP-9 in the two groups was detected by ELISA. It was found that the MMP-2 and MMP-9 in the hydrogel sustained release within 72 h, the release concentration in group A was significantly higher than that in group B Statistical significance (P <0.05). Fluorescence detection of hydrogel degradation found that starting from the first day, A group of hydrogel degradation percentage increased rapidly, then the growth rate slowed down, while the B group continued to grow trend, the difference was statistically significant. The results of quantitative RT-PCR showed that the Ct of group A was higher than that of group B, and the difference was statistically significant (P <0.05). The results of CAM technique showed that the total number of neovascularization in group A (blank group), group B (excluding b FGF and VEGF group) and group C (group b FGF and VEGF) was 9, 25 and 36, respectively There was statistical significance (P <0.05). Conclusion In vitro studies demonstrate that sustained release of bFGF and VEGF can not only promote the proliferation, differentiation, maturation and vascularization of EPCs in hydrogels, but also stimulate angiogenesis.