共价结合生长因子胶原补片对人骨髓间充质干细胞增殖和分化的影响  

Covalent binding of growth factor collagen patch on human bone marrow mesenchymal stem cell proliferation and differentiation

作　　者：卢耀军[1] 赵瑞平[1] 孙凯[1] 乌日娜[1] 韩瑞娟[1] 

机构地区：[1]包头市中心医院心内科,内蒙古包头014040

出　　处：《中国医学前沿杂志（电子版）》2016年第3期108-112,共5页Chinese Journal of the Frontiers of Medical Science(Electronic Version)

摘　　要：目的制备与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)共价结合的胶原蛋白海绵补片,探讨其对人骨髓间充质干细胞增殖和分化的影响。方法制备胶原补片,随机分为空白对照组和实验组。空白对照组胶原补片在无菌条件下经碳化二亚胺处理,实验组胶原补片于室温条件下利用N-羟基硫代琥珀酰亚胺+磷酸盐缓冲液+1-(3-二甲基丙基)-3-乙基碳二亚胺盐酸盐溶液处理,并与b FGF和VEGF共价结合。保存后1、3、7、14、21、28天采用酶联免疫吸附测定(ELISA)检测上清液中b FGF和VEGF含量。将人骨髓间充质干细胞均匀接种于两组胶原补片上,分别进行细胞计数、MTT检测以及溴脱氧尿苷单克隆抗体增殖实验,并采用SMA免疫荧光染色检测细胞分化情况,采用逆转录-聚合酶链反应(RT-PCR)检测细胞Ⅰ、Ⅲ型胶原表达情况。结果实验组胶原补片与VEGF和b FGF结合率均显著高于空白对照组(P<0.05)。绘制释放曲线并分析发现,实验组生长因子处于稳定状态,仅有极少数发生解离释放。制备后28天,大部分生长因子存留于胶原补片上。随时间推移,两组胶原补片上骨髓间充质干细胞均呈增长趋势。接种后3天、7天比较显示,实验组胶原补片上碳化二亚胺数量均显著多于对照组(P<0.05)。经RT-PCR检测和SMA免疫荧光染色发现,与空白对照组相比,实验组胶原补片上细胞Ⅰ、Ⅲ型胶原表达水平均显著下调(P<0.05)。结论 b FGF和VEGF共价结合的胶原补片可以对人骨髓间充质干细胞产生较大影响,有效促进其增殖并抑制分化。Objective To prepare the basic fibroblast growth factor（b FGF） and vascular endothelial growth factor（VEGF） collagen covalently bonded patch, and discusse the effects of human bone marrow mesenchymal stem cell proliferation and differentiation. Method Collagen patch was prepared and were randomly divided into control group and experimental group. Control group collagen patch treated by carbodiimide under sterile conditions. Experimental group collagen patch at room temperature under the condition of used N-hydroxy succinimide and ＋ phosphate buffered saline ＋ EDC.HCL and with b FGF and VEGF of covalent binding. Saved after 1, 3, 7, 14, 21, 28 days, the concent of b FGF and VEGF in the supernatant fluid were detected by ELISA. The human bone marrow mesenchymal stem homogeneous cell were seeded on two groups of collagen patch, cell counts and MTT assay and bromodeoxyuridine monoclonal antibody proliferation assay were performed, and the SMA immunofluorescence staining method was used to detect cell differentiation, the expression of collagen Ⅰ and collagen Ⅲ were detected by RT-PCR. ResultExperimental group collagen patch with VEGF and b FGF binding were significantly higher than control group（P 0.05）. Draw the release curve and analysis found that the experimental group growth factor in a very stable state, only a handful of dissociated release. 28 days after preparation, most growth factor remained in the collagen patch. Two groups of collagen patch with the passage of time, bone marrow mesenchymal stem cells showed a growth trend. 3 and 7 days after inoculation comparison showed that experimental group collagen patch on carbodiimide were significantly more than control group（P 0.05）. Compared with control group, experimental group of collagen supplement cell sheet on the expression levels of collagen Ⅰ and collagen Ⅲwere significantly reduced by RT-PCR detection and SMA staining found（P 0.05）. Conclusion b FGF and VEGF covalent binding of collagen patch has a greater impact on hum

关 键 词：胶原蛋白海绵 生长因子 碱性成纤维细胞生长因子 血管内皮细胞生长因子 人骨髓间充质干细胞 增殖 分化 

分 类 号：R54[医药卫生—心血管疾病]