CD31抗体耦联脾脏脱细胞支架的构建及再内皮化  

Construction of anti-CD31 antibody-conjugated decellularized spleen scaffolds

作　　者：万建 袁骁祺 周元[2] 陈春球[1] 尹路[1] Wan Jian;Yuan Xiaoqi;Zhou Yuan;Chen Chunqiu;Yin Lu(Center for Difficult and Complicated Abdominal Surgery,the Tenth Hospital Affiliated Shanghai Tongji University,Shanghai 200072,China;Department of Hepatobiliary Surgery,Nantong Tumor Hospital,Nantong 226361,China)

机构地区：[1]上海同济大学附属第十人民医院腹部外科疑难诊治中心,200072 [2]南通市肿瘤医院肝胆外科,226361

出　　处：《中华实验外科杂志》2020年第5期847-849,共3页Chinese Journal of Experimental Surgery

基　　金：南通市卫计委青年科研基金项目(WKZL2018047)。

摘　　要：目的:构建具有良好组织相容性的CD31抗体耦联脾脏脱细胞支架,经内皮细胞再种植实现支架脉管系统预血管化。方法:经脾动脉灌注1%曲拉通X-100(Triton X-100)/0.1%氨水制备大鼠脾脏脱细胞支架,苏木精-伊红(HE)染色和糖胺多糖(GAG)含量检测评价脱细胞效率, t检验分析脱细胞后DNA去除的效果。利用(3-二甲基氨丙基)-碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将CD31抗体耦联到脾脏脱细胞支架,再种植人脐静脉内皮细胞(HUVECs)。继续培养3 d后,免疫荧光检测评价细胞黏附。体内移植2周后,经HE染色、免疫组织化学染色,评价支架体内血管生长及组织相容性。 结果:脾脏脱细胞支架HE染色未见明显细胞残留,脱细胞支架DNA含量[(43.26±5.14) ng/mg]较正常脾脏组织DNA含量[(5 896.00±393.90) ng/mg]明显减少( t=14.860, P<0.05)。脱细胞支架GAG含量[(30.92±1.70) ng/mg]较正常脾脏组织GAG含量[(42.37±1.77) ng/mg]保留了70%以上。免疫荧光结果显示CD31成功结合到脱细胞支架中,且HUVECs定植于支架血管腔内,血管性血友病因子(vWF)呈阳性表达。支架体内移植2周后,HE染色、免疫组织化学结果显示CD31修饰脾脏脱细胞支架组血管生成数目明显多于未修饰组,且具有良好的组织相容性。 结论:大鼠脾脏脱细胞支架保留了基本的脉管结构及细胞外基质成分,CD31耦联修饰的脾脏脱细胞支架支持HUVECs的定植生长,且体内具有促血管生成作用,具有良好的组织相容性。Objective CD31 antibody modification can promote the re-endothelialization of splenic decellularized scaffolds(DSSs),and improve the histocompatibility of DSSs.Methods DSSs were obtained by perfusion of detergent through the splenic artery,and the hematoxylin-eosin(HE)staining and glycosaminoglycan(GAG)were adopted to evaluate the results.DNA contents of DSSs and normal spleen were compared by t test.CD31 antibody was conjugated to the DSSs according to the activating of N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS).Human umbilical vein endothelial cells(HUVECs)were replanted on scaffolds and cultured for 3 days.Immunofluorescence(IF)was performed to detect cell adhesion and proliferation.At 2nd week after transplantation,HE staining and immunohistochemistry(IHC)were used to evaluate vascular growth in vivo.Results HE staining demonstrated no residual cells remained in DSSs.The DNA content of DSSs[(43.26±5.14)ng/mg]was significantly lower than that of normal spleen[(5896.00±393.90)ng/mg](t=14.860,P<0.05).The GAG content of DSSs[(30.92±1.70)ng/mg]was at least 70%of normal spleen tissue[(42.37±1.77)ng/mg].IHC staining showed not only CD31 antibody adhered to the surface of the scaffolds,but the HUVECs grew well in the scaffolds after culture for 3 days.The CD31-conjugated DSSs implanted with HUVECs were transplanted in vivo for 2 weeks.The results of HE staining and IF showed that the number of angiogenesis in the CD31-conjuncted DSSs was significantly greater than that in the DSSs.Conclusion The DSSs preserved the structure of vascular and extracellular matrix.CD31-conjuncted DSSs could not only support the growth of HUVECs in vitro,but had the effect of promoting angiogenesis in vivo and showed good histocompatibility.

关 键 词：脾脏 脱细胞支架 CD31 再内皮化 组织相容性 

分 类 号：R654[医药卫生—外科学] R318.08[医药卫生—临床医学]