构建高生物相容性干细胞心脏补片的研究

Construction of high biocompatible stem cell cardiac tissue patch

目的:去细胞化处理脐动脉构建生物支架,将小鼠脐带胶样组织间充质干细胞(MCS)种植到生物支架上构建心脏补片,移植到小鼠心肌梗死区域观察其生物相容性。方法:1联合应用胰酶和胶原蛋白酶处理小鼠脐带动脉组织构建生物支架;2体外分离并培养小鼠脐带MCS至第三代,种植到生物支架构建心脏补片;3移植到野生型小鼠皮下3天后观察免疫反应;4移植到小鼠心肌梗死区域,观察其与原位心肌的融合和血管新生。结果:脱细胞化处理后的脐带动脉组织在电镜观察下显示为富含多孔隙的纤维支架;荧光染色观察MSC种植到支架上深入生长到支架的内部并黏附生长;去细胞化处理未明显改变脐动脉组织的含水比例[(95.3±1)vs.(94.9±0.6),P>0.05];与对比组比较,去细胞化处理导致组织支架的断裂牵张力显著降低[(0.24±0.0)vs.(0.15±0.06)m Pa,P<0.05]。但是去细胞化处理未明显改变脐动脉组织的断裂变形率[(45±15)vs.(53±10)%,P>0.05];将裸支架和种植了干细胞的组织补片分别种植于C57野生小鼠的皮下,3日后切片染色,荧光显微镜下观察裸支架移植后局部CD+4淋巴细胞为(15±2.4)%,而干细胞补片内仅见少量CD+4的细胞浸润[(1±0.4)%,P<0.05]。将干细胞补片移植到小鼠急性心肌梗死区1周后,发现补片与原位心肌的融合性良好,补片内部有大量的新生血管生成。结论:成功构建心脏补片;移植到小鼠具有较高的生物相容性,并与原位心肌融合性好并有新生血管形成,为急性心肌梗死的干细胞治疗提供了实验依据。

Objectives： To construct the mesenchymal stem cells（ MSCs）-cardiac patch using the decellularized umbilical artery and the MSCs. To study the biocompatibility of the MSC patch when transplanted in the infarct heart. Methods： The umbilical artery was decellularized by enzymatic digestion. The mice umbilical MSC were isolated and cultured untile the 3nd generation then cultured on the decellularized umbilical artery scarffold. Then the MSC-tissue patchs were transplanted to the subcutaneous tissue and the infracted heart area,the immunology reaction and the tissue intergration were observed. Results： The scaffold maintained the interconnected porous structure. It remained the similar high water content（ 95. 3 ± 1） % compared to（ 94. 9 ± 0. 6） % in the intact umbilical artery（ P 〉0. 05）. The decellularization process decreased the stress from（ 0. 24 ± 0. 05） to（ 0. 15 ± 0. 06） mPa（ P〈0. 05）. However the decellularization did not change the strain force of the umbilical artery（ 45 ± 15） % vs.（ 53 ± 10） %（ P 〉0. 05）. When the scaffold was transplanted to the subcutaneous tissue in the wild type mice,there were less T cells appeared in the surrounding tissue which mean the decreased of the immunogenicity by decellularization. One week after the transplantation on the infracted heart,we observed the stem cell cardiac patch integrate with the in-situ heart tissue and the neovasculars appeared in the patch. Conclusion： In this study,we constructed a biologicalcompatible porous cardiac patch combining the decellularized umbilical artery and the MSC. The MSC-cardaic patch can intergrate well with the in situ cardiac tissue and perform the neovasculars in the patch.