兔骨髓间充质干细胞同种异体皮下移植研究

Transplantation of allogeneic mesenchymal stem cells into rabbit subcutaneous tissue

目的研究兔骨髓间充质干细胞(mesenchymalstemcells,MSCs)同种异体皮下移植后的存活与分布情况,为拓展MSCs应用提供理论基础。方法采用绿色荧光蛋白(EGFP)或5-溴-2-脱氧尿苷(BrdU)标记细胞,与明胶海绵复合后植入兔异体或自体背部皮下,观察术后3d、1,3,5周标记细胞存活、分布和免疫反应等。结果异体和自体MSCs植入皮下后均表现出较强的迁移能力,部分进入宿主组织内。随时间延长,术后植入区域的标记细胞和炎性细胞逐渐减少,3周时异体和自体标记细胞在局部的分布出现差异(P<0.05),但是5周时仍可以在异体组和自体组观察到较多的细胞表达EGFP和含有BrdU。结论兔骨髓MSCs在异体皮下组织中至少可以存活5周,并表现出较强的迁移能力,从而提示异体MSCs可能具有一定的应用价值。

Objective To study the short-term fate of labeled allogeneic mesenchymal stem cells (MSCs) after implantation into rabbit subcutaneous tissue, and provide insights into the application of allogeneic MSCs for tissue regeneration. Methods First of all MSCs isolated from rabbit femur marrow were culture-expanded. Next MSCs were labeled by two different methods, one was that MSCs were transfected with the recombinant retrovirus containing enhanced green fluorescent protein (EGFP) and the other was that monolayers of MSCs were grown in medium containing 5-bromo-2-deoxyuridine (BrdU). After labeled MSCs were incubated with gelatin sponge and non-adherent cells were washed away, autologous MSCs-Gelatin constructs and allogeneic MSCs-Gelatin constructs were implanted into subcutaneous tissue of 24 rabbits. The constructs were analyzed for the survival and migration of labeled MSCs at 3 days, 1 week, 3 weeks and 5 weeks post-implantation. Results EGFP was successful expressed after MSCs were transfected with the retroviral vector pLEGFP-N1. At early time points of three days and one week, inflammatory reaction was significant and then inflammatory cells diminished. By three and five weeks, the labeled cells were extensive on the surface of gelatin sponge and gradually retained into host tissue. There was significant difference between two groups (P<0.05). Fluorescence microscope showed that cells expressed EGFP, and BrdU was detected by immunohistochemical method during the initial five weeks after allogeneic or autologous MSCs-Gelatin constructs had been implanted. Conclusions The rabbit allogeneic MSCs can survive for at least five weeks after implantation into subcutaneous tissue and maintain strong ability of migration, as indicates that allogeneic MSCs have certain clinical application value.