宫内移植骨髓间充质干细胞对显性脊柱裂胎鼠神经元重建的作用研究

Neuron regeneration of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta

目的胚胎期移植骨髓间充质干细胞（bonemarrowstemcell，BMSCs）至先天性脊柱裂模型胎鼠脊髓中，观察其在显性脊柱裂胎鼠脊髓中存活及分化情况，并探讨其对脊髓神经元修复的治疗潜能。方法利用胎仔外科和显微注射技术将绿色荧光蛋白（eGFP）转染的BMSCs分别在胚胎第16、17和18天移植到83只脊柱裂和20只正常大鼠胚胎中（groupE16、E17andE18），胚胎20d的胎鼠脊髓被取出并固定。经冰冻切片后，计数eGFP阳性细胞数目，并利用免疫荧光方法检测脊髓中eGFP阳性的BMSCs中神经干细胞（Nestin）、神经元（Neurofilament）和神经胶质细胞（GFAP）的分布与表达。结果本研究成功的将MSC在宫内移植入胎鼠体内，孕鼠的存活率为94％（81／86），胎鼠的存活率为78．1％（136／174）；实验中孕16d的胎鼠存活率为75．8％（91／120）低于孕·17d的80．6％（25／31）和孕18d的87．O％（20／23）。计数脊髓中eGFP阳性细胞数，范围在166～735个／根，其中P0代MSCs存活率最低，为10．8％；P3～6代为22．6％、P10～14代为20．1％；P10～14代MSC在母鼠孕16d时移植得到最佳分化率；统计eGFP阳性细胞表达Nestin、Neurofilament和GFAP的阳性率分别为4％～5％、1％～2％和5％～7％。结论BMSCs移植到显性脊柱裂胎鼠脊髓中能够存活，并在脊髓中表达Nestin、Neurofilament和GFAP，提示移植骨髓间充质干细胞具有向神经干细胞、神经元和神经胶质细胞分化的可能。

Objective To observe the survival and differentiation status of transplanted bone marrow mesenchymal stem cells （BM-MSCs） in spinal cord of dominant spina bifida fetal rats and discuss their therapeutic potentials for repairing spinal cord neurons. Methods Fetal surgery and micro-injection technology were employed to transplant green fluorescent protein （eGFP）-transfected BMSCs into 83 spina bifida and 20 normal rat embryos respectively at embryonic days 16,17 and 18 （groups E16,E17 ＆ El8）. And,at Day 20,spinal cord was removed and fixed. The number of eGFP- positive cells was counted. And immunofluorescence method was used to detect the distribution and expression of Nestin, Neurofilament and GFAP in eGFP-positive MSCs. Results After transplantation,the maternal survival rate was 94% and the fetal survival rate 78. 1%. The overall survival rates were tabulated in different operative groups. Better survival was achieved in the operative groups performed during later development stages （E17/E18） as compared with E16 group. The count of eGFP-positive cells in each spinal cord was 166-735. Secondly, we investigated if prolonged culture of MSCs affected the survival of transplanted MSCs. P3-P6 and P10-P14 cells had similar survival （22. 6% vs. 20. 1%） and it was higher than that of P0 cells （10. 8%）. In line with the increased neuronal differentiation potentials of MSCs in prolonged culture, MSCs generated more neurons in spinal column. Development stages of recipients also affected neural differentiation of MSCs. MSCs in El6 spinal column displayed significantly higher neuronal marker expression as compared with E18 and E17 groups. The eGFP-positive cells expressed nestin, neurofilament and glial fibrillary acidic protein at a oositive rate of 4-5 %, 1-2 % and 5-7 % resoectivelv. Conclusions After transplantation, the eGFP-positive BMSCs survive and differentiate in spinal cord of dominant spina bifida fetal rats. And they may express Nestin, Neurofilament and GFAP.