骨髓间充质干细胞在先天性脊椎裂胎鼠脊髓中的分化

Differentiation of Bone Marrow Mesenchymal Stem Cells in Congenital Spina Bifida of Fetus Rats

目的观察大鼠骨髓间充质干细胞(MSC)在先天性脊椎裂胎鼠脊髓中的存活与分化,探寻细胞替代治疗先天性脊椎裂的方法。方法用贴壁培养法进行大鼠MSC原代培养并传代,携带增强型绿色荧光蛋白(EGFP)基因的腺病毒转染第3代骨髓MSC。将转染的MSC注射到先天性脊椎裂胎鼠的脊髓中。母鼠孕20 d时取先天性脊椎裂胎鼠的脊椎,固定脱水后做冷冻切片,免疫荧光方法检测脊髓中EGFP阳性MSC中巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)的表达。结果骨髓MSC中CD90阳性细胞占99%,CD44阳性细胞占95%,不表达CD34。腺病毒-EGFP转染骨髓MSC的转染率为61%。致畸组显性脊椎裂胎鼠占64.3%。移植的MSC存在于脊髓表面或进入脊髓中,部分细胞变为长梭形或多角形。免疫荧光法检测结果显示移植到脊髓中的MSC可表达神经干细胞的标志物Nestin和神经胶质细胞的标志物GFAP。结论带有EGFP的MSC经细胞移植后能在胎鼠脊髓内存活,并能分化为神经干细胞和神经胶质细胞。

Abstract： Objective To investigate the bone of survival and differentiation of mesenchymal stem cells （MSC） in congenital spina bifi- da of fetus rats and to explore the theoretical clues for cell replacement of neural tube defects. Methods In sterile condition, rat bone marrow was flushed and cells were seeded in culture flask. Passage 3 of MSC were transfected by adenovirus enhanced green fluorescent protein （EGFP） gene and then were injected into congenital spina bifida of fetus rats through fine glass needle. On 20 days of gestation,fetus rats un- derwent operation and spinal cords were removed for fixation and dehydration and got frozen sections. Expressions of Nestin（ marker of neural stem cells） and glial fibrillary acidic protein（ GFAP,marker of neuroglioeyte） in transplanted MSC with EGFP were tested by immunofluores- eence. Results MSC surface antigen by flow cytometry showed CD34 （ - ） ,CD44 occupying 95% ,and CD9o occupying 99%. Transfection effi- ciency of adenovirus- EGFP to MSC was 61%. The rate of dominant spina bifida was 64.3% in teratogenic group. MSC distributed on the surface of spinal cord, or migrated into it. Some MSCs displayed different cell shapes such as long spindle or muhiangle. Expressions of Nestin and GFAP were present in transplanted MSC by immnnofluoreseence assay. Conclusion MSC with EGFP can survive in fetus spinal cord af- ter transplantation and differentiate into neural stem cells and neurogliocytes.