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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。
OBJECTIVE: To observe the survival and differentiation of rat bone marrow mesenchymal stem cells (MSCs) in the spinal cord of congenital spinal fissured rat, and to explore the method of cell replacement in the treatment of congenital spinal fractures. Methods Primary cultured rat MSCs were cultured in adherent culture and passaged. The adenovirus carrying enhanced green fluorescent protein (EGFP) gene was transfected into the third generation bone marrow MSCs. Transfected MSCs were injected into the spinal cord of a congenital spinal fissured rat. Spleen of congenital vertebral fissure rat was taken 20 days after pregnancy, fixed frozen and dehydrated, frozen sections were made. The expression of Nestin and glial fibrillary acidic protein (GFAP) in EGFP positive MSC in spinal cord was detected by immunofluorescence. Results The percentage of CD90 positive cells in bone marrow MSCs was 99%, while that of CD44 positive cells was 95%. The transfection rate of adenovirus-EGFP transfected bone marrow MSCs was 61%. Teratogenic group dominant spina bifida accounted for 64.3%. Transplanted MSC is present on the surface of the spinal cord or into the spinal cord, and some cells become long fusiform or polygonal. Immunofluorescence results showed that MSC transplanted into the spinal cord could express Nestin, a neural stem cell marker, and GFAP, a marker of glial cells. CONCLUSION: MSC with EGFP can survive in spinal cord of fetal rat after cell transplantation and can differentiate into neural stem cells and glial cells.