Mash-1转染对胚胎干细胞在小鼠脊髓损伤部位向神经细胞分化的促进作用

Effects of Transfection of Mash-1 Gene on Neural Differentiation of Embryonic Stem Cell in Spinal Cord Injury Mice

目的观察过表达Mash-1基因的胚胎干细胞在脊髓损伤部位向神经细胞分化的情况及其对脊髓神经损伤的修复作用。方法采用鼠干细胞病毒将Mash-1基因转染CE3小鼠胚胎干细胞稳转株。4周龄雄性SPF级昆明小鼠钳夹法建立急性脊髓损伤模型。造模后3 d向脊髓损伤部位注射生理盐水(模型组,n=12)、CE3胚胎干细胞(CE3组,n=12)、转染Mash-1基因的CE3小鼠胚胎干细胞(CE3-Mash-1组,n=12)。术后1 d、7 d、14 d、21 d、28 d检测小鼠后肢功能评分。术后14 d、28 d分别处死小鼠,HE染色观察脊髓剩余面积;CE3组、CE3-Mash-1组行Oct3/4、nestin、β-tubulinⅢ、神经胶质酸性蛋白(GFAP)免疫荧光染色,观察移植细胞的分化。结果与模型组比较,移植CE3和CE3-Mash-1细胞的小鼠运动功能均提高(F>84.471,P<0.05),脊髓剩余面积增加(F>49.990,P<0.05)。与移植CE3细胞相比,移植CE3-Mash-1细胞在损伤的脊髓部位Oct3/4阳性细胞数显著减少(t=5.439,P<0.001),而nestin(t=-7.536,P<0.001)和β-tubulinⅢ(t=-9.941,P<0.001)阳性细胞数显著增加,GFAP阳性细胞数无显著性差异(t=1.701,P=0.120)。结论过表达Mash-1基因可促进CE3细胞在脊髓损伤部位分化成神经元,促进小鼠后肢运动功能的恢复。

Objective To investigate the effects of overexpression of Mash- 1 gene on functional recovery and neural differentiation of embryonic stem cells in spinal cord injury mice. Methods CE3 cell line with overexpression of Mash-1 gene was generated with murine stem cell virus. Spinal cord injury model was established with forceps compression in 4-week-old KM mice. Normal saline （model group, n= 12）, CE3 cells with or without overexpression Mash-1 gene （CE3-Mash-1 and CE3 groups, n=l 2 respectively） were transplanted into the ar- eas of injury 3 days after injury. They were assessed with the Basso Mouse Scale （BMS） 1, 7, 14, 21, and 28 days after injury. 6 mice from each group were sacrificed 14 and 28 days after injury respectively. The spinal cord area remained were observed with HE stained, and the expression of Oct3/4, nestin, ^-tubulin III and glial fibrillary acidic protein （GFAP） were detected with immunofluorescence in the injured spinal cord in the CE3 and CE3-Mash-1 groups. Results The score of BMS significantly improved in CE3 and CE3-Mash-1 groups com- pared with that of the model group （F〉84.471, P〈0.05）, with the more area of spinal cord remained （F〉49.990, P〈0.05）. There were less Oct3/4 positive cells in the CE3-Mash-1 group than CE3 group （t=-5.439, P〈0.001）, with more nestin （t=--7.536, P〈0.001） and β-tubulin IIl （t=-9.941, P〈0.001） positive cells. However, there was no significant difference in GFAP positive cells between CE3-Mash-1 and CE3 groups （t=1.701, P〉0.05）. Conclusion Overexpression of Mash-1 gene promotes CE3 cells to differentiate into neurons in spinal cord injury mice, and improve the motor function recovery.