目的建立一种先天性显性脊椎裂胎鼠宫内干细胞移植的新方法。方法选择孕16、17、18天的雌性大鼠各30、30、26只,制备先天性显性脊椎裂胎鼠模型。采用胎仔外科、显微外科和显微注射技术,将转染绿色荧光蛋白基因的骨髓间充质干细胞(MSCs)移植到脊椎裂胎鼠脊髓中。大鼠孕20天时剖腹、取胎,计算胎鼠成活率;制作胎鼠脊髓切片,荧光显微镜下观察胎鼠脊髓干细胞移植情况。结果接受手术的孕鼠86只,孕20天成活81只,成活率为94.2%。行干细胞移植的先天性显性脊椎裂胎鼠258只,共取出198只,成活率为76.7%(198/258),其中胎龄为16、17、18天的胎鼠成活率分别为72.1%(80/111)、77.3%(78/101)、87.0%(40/46)。荧光显微镜下观察胎鼠脊髓切片,可见呈现绿色荧光的MSCs;干细胞移植成活的胎鼠195只,成活率为98.5%(195/198)。结论成功建立先天性显性脊椎裂胎鼠宫内干细胞移植的新方法;该方法干细胞成活率较高,可用于神经管畸形治疗。
Objective To establish a novel method of stem cell transplantation in utero for rats with congenital dominant spine bifida. Methods Thirty pregnant female rats( 16 days,E16),30 pregnant female rats( 17 days,E17) and26 pregnant female rats( 18 days,E18) were selected to establish the animal models of congenital dominant spine bifida.The bone marrow mesenchymal stem cells( MSCs) transfected with green fluorescent protein( GFP) gene were subsequently transplanted into spinal cord of pregnant rats with congenital dominant spine bifida by fetal surgery,microsurgery and microinjection. The E20 pregnant rats were killed and embryo biopsy was performed to count fetal rat survival rate. The spinal cord stem cell transplantation in fetal rat was observed under the fluorescence microscope. Results The survival rate was94. 2%( 81 /86) for E20 pregnant rats receiving surgery. One hundred ninety-eight embryo rats with stem cell transplantation survived in 258 rats with congenital spinal bifida fetal,whose survival rate was 76. 7%( 198 /258). The survival rates were 72. 1%( 80 /111),77. 3%( 78 /101) and 87. 0%( 40 /46) in E16,E17,and E18 fetal rates receiving stem cell transplantation. The GFP was observed in embryo spinal cord under fluorescence microscopy. The survival rate of MSCs was 98. 5%( 195 /198). Conclusions We successfully establish an new method of stem cell transplantation in utero for rats with congenital dominant spine bifida and its stem cell survival rate is high. This method can be used as one of the methods in treatment of neural tube defects.