体外转染绿色荧光蛋白基因的肌源性干细胞移植修复大鼠脊髓损伤

Transplantation of muscle-derived stem cells in vitro transfected with green fluorescent protein gene for repairing spinal cord injury in rats

背景：肌源性干细胞易于提取、分离及扩增,在特定条件下可分化为骨、软骨、肌肉等中胚层组织细胞,还可以跨胚层分化为神经细胞等,是组织工程临床用于脊髓损伤修复的理想种子细胞。目的：观察肌源性干细胞移植对脊髓半切损伤大鼠运动功能的修复作用。方法：40只成年SD大鼠随机数字表法分为移植组和对照组,每组20只。均进行脊髓半切损伤,伤后9d,移植组于伤处移植体外转染绿色荧光蛋白基因的大鼠肌源性干细胞,而对照组仅注射等量PBS,于移植后1,2,3,4周用斜板实验和BBB评分测大鼠的运动功能,同时进行损伤脊髓取材、快速冰冻切片进行荧光显微镜观察。结果与结论：所有大鼠脊髓半切损伤手术均成功,术后无动物死亡。肌源性干细胞移植后1周,移植组与对照组均有所恢复,斜板实验和BBB评分差异无显著性意义（P〉0.05）;2~4周移植组恢复明显较好,斜板实验和BBB评分显著高于对照组（P〈0.05）,移植组后肢活动与前后肢活动的协调性明显优于对照组。荧光显微镜观察经诱导分化和基因标记的肌源性干细胞在损伤脊髓组织局部生长良好,并且有沿着脊髓神经束向头尾两侧迁移的趋势。提示脊髓半切损伤大鼠经肌源性干细胞移植后能在损伤脊髓组织局部长期存活并明显改善其运动功能,肌源性干细胞移植对脊髓半切损伤大鼠有修复作用。

BACKGROUND：Muscle-derived stem cells（MDSCs） are easily extracted,isolated and amplified and have a pluripotent ability to differentiate into a variety of cells under certain conditions such as myocytes,cartilage cells and osteoblasts,even neural cells.Thus,MDSCs are ideal seed cells for repairing spinal cord injury in tissue engineering.OBJECTIVE：To observe the effects of MDSC transplantation on the locomotor recovery of hemisectioned spinal cord injury in rats.METHODS：A total of 40 adult Sprague-Dawley rats were randomly divided into transplantation group（n = 20） and control group（n = 20）.Spinal cord hemisection was performed in all rats.At 9 days post-injury,MDSCs with green fluorescence protein（GFP） gene were transplanted into the injured spinal cord in transplantation group,while control group was treated with injection of an equal volume of phosphate buffered saline.The rat behaviors were assessed by slope test and Basso,Beattie,and Bresnahan（BBB） scale at 1,2,3 and 4 weeks after transplantation respectively.At the same time,the injured spinal cords were made into frozen sections and were observed through fluorescence microscope.RESULTS AND CONCLUSION：Spinal cord hemisection was successful in all rats,and no death was determined.At 1 week following MDSC transplantation,locomotor recovery was found in the transplantation and control groups.No significant difference was detected in tiltboard test and BBB scale（P 0.05）.At 2-4 weeks,the recovery was good in the transplantation group.Scores of tiltboard test and BBB scale were significantly greater in the transplantation group than in the control group（P 0.05）.Coordination in hindlimb activity,as well as forelimb and hindlimb activities were obviously better in transplantation group than in the control group.Under fluorescence microscope,MDSCs that were differentiated and marked with GFP grew well at the injured myeloid tissue and migrated following spinal nerve tract.Above-described results have suggested that MDSCs can grow well at the injured myeloid tissue and improve the locomotor recovery in rats with hemisectioned spinal cord injury.MDSC transplantation has therapeutic effect on hemisectioned spinal cord injury in rats.