静脉移植骨髓间充质干细胞对大鼠脊髓损伤后生长相关蛋白43、神经丝蛋白及巢蛋白的调节

Regulatory effect of intravenous transplantation of bone marrow mesenchymal stem cells on the expression of growth-associated protein-43,neurofilament protein and Nestin in rats after spinal cord injury

目的:已证实骨髓间充质干细胞不仅具有向中胚层间质细胞分化的能力,在一定条件下或微环境中还能够向外胚层和内胚层细胞分化。为此观察静脉移植骨髓间充质干细胞对脊髓损伤后生长相关蛋白43、神经丝蛋白及巢蛋白的影响。方法:实验于2004-12在四川大学华西医院组织工程实验室完成。①实验动物:雌性SD大鼠66只,随机数字表法分为3组:细胞移植组、注射液对照组、模型对照组,22只/组。另取6周龄SD大鼠10只用于骨髓间充质干细胞的分离培养。②实验方法:应用密度梯度离心、贴壁培养和酶消化控制相结合,分离纯化鼠骨髓间充质干细胞。传至第6代在移植前1d予以BrdU标记,制成浓度约为2×109L-1细胞悬液。各组大鼠均建立脊髓损伤模型,造模后1周作尾静脉穿刺,细胞移植组注入骨髓间充质干细胞悬液1mL,注射液对照组注入等量的无血清低糖培养基,模型对照组未作处理。③实验评估:分别于细胞移植后2,3,6周取材,免疫组织化学检测骨髓间充质干细胞在损伤段脊髓的存活情况,观察损伤段脊髓内生长相关蛋白43、神经丝蛋白及巢蛋白的表达。结果:①骨髓间充质干细胞存活及计数:移植后2,3,6周BrdU阳性的骨髓间充质干细胞主要分布于损伤段及邻近节段脊髓内,计数分别为(9830±874)个,(8780±720)个,(5700±420)个,各占移植细胞总量的4.9%,4.4%,2.9%。②生长相关蛋白43及神经丝蛋白的表达:细胞移植后2,3,6周,细胞移植组生长相关蛋白43及神经丝蛋白的表达面积均明显高于其余两组(P<0.05)。③巢蛋白的表达:细胞移植后2,3周,细胞移植组的巢蛋白阳性细胞数均明显高于其余两组(P<0.05)。至第6周细胞移植组巢蛋白阳性细胞数明显降低(P<0.05),其余两组不表达。结论:脊髓损伤后静脉移植的骨髓间充质干细胞能向脊髓损伤灶内迁徙、存活,上调生长相关蛋白43、经丝蛋白及巢蛋白的表达。

AIM： It has been proved that bone marrow mesenchymal stem cells （BMSCs） could not only differentiate towards mesodermal mesenchymocyte, but also ectoderm and endoderm cells under specific condition or microenvironment. In this study, the influence of intravenous transplantation of BMSC on the expression of growth-associated protein-43 （GAP-43）, neurofilament protein （NF200） and Nestin in rats after spinal cord injury was observed. METHODS： The experiment was conducted in the Laboratory of Tissue Engineering, West China Hospital of Sichuan University in December 2004. （1）Sixty female SD rats were randomly divided into cell transplantation group, injection group and model control group with 22 rats in each group. （2）BMSCs were harvested and isolated from 10 donor 6-week-old SD rats with density gradient centrifugation method, cultured with attachment, and digested. The sixth generation cells were labeled with bromodeoxyuridine （BrdU） and prepared suspension of 2 ×10^9 L^ -1 before transplantation. All rats were subjected to weight-drop impact to establish spinal cord injury models. One week later, 1 mL BMSC suspension was injection in transplantation group by tail vein; serum-free low glucose medium in injection control group, while the model control group was not given any treatment. （3）The rats were samples 2, 3 and 6 weeks after transplantation to detect the survival of BMSCs in injured spinal cord with immmohistochemical staining. The expressions of GAP-43, NF200 and Nestin around the injured spinal cord were also measured. RESULTS： （1）After 2, 3 and 6 weeks transplantation, BrdU positive cells mainly distributed in the injured spinal cord and the surrounding regions of spinal cord （9 830±874） cells, （8 780±720） cells and （5 700±420） cells, respectively, accounted for 4.9%, 4.4% and 2.9% of the total BMSCs. （2）The expression of GAP-43 and NF200 in cell transplantation group was significantly higher than the other groups 2, 3 and 6 weeks after treatment （P 〈 0.05）. （3）Nestin positive cells in cell transplantation group was significantly higher than the other groups 2 and 3 weeks after treatment （P 〈 0.05）. But at the 6^th week, Nestin positive cells in cell transplantation group were remarkably decreased （P 〈 0.05）, even no cell in the other groups. CONCLUSION： BMSC migrates and survives into spinal cord with intravenous transplantation after spinal cord injury. It can upregulate the expression of GAP-43, NF200 and Nestin.