转基因骨髓间充质干细胞促进轴突生长的实验研究

The spinal cord injury is highly concerned for it poor treatment effective and heavy burden to the society. Up to now there is no effective method to cure spinal cord injury, the reasons for the blackage of the regeneration of the spinal cord are most complicated, and mainly related to the glial scar and Nogo kinds molecules after spinal cord injury, which block the grown of axon. So the key point of the regeneration of the spinal cord is suppressing the effect of Nogo kinds molecules and promote the grown of axon. The purpose of this study is to clone the gene of NEP1-40, which is the competitive antagonist of the receptor of Nogo kinds molecules, and contrast the slow virus vector, transfect the bone marrow mesenchymal stem cells, finally contract the tissue engineering cells which can express NEP 1-40 gene. Study the safety and effective of the tissue engineering cell to promote the grown of the axon by transplant in situ for the spinal cord injured rat models. The creative point of the study is applying the engineering mesenchymal stem cell, supplying the neuro stem cells and suppressing Nogo kinds molecules at the same time to promote the axon grown..With this study the build of the engineering cell based on mesenchymal stem cell will be more mature. And the feasibility of promoting axon grown with engineering cells will be tested.

脊髓损伤所引起的重大社会和经济问题已经引起人们的高度重视。目前对于脊髓损伤尚无有效的治疗方法，脊髓损伤再生困难的原因复杂，主要是因为脊髓组织在受到损伤后损伤局部形成胶质瘢痕，以及Nogo类分子的存在抑制并阻碍轴突再生。因此，修复脊髓损伤的关键在于克服Nogo类分子对轴突生长的抑制作用，促进轴突生长。本项研究拟克隆Nogo受体竞争性拮抗物NEP1-40的基因，构建慢病毒载体，转染骨髓间充质干细胞，从而构建能够稳定表达NEP1-40的组织工程细胞。通过病灶原位接种的方式研究其治疗脊髓损伤的安全性和有效性，并研究其发挥治疗作用的机制。本项研究的创性点在于采用转基因骨髓间充质干细胞移植的方式，从补充外源性神经干细胞和抑制Nogo受体，促进神经元轴突生长两个方面促进脊髓损伤神经再生。.通过本项研究可以为组织工程细胞的构建以及临床应用组织工程细胞治疗脊髓损伤提供理论依据。

目的：将含有NEP1-40 基因的慢病毒载体转染骨髓间充质干细胞，评价基因转染后骨髓间充质干细胞生物学功能变化，观察NEP1-40 在骨髓间充质干细胞中的表达。方法：体外分离和培养骨髓间充质干细胞，流式细胞仪检测细胞表面标记，并对其生物学特性进行鉴定。同时将NEP1-40 基因克隆入慢病毒载体，包装出病毒上清，以不同拷贝数转染骨髓间充质干细胞。结果与结论：实验通过密度梯度离心法成功在体外分离和培养骨髓间充质干细胞，诱导其向脂肪细胞分化，流式细胞仪检测结果显示脐血间充质干细胞中CD90、CD73 及CD105 蛋白阳性，不表达CD14、CD34、CD45、CD19、HLA-DR、Stro-1 及CD106 蛋白；real-time PCR 检测发现其NEP1-40 mRNA 表达水平与转染拷贝数有关，转染拷贝数越高，NEP1-40 的表达量越高，此外转染NEP1-40 后的骨髓间充质干细胞中可检测到NEP1-40 蛋白，提示NEP1-40 基因转染后骨髓间充质干细胞原有的生物学功能无明显影响。