摘要
各种原因所致中枢神经损伤后的预后不良,主要是由于神经抑制因子抑制神经再生的作用以及微环境缺乏神经生长因子所导致。神经再生治疗一直是神经损伤研究的热点和前沿,其中对神经干细胞的起源、培养条件以及定向分化调控的研究更是其中的焦点问题。研究发现,成年哺乳动物中枢神经系统内一些非神经发生的部位如脊髓,存在有成体神经干细胞,为脊髓源性神经干细胞(Neural stem cells,NSCs),这为神经再生提供新的易于获得的种子细胞来源,并为神经系统损伤的治疗带来了新的思路和手段。
本实验主要研究胰岛素样生长因子-1(Insulin like growth factor-1,IGF-1)对脊髓源性神经干细胞定向分化的调控作用及初步探索其调控机制。IGF-1是一种与组织代谢和细胞分化、增殖有关的细胞因子,对维持神经细胞的生存、生长和损伤后的修复具有极其重要的作用。本实验在体外分离并培养了新生大鼠脊髓源性NSCs;利用脂质体介导法将带有IGF-1基因的质粒载体转入NSCs,经G418筛选,使用RT-PCR、免疫荧光、Western blotting鉴定IGF-1基因的表达,并利用免疫细胞化学法对神经干细胞的分化类型进行鉴定;使用细胞外信号调节激酶(Extracellular signal regulated kinase,ERK)的特异性抑制剂PD98095阻断IGF-1诱导神经干细胞向少突胶质细胞分化,观察神经干细胞的生长状态的变化,使用Western blotting、RT-PCR检测ERK的磷酸化和转录因子c-fos、c-jun、c-myc的表达情况,进而探讨ERK信号途径在IGF诱导神经干细胞分化中的作用和机制。
本研究选择IGF-1对脊髓神经干细胞进行干预,观察其作用效果及机制,从而更加深入的探讨脊髓源性神经干细胞在脊髓损伤后的转化方向和影响因素,加深对脊髓损伤后神经再生不良机制的研究,从而为探索治疗脊髓损伤的新方法奠定基础。
Prognosis mala often occurred after central nerve damage because of the affection of nerve inhibiting factor and lack of neurotrophic factor. Nerve regeneration therapy is an very interesting area of nerve injury research.And the source,culture condition and directional differentiation regulation is the hottest area.This study is about the directional differentiation regulation of IGF-1 to neural stem cells from spinal and discuss the convert directions and influence factor.This study provide new foundation for spinal injure thrapy.
1. The separation, culture and identification of neural stem cells from new born rat’s spinal marrow
The perfusion method and trypsinization method was used to sepetate the stem cells.And the cells can be separated by both method..The perfusion can easily damage the cells because of crushing, and it need gental operation.And the trypsinization method should control the digesting time.
The machinery method was used to generate of the cells. The different size pipette was used to thresh the cells, turned from big to small. The cells ball was separated from outside to inside. And this kind of method can reduce the damage to the cell, and enhance the proliferation ability.
The cells were transfer to the culture disk which was covered by polylsine first. The cell start to stick to the bottom of the 1d after generation. The cells began to ecphyma grow ing after 3d. The cells turned to different morphous after 7d, and showed special cell morphous. The cel of every dish can evection growth. Some cells were lage, and it had two long ecphyma, and some little ecphyma, and this cells is the nerve cell. Some cells had middle long range ecphyma, and this cells were horizontal cell. And some cells have rarely the ecphyma were oligodendroglial cell cell. And the amout of the horizontal cells were more than other cell types. The Nestin action is positive using immunal cell chemistry of the neural stem cells ball. The serum was used to induce the NSEs, and the NSE became the neuron which the result of NSEs is positive, the oligodendroglial cells is MBP positive, and the horizontal cell was GFAP positive. It was confirmed stem cells can remain its phenoty.
2. Recombination of pcDNA3.1-IGF-1 and expressed in the NSCs
The Enzyme cutting was used to obtain the GFP fragment. And it was link to the plasmid pcDNA3.1-IGF-1 to recombinate pcDNA3.1-GFP-IGF-1. The recombination express plasmid was identified by the enzyme BamHⅠand HindⅢ, and it was amplificated and purificated. G418 was used to assessment the least fatal dose of the NSCs. The plasmid was transferred the plasmid to the NSCs disk. And the RT-PCR,Western blotting was used to assess the expression of the IGF-1.
The pcDNA3.1-GFP-IGF-1 was cutted by BamHⅠand turned to linearization. The pcDNA3.1-GFP-IGF-1 was cutted by HindⅢand turned to two fragment, which were about 3023bp, 5127bp. The result showed the direction and magnitude of the recombinated plamid was right. The screening concentration of G418 was 0.4μg/ml. The expression of IGF was observed under fluorescend microscope. IGF-1 began to express 24h after transfection,and turned to weak. After RT-PCR test, a fragment of 407bp is obtained,and the length is according to the target gene.
And the result of the Western blotting of the IGF-1 was positive. The survival time and the amout of the NSCs which transfected with recombinated plasmid is prolonged.The large amount of the cell became oligodendrocyte status after 7-10days. The rate of the MBP immunocytochemical stain was increased obviously.
3. The Regulation of Insulin-like Growth Factor-1 (IGF-1) on Spinal Marrow-derived Neural Stem Cells
Western blotting was used to assess the degree of EKR1/2’s phosphorylation of the differentiated NSEs. And the level of the phosphorylated ERK was raise according to the time and went it’s highest level at 2h and decreased after that time. The different fina concentration of the ERK1/2 inhibitor PD98059 was added to the disk to co-cultured for 2h.And after 2h the protein was extract.And the Western blottin was used to assess the the degree of phosphorylated ERK.The degree of the PD98059 to inhibite the phosphorylated ERK raised by concentration. And when the PD98059 is 20μM, it reached it’s top.
RT-PCT was used to test the expression of c-fos、c-jun and c-myc gene at different time.And the result is the level of c-fos、c-jun was raised obviously at 0.5h and 1h, and the expression level is almost the same at the different time. RT-PCR was used to assess the expression level of c-fos、c-jun after PD98059 was added to the disk with the cells. The result was PD98059 can strongly inhit the mRNA expression of c-fos and c-jun.
Above all, there has central NSCs in spinal, and it can be induced to differentiate by some method. This study used lipidosome to transfect the plasmid into the NSCs. The result showed IGF-1 secreted from NSCs can contribute to the cells by paracrine secretion, activate the MAK- ERK signal transduction pathway to send the outside signal into the cell. And after that some protein associated with the cell proliferation and differention became expressed,at last NSCs turned to OLs. IGF-1 switch on the ERK signal pathway, and passed the signal to the IGEs gene c-fos、c-jun, and c-fos、c-jun began to express. The result is NSCs turned to OLs.
引文
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