肌源性干细胞在修复大鼠脊髓损伤过程中Notch1基因的表达差异

发布时间：2018-08-07 16:14

【摘要】：目的 探讨肌源性干细胞(Muscle derived stem cells,MDSCs)在修复大鼠脊髓损伤过程中Notch1基因的表达差异。 方法 1、采用机械-混合酶消化法分离培养大鼠MDSCs,结蛋白(Desmin)细胞免疫化学鉴定其为肌源性干细胞。NFG诱导MDSCs并采用免疫组织化学法检测诱导后的细胞表达NSE特异性标志物。 2、根据实验要求,选取120只大鼠样本,动物按随机数目表法分为:A组:NGF诱导后且BrdU标记的MDSCs组(n=30);B组:未诱导且BrdU标记的MDSCs组(n=30);C组:对照组(n=30)及D组:空白对照组(n=30)。A、B、C组按取材时间不同分为1周、4周、8周。 3、手术方法:制作脊髓半横断模型。脊髓损伤后使用微量注射器分别向A组注射进行NGF诱导分化后且BrdU标记的MDSCs培养液,向B组注射等量未经诱导分化但经BrdU标记的MDSCs培养液,C组注入相同剂量的PBS液。 4、各组大鼠在存活期间按不同时间点测定各组大鼠后肢运动功能(BBB运动功能评分及斜板实验)。 5、免疫组化染色检测观察MDSCs在大鼠体内的存活、迁移及分化情况。 6、RT-PCR和Western技术检测肌源性干细胞植入后Notch通路中关键基因的表达情况。 结果 1、经过机械-混合酶消化和差速贴壁后,成功培养出高纯度、高活性的MDSCs,细胞免疫化学结果为Desmin(+)(阳性率80%)。 2、诱导过程中,细胞生长状态良好,形态呈渐进性变化,未见神经球形成;诱导组7d后神经样细胞的胞体和突起NSE染色呈强阳性(37.29±1.59)%,而未诱导组的细胞阳性细胞率极低(4.26±0.34)%,两组之间差异明显(P0.01);诱导组的MDSCs样品中检测到NSE表达水平高于未诱导组(P0.01)。 3、行为观察结果:伤后1周时A组(诱导组)的评分略高于B组(未诱导组)但均高于C组(对照组),随着时间的延长,各组差异逐渐明显,A组的评分明显高于B组、明显优于未加干预的C组。结果显示,经NGF诱导肌源性干细胞移植治疗脊髓损伤的疗效明显优于单独应用肌源性干细胞移植的方法。 4、组织学结果:观察BrdU标记后移植的MDSCs在大鼠体内的存活、迁移及分化情况。A组损伤修复尤其明显,细胞数量多于B组。结果显示,经NGF诱导后的MDSCs在大鼠体内修复脊髓损伤效果优于单独应用的MDSCs。 5、RT-PCR及Western检测结果显示:在大鼠脊髓损伤过程中Notch信号通路中的Notch1节点基因mRNA及Notch1蛋白均呈现低表达,诱导组和未诱导组有显著差异(P0.05)。 结论 1、本实验采用机械-混合酶消化法进行体外培养MDSCs的方法原代培养获得了高纯度、高活性的MDSCs,Desmin细胞免疫化学方法鉴定证明。 2、本实验采用NGF对MDSCs进行体外诱导分化成神经样细胞植入大鼠脊髓损伤处进行治疗,取得一定效果,这对MDSCs做为种子细胞修复脊髓损伤有重要意义。 3、在大鼠脊髓损伤过程中Notch信号通路中的Notch1mRNA及Notch1蛋白均呈现低表达,Notch信号分子低表达可促进MDSCs分化为神经样细胞。
[Abstract]:Objective to investigate the difference of expression of Notch1 gene in the repair of spinal cord injury (sci) by myogenic stem cells (Muscle derived stem cells). Methods 1. Rat MDSCs were isolated and cultured by mechanical-mixed enzyme digestion. The desmin (Desmin) cells were identified by immunocytochemistry as myogenic stem cells. NFG-induced MDSCs and the expression of induced MDSCs were detected by immunohistochemistry. NSE specific markers. 2, according to the experimental requirements, 120 rat samples were selected. Animals were randomly divided into two groups according to the random number table: group A, induced by BrdU and labeled with BrdU, group B, MDSCs group without induction and labeled with BrdU, group C, group C: control group (nong30) and group D, group D: blank control group (nong30). Group A, B, C were divided into 1 week, 4 weeks and 8 weeks, according to the time of sampling, they were divided into 1 week, 4 weeks and 8 weeks, according to the time of sampling, they were divided into two groups: 1 week, 4 weeks and 8 weeks, respectively. 3. Operative method: the model of spinal cord hemitransection was made. After spinal cord injury, microsyringes were injected into group A with BrdU labeled MDSCs medium after NGF induction. The same dose of PBS solution was injected into group B with the same dose of PBS in group C, which was not induced to differentiate but was labeled by BrdU. 4. The hind limb motor function (BBB) was measured at different time points during the survival of rats in each group. (5) Immunohistochemical staining was used to observe the survival of MDSCs in rats. The expression of key genes in the Notch pathway after myogenic stem cell implantation was detected by RT-PCR and Western. Results 1. After mechanically mixed enzyme digestion and differential adherence, high purity and high activity MDSCs were successfully cultured. The positive rate of Desmin () (was 80%. 2. During induction, the cells grew well. The morphology showed progressive changes, and no neurosphere formation was found. After 7 days, the NSE staining of neuronal cells and neurites was strongly positive in the induction group (37.29 卤1.59), but the positive cell rate in the uninduced group was very low (4.26 卤0.34), the difference between the two groups was significant (P0.01), and the NSE expression level in the MDSCs samples of the induced group was higher than that in the uninduced group (P0.01). The scores of group A (induction group) were slightly higher than group B (no induction group) but higher than group C (control group) at 1 week after injury, and the scores of group A were higher than those of group C (control group) at 1 week after injury (P0.01), and the scores of group A were higher than those of group C (control group). The scores of group A were significantly higher than that of group B, and the scores of group A were better than those of group C without intervention. The results showed that the effect of NGF induced myogenic stem cell transplantation on spinal cord injury was significantly better than that of myogenic stem cell transplantation alone. 4. Histological results: the survival of BrdU labeled MDSCs in rats was observed. Migration and differentiation were more obvious in group A than in group B. the number of cells in group A was more than that in group B. The results show that The effect of MDSCs induced by NGF on the repair of spinal cord injury in rats was better than that of MDSCs.5 RT-PCR and Western alone. The results showed that the expression of Notch1 node gene mRNA and Notch1 protein in Notch signaling pathway was low in the process of spinal cord injury in rats. There was significant difference between the induced group and the uninduced group (P0.05). Conclusion 1. The method of mechanical-mixed enzyme digestion was used to culture MDSCs in vitro. The identification of high activity MDSCS Desmin cell immunocytochemistry proved that. 2. NGF was used to treat rat spinal cord injury induced by MDSCs in vitro. This is of great significance for the repair of spinal cord injury by MDSCs as seed cells. 3. In the process of spinal cord injury in rats, the low expression of Notch1mRNA and Notch1 protein in Notch signaling pathway can promote the differentiation of MDSCs into neuron-like cells.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】

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1 邢莹;白瑞樱;鄢文海;韩雪飞;段萍;许燕;樊志刚;;人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化[J];生理学报;2007年03期

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