【作者】 杨晓苏；

【导师】 杨期东；

【作者基本信息】 中南大学 ， 神经病学， 2007， 博士

【摘要】 脊髓性肌萎缩症是一种常染色体隐性遗传性神经变性疾病，其特征是脊髓前角的α运动神经元变性，临床表现为肢体近端肌无力和肌萎。SMN基因是SMA的致病基因，该基因有两个结构基本相同的拷贝，分别称SMN1和SMN2，SMN1编码全长SMN(fl-SMN)蛋白，SMN2编码△7-SMN蛋白。研究表明，SMN1基因的功能缺陷是大多数SMA患者的发病原因，且SMA临床表型的严重程度与全长SMN蛋白的含量密切相关。我们设想，fl-SMN蛋白与脊髓前角的α运动神经元的凋亡调控相关，fl-SMN蛋白含量下降可能诱导α运动神经元凋亡，进而出现细胞的变性。本研究拟利用RNAi技术抑制非SMA患者的骨髓问质干细胞SMN1基因的表达，将其诱导分化为神经元样细胞建立SMA细胞模型，并通过一系列分子生物学方法观测其凋亡情况，以探讨SMN蛋白对神经元的保护作用及其机制。第一章RNA干扰对人骨髓间充质干细胞SMN1，基因表达抑制的实验研究目的：构建能在哺乳动物细胞中表达SMN1 shRNA的质粒，并初步探讨其对人骨髓间充质干细胞SMN1 mRNA及蛋白的抑制作用。方法：取非SMA病人(且无血液疾病及骨肿瘤)骨髓，分离、纯化得到骨髓间充质干细胞(MSCs)。根据Genbank中SMNl eDNA序列设计5条SMN1靶向的shRNA并克隆得到相应的DNA序列；将此DNA序列克隆至pRNAT-U6.1／Neo载体中U6启动子的下游，形成能在细胞内表达SMN1特异性的短发夹状RNA的重组质粒pshRNA-SMN1；在脂质体介导下将重组质粒pshRNA-SMN1转染人骨髓间充质干细胞，通过RT-PCR、Western-Blot、流式细胞分析技术检测转染后MSCs的fl-SMNmRNA及SMN全长蛋白的表达。结果：琼脂糖凝胶电泳及测序证实了重组质粒pshRNA-SMN1构建成功，转染后不同程度地抑制人骨髓间充质干细胞的fl-SMNmRNA及其蛋白的表达，其中以pshRNA-SMN1-1组和pshRNA-SMN1-4组最明显，mRNA水平上干扰效率分别为64.87％、61.45％，蛋白水平上干扰效率分别为60.25％和54.05％，流式细胞分析显示干扰效率分别为96.00％、94.29％，三项与对照组相比，差异均有统计学意义(P＜0.05)。结论：构建的pshRNA-SMN1-1和pshRNA-SMN1-4重组质粒能有效地抑制人骨髓间充质干细胞f1-SMN mRNA及其蛋白的表达。第二章SMA细胞模型的建立目的：应用RNAi沉默SMN1基因的MSCs建立SMA细胞模型。方法：根据实验一的RT-PCR、Western-Blot分析结果，选择能在最大程度上抑制SMN1基因表达的重组质粒pshRNA-SMN1-1，将此重组质粒转染人骨髓问充质干细胞，经G418筛选得到能稳定表达目的shRNA的单克隆细胞系后并诱导其分化为神经元样细胞。后者通过细胞形态学观察及免疫细胞化学染色分析神经元样细胞NSE、NF蛋白表达进行鉴定。再次用RT-PCR，Western-Blot方法检测神经元样细胞SMN mRNA及其蛋白的表达，并与诱导前进行比较。结果：诱导后的细胞呈典型的神经元样细胞形态，免疫细胞化学染色显示细胞膜NSE、NF蛋白表达阳性；其fl-SMNmRNA、△7-SMNmRNA及fl-SMN蛋白表达均较诱导前增加(P＜0.05)，但PshRNA-SMN-1组的fl-SMN mRNA及蛋白表达仍明显低于对照组，差异有统计学意义(P＜0.05)；而诱导后△7-SMN mRNA的表达在各组间差异无统计学意义(P＞0.05)。结论：SMNI mRNA及其蛋白被抑制的MSCs诱导分化为神经元样细胞后可以作为SMA的细胞模型。第三章SMA模型细胞自发性凋亡与SMN蛋白的保护作用及其机制的研究目的：初步研究SMA模型细胞的自发性凋亡和丙戊酸对其凋亡的影响及其机制，以探讨SMN蛋白对神经元的保护作用及其机制。方法：实验以SMA模型细胞、正常神经元样细胞为研究对象，以1μmol／ml丙戊酸干预，共分为无干预正常神经元样细胞组、无干预SMA模型细胞组、丙戊酸干预正常神经元样细胞组、丙戊酸干预SMA模型细胞组等4个实验组，①绘制细胞生长曲线，观察各组细胞增殖趋势及速度；②采用噻唑兰比色实验(MTT)检测各实验组细胞的细胞活力变化；③Tunel荧光染色比较各实验组凋亡细胞比率；④流式细胞分析Annexin V法检测各实验组细胞凋亡及坏死情况；⑤RT-PCR检测各实验组细胞Caspase-3 mRNA的表达；⑥以不同剂量的丙戊酸分别对SMA模型细胞进行干预，RT-PCR检测各实验组细胞fl-SMNmRNA及Caspase-3 mRNA的表达；⑦Western-Blot检测各实验组细胞fl-SMN蛋白及Caspase-3蛋白的表达；⑧共聚焦显微镜共定位fl-SMN蛋白和Caspase-3蛋白。结果：①细胞生长曲线结果显示，各组细胞数均出现2次负增殖趋势：在第2次细胞负增殖期内(在诱导结束后第4、5、6天时)，不同时间点的细胞数目有统计学差异(时间主效应：P＜0.05)；且SMA模型细胞负增殖较正常神经元样细胞明显，两者之间差异有统计学意义(RNA干扰主效应：P＜0.05)；而丙戊酸干预后负增殖减小，与无干预组相比差异有统计学意义(丙戊酸主效应：P＜0.05)；②MTT结果显示，各组MTT曲线均出现2次细胞活力下降趋势；在第二次细胞活力下降期，各组细胞在诱导结束后第3、4、5、6、7天时的MTT值比较差异均有统计学差异(时间主效应：P＜0.05)，且SMA模型细胞的活力明显低于正常神经元样细胞(RNA干扰主效应：P＜0.05)，但丙戊酸干预与否差异无统计学意义(丙戊酸主效应：P＞0.05)；③Tunel荧光染色显示，SMA模型细胞组的凋亡细胞率为(40.824±3.66)％，明显高于正常神经元样细胞(22.98±2.04)(P＜0.05)：在丙戊酸干预后细胞凋亡率降低(31.69±2.53)％，而正常对照组却有增加(26.62±2.62)％：各实验组之间比较差异均有统计学意义(P＜0.05)；④流式细胞分析Annexin V结果亦显示，SMA模型细胞组的细胞凋亡率(41.39％)较正常神经元样细胞组(32.93％)高，丙戊酸干预后细胞凋亡率减少(36.88％)，但正常对照组有增加(34.67％)：⑤RT-PCR凝胶电泳半定量结果表明，SMA模型组细胞Caspase-3 mRNA的表达量(0.85583±0.05241)明显高于正常对照组(0.14975±0.03093)(P＜0.05)，丙戊酸干预后表达量降低(0.61311±0.05093)(P＜0.05)，但正常神经元样细胞的Caspase-3 mRNA表达量升高(0.21796±0.03354)(P＜0.05)；⑥随着VPA的干预浓度增加，SMA模型细胞的fl-SMN mRNA及蛋白的表达增加，两者呈S形量一效相关(R=0.9424，P=2.047e-31；R-0.9424，P=5.682e-30)：而Caspase-3mRNA及蛋白的表达在VPA的干预浓度由1μmol／ml加至5μmol／ml时，是逐渐降低，两者呈S形量-效相关(R=0.959，P=4.510e-21；R=0.942，P=8.651e-19)，继续加大VPA的干预浓度，Caspase-3 mRNA的表达又逐渐回升，曲线成钩状；⑦共聚焦显微镜结果显示，fl-SMN蛋白和Caspase-3蛋白并无直接的相互作用。结论：①SMA细胞模型易发生凋亡，其可能与细胞内fl-SMN蛋白表达减少和Caspase-3蛋白表达增多有关。②一定剂量范围内的丙戊酸增加fl-SMN蛋白和减少Caspase-3蛋白表达后能减少SMA细胞模型的凋亡，提示SMN蛋白有神经元保护作用，但其与Caspase-3蛋白者间可能不是直接作用的结果。更多还原

【Abstract】 Spinal muscular atrophy （SMA） is an autosomal recessive disease which affects motor neurons of spinal cord, and SMN gene is the virulence gene of SMA. SMN gene has two copies called SMN1 andSMN2, of which products are fl-SMN protein and△7-SMN protein. The dysfunction of SMN1 gene is the cause of SMA, and the quantity of fl-SMN protein determinate its clinical phenotype. We assumed that fl-SMN protein is related with the apoptosis process of motor neurons, that is to say, the reduction of fl-SMN protein may induce the apoptosis of motor neurons.In this research, the expression of SMN1 gene was inhibited in MSCs by RNAi, and then MSCs were differentiated to neuron like cells which were called SMA cell model. To explore the effect of fl-SMN protein toαmotor neuron, the apoptosis of SMA cell model were investigated by a series of molecular biological methods.Chapter 1 The Effect Of shRNA to the Expression of SMN1 Gene in Human Mesenchymal Stem Cellsobjective: To construct the plasmid containing short hairpin RNA （shRNA） of SMN1 expression vector and to investigate the effect of shRNA to the expression of SMN1 gene in human mesenchymal stem cells （hMSCs）.Methods: The hMSCs come from the patients without SMA. Short chain oligonucleotide was designed according to the SMN1 mRNA sequence provided by Genebank, and then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pRNAT-U6.1/Neo vector. The recombinant SMN1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed SMN1 expression vector was transfected into MSCs by Lipofectomine TM 2000, and its effect on SMN1 expression was observed by RT-PCR, Western-Blot, and flow cytometry.Results: Successful construction was identified by enzyme cutting and the constructed plasmid was called pshRNA-SMN1. Expression of SMN1 mRNA and protein can be effectively inhibited by pshRNA-SMN1-1 and pshRNA-SMN1-4, of which the ratio of interference was 64.87%,61.45% on the level of mRNA, 60.25%,54.05% on the level of protein and 96.00%,94.29% indicated by flow cytometry analysis. Compared with control groups, the differences showed the statistical significance （P＜0.05）.Conclusion: The vector constructed by SMN1 shRNA can block the expression of SMN1 mRNA and their protein in mesenchymal stem cells. Chapter 2The Establishment of SMA Experiment Cell Model by RNAiObjective: To establish SMA experiment cell model by blocking the expression of SMN1 gene with shRNA.Methods: The pshRNA-SMN-1 vector which could inhibit the expression of SMN1 furtherest was choosed according to the experiment, and it was transfected into mesenchymal stem cells by Lipofectomine TM 2000. The clones of transfected cells were selected in G418 medium, and they were differentiated to neuron like cells. Immunocytochemical analysis was used to check the expression of NSE, NF on the neuron like cells, and the expression of fl-SMN gene and protein were observed by RT-PCR and Western-Blot analysis.Results: After differentiation, NSE, NF protein was detected on neuron like cells. After induction, the expression of fl-SMN,△7-SMN mRNA and protein of SMA model group was upregulated （P＜0.05）, but the expression in PshRNA-SMN-1 group is less than that in control group （P＜0.05）, and the differences have the statistical significations. The expression of△7-SMN mRNA between the groups have no statistical difference （P＞0.05）.Conclusion: The neuron like cells, which recombinant SMN1 shRNA expression vector was transfected into, can be regarded as SMA experiment cell model. Chapter 3The Spontaneous Apoptosis in SMA Experiment Model Cell and the Effect of SMN ProteinObjective: To investigate the spontaneous apoptosis in SMA experiment model cell and the effect of SMN protein.Methods: By using SMA experiment model cell and normal neuron like cell as research target, and cells were divided into SMA model group, normal control group and VPA intervention group.①Cell proliferation was observed by drawing cellular growth curve;②The activity of cells was detected using the method of MTT spectrography;③The ratio of apoptosis cells was investigated by Tunel fluorescent staining;④Using PE-Annexin V and 7-AAD double staining, apoptosis or necrosis was detected by flow cytometry;⑤The expression of Caspase-3 mRNA was detected by RT-PCR;⑥After intervention by different dose of VPA, the expression of Caspase-3 mRNA and fl-SMN mRNA was detected by RT-PCR;⑦The expression of Caspase-3 protein and fl-SMN protein was detected by Western-Blot.⑧The position of fl-SMN protein and Caspsase-3 protein was detected by confocal microscopy.Results:①Cellular growth curve: Two negative proliferations were showed in all groups. During the phase of the second negative proliferations (at the 4^th, 5^th and 6^th day), the difference of cells numbers was showed between different time point （principle effect of time: P＜0.05）; the negative proliferation of cells in SMA model cell group was more obvious than normal NLC group （principle effect of RNAi: P＜0.05）, and the negative proliferation of cells was decreased after the intervention by VPA （principle effect of VPA: P＜0.05）.②MTT spectrography: Two downtrends of cellular activity was observed in all groups, and the difference of their MTT value had statistic significance （P＜0.05） at the 3^rd, 4^th, 5^th, 6^th and 7^th day in the second downtrend. In addition, the cellular activity of SMA model cell group was lower than normal NLC （P＜0.05）, but no difference after the intervention by VPA （P＞0.05）.③Tunel fluorescent staining: The ratio of apoptosis cells was （40.82±3.66）% in SMA experiment model groups, （31.69±2.53）% after VPA intervention, （22.98±2.04）% in normal control groups, （26.62±2.62）% after VPA intervention, and the difference in all groups had statistic significance （P<0.05）.④Flow cytometry: The ratio of apoptosis was 41.39% in SMA experiment model group, 36.88% after VPA intervention group, 32.93% in normal control group, 34.67% after VPA intervention group and the difference in all groups had statistic significance （P＜0.05）.⑤RT-PCR: The expression of Caspase-3 mRNA in SMA model cell group （0.85583±0.05241） was more than normal control group （0.14975±0.03093） （P＜0.05）. After intervention by VPA, the expression of Caspase-3 mRNA in SMA model cell group tend to downregulate （0.61311±0.05093） （P＜0.05）, but the expression in normal NLC group tend to upregulalte （0.21796±0.03354） （P＜0.05）.⑥With the addition of VPA concentration, the expression of fl-SMN mRNA and protein in SMA model cell group tend to upregulate, and quantity-effect correlation was elucidated （R=0.9424, P=2.047e-31; R=0.9424, P=5.682e-30）. The expression of Caspase-3 mRNA and protein tend to downregulate with the concentration of VPA from 1μmol/ml to 5μmol/ml, and quantity-effect correlation was elucidated （R= 0.959, P= 4.510e-21; R=0.942, P=8.651e-19）. When the concentration of VPA exceed 5μmol/ml, the expression of Caspase-3 mRNA came back to unregulated, and hook-like curve was elucidated.⑦Confocal microscopy: The direct interaction between fl-SMN protein and Caspase-3 protein was not identified.Conclusion:①The process of apoptosis of SMA experiment model cell is earlier than normal neuron like cell, which may relate with the reduction of fl-SMN protein and the increase of Caspase-3 protein.②VPA can decrease the apoptosis of SMA experiment model cell at the certain dose range by increaseing the expression of fl-SMN protein an reducing the expression of Caspase-3 protein, which suggested SMN protein have the effect of neuron protection, but it is not the result of the direct interaction between fl-SMN protein and Caspase-3 protein.更多还原