【作者】 王正锋；

【导师】 雷霆；

【作者基本信息】 华中科技大学 ， 神经外科， 2010， 博士

【摘要】 第一部分SUMO2／3基因的特异性MicroRNA干扰表达载体的构建目的：构建SUMO2/3 (small uniquitin-like modifier 2/3)基因的MicroRNA特异性干扰质粒,为探讨抑制SUMO2／3基因在细胞中表达对B35细胞的影响的研究奠定基础。方法：根据GeneBank数据库提供的SUMO2和SUMO3的DNA序列,首先克隆出SUMO2和SUMO3的功能性基因片段,并将基因片段连接到pcDNA3.1载体；然后分别设计对SUMO2和SUMO3基因特异的miRNA片段,分别命名为miR2和miR3,同时设计一条非特异性序列作为阴性对照,命名为negative-miR,并将这些片段克隆到pcDNA6.2-GW/EmGFP-miR载体上。通过转化感受态大肠杆菌,挑选阳性克隆,提取重组质粒,并对抽提的质粒进行限制性酶切反应,DNA测序鉴定。然后将pcDNA6.2-GW/EmGFP-miR2和pcDNA6.2-GW/EmGFP-miR3通过酶切和连接反应将miR2和miR3同时连接到同一pcDNA6.2-GW/EmGFP-miR载体上,称之为pcDNA6.2-GW/EmGFP-miR2/3,同时抑制SUMO2和SUMO3的表达。分别将相应的功能基因片段和其对应的抑制性质粒共转染到HT22细胞中,并用50μM H2O2作用转染的细胞10 min,激活SUMO化通路的表达,用Western-Blot在蛋白水平检测SUMO2和SUMO3化蛋白的表达。结果：重组质粒成功转化感受态大肠杆菌,经酶切和电泳证明序列成功插入到pcDNA6.2-GW/EmGFP-miR载体预计位点,经DNA测序进一步证明序列完全正确。Western-Blot结果证明pcDNA6.2-GW/EmGFP-miR2和pcDNA6.2-GW/EmGFP-miR3分别成功沉默SUMO2和SUMO3基因的表达,并且pcDNA6.2-GW/EmGFP-miR2/3能够同时沉默SUMO2／3基因的表达。结论：成功构建针对SUMO2／3基因特异性的miRNA真核表达载体,转染细胞后能够明显抑制SUMO2／3基因的表达,为进一步研究其基因功能奠定了基础。第二部分SUMO2／3基因沉默对B35细胞生长的影响和OGD作用后细胞的SUMO化蛋白表达的变化目的：构建稳定转染pcDNA6.2-GW/EmGFP-miR2/3及pcDNA6.2-GW/EmGFP-negative-miR质粒的B35细胞系,观察SUMO2／3基因沉默对细胞生长的影响,并用OGD体外缺血模型对细胞进行处理,然后根据不同的再灌注时间观察SUMO家族蛋白表达的变化。方法：用携带CMV的pcDNA6.2-GW/EmGFP-miR2/3质粒和对照质粒pcDNA6.2-GW/EmGFP-negative-miR转染B35细胞系,用blasticidin筛选出稳定株,用Western-Blot检测SUMO2／3基因表达的改变,然后用CellTiter-Blue reagent试剂盒检测细胞的生长速度。将稳定转染的细胞用体外缺血模型OGD干预后,用Western-Blot检测SUMO家族蛋白表达的变化。结果：稳定转染pcDNA6.2-GW/EmGFP-miR2/3质粒的B35细胞在受到应激刺激后,SUMO2／3蛋白表达和对照组相比明显降低；并且其细胞生长速度和对照组相比3天后降低为对照组的64%。细胞经过OGD作用后,对照组SUMO1蛋白的表达在0min时降低,然后升高,30min时到达高峰,然后有所降低,但趋势并不是很明显；SUMO2／3沉默组和对照组相比没有明显差别。对照组和正常细胞组SUMO2／3化蛋白表达变化为：在0 min时先降低,然后逐渐升高,30 min达到高峰,然后逐渐降低,至3h后基本恢复正常。和对照组及正常细胞组相比,SUMO2／3沉默组的每个时间段的SUMO2／3化蛋白表达均明显降低,各时间段表达变化不明显。结论：SUMO2／3基因沉默后可以明显降低细胞的生长速度；稳定转染对照载体的细胞和稳定转染pcDNA6.2-GW/EmGFP-miR2/3质粒载体的细胞经OGD作用后SUMO1蛋白表达没有明显区别；SUMO2／3在稳定转染pcDNA6.2-GW/EmGFP-miR2／3质粒的B35细胞中表达相对正常细胞组及对照组明显降低,而对照组和正常细胞组相比没有明显区别。第三部分SUMO基因的表达在脊髓缺血性损伤中的改变及机制研究目的：建立小鼠脊髓缺血的简单易重复的动物模型,并检测SUMO蛋白在其中表达的变化。方法：将成年雌性C57B1／6J小鼠用异氟咪麻醉后,取右侧卧位,保持肛温在37.0±0.5℃,于胸8肋间切开1 cm的切口,将一个小动脉瘤夹放置在胸主动脉的胸8部位,根据夹闭时间的不同,将小鼠分为对照组、8 min、10 min和12 min夹闭组,用激光多普勒探头检测夹闭后小鼠腰段脊髓表面血流的变化；同时对各组小鼠在缺血后1h、1d、3d、5d、7d进行BBB评分检查和Rotarod检测；在手术7天后将腰段脊髓取出包埋固定后行HE染色,对脊髓前脚的存活神经元进行计数；同时将另外一批10 min夹闭组和对照组的腰段脊髓组织,用Western-Blot检测脊髓损伤后SUMO蛋白表达的变化。为了研究本模型对小鼠长期存活的影响,我们使另一10 min夹闭组和对照组存活至术后28天,并对其病理生理功能进行检测。结果：在8 min缺血组,BBB评分在1h后下降到15分,24h基本恢复正常,而10 min和12 min组,BBB评分在7天时与对照组和8 min组相比均明显降低,差距具有显著的统计学意义；Rotarod检测结果和BBB评分相似,在7天时,8min组和对照组相比没有明显区别,而10 min或12 min组与对照组或8 min组相比差别有显著统计学意义(p<0.01,10 min or 12 min vs.0 min or 8 min)；胸段脊髓夹闭后可以引起腰段脊髓表面血流降低到夹闭前的10%；神经功能障碍和神经元细胞的死亡和缺血时间密切相关。长时程10min夹闭检测组的小鼠有80%存活到28天,BBB评分和Rotarod检测和对照组相比差异显著的统计学意义(p<0.01)。结论：成功建立了小鼠的脊髓缺血模型,脊髓缺血后SUMO2／3蛋白表达和对照组相比明显升高,这和脑缺血后SUMO化蛋白表达变化相似。更多还原

【Abstract】 PartⅠConstruction of the SUMO2/3 Gene specific MicroRNA expression vectorObjectives To Construct the SUMO2/3(small uniquitin-like modifier 2/3) gene specific MicroRNA expression vector, and prepare well for investigating the effect of silencing the SUMO2/3 gene in the B35 cell line.Methods Genomic sequences of SUMO2 and SUMO3 gene were retrieved from Genbank. Firstly the functional sequence of SUMO2 and SUMO3 gene were cloned and then they were inserted to pcDNA3.1 vector. Secondly miRNA specific silencing the SUMO2 and SUMO3 gene were designed which were called miR2 and miR3, at the same time we dedigned a negative control called negative-miR. All these sequences were inserted to pcDNA6.2-GW/EmGFP-miR. Recombinant vectors were then transformed to E-coli, The positive colony were selected and recombinant plasmids were extracted. After restricted enzyme digestion and sequencing, the plasmids were identified correctly. Then miR2 and miR3 were colonied into the same pcDNA6.2-GW/EmGFP-miR vector called pcDNA6.2-GW/EmGFP-miR2/3.The pcDNA6.2-GW/EmGFP-miR2/3 and counterpart pcDNA3.1-SUMO2 or SUMO3 vector were co-transfected into HT22 cells. After co-transfection, the cells were treated with 50μM H2O2, for 10 min. The cells were extracted with lysis buffer and runned Western-Blot to test expression of the SUMO2and SUMO3 proteins Results Recombinant plasmids were successfully transformated to E-coli cells, using the restricted enzyme and electrophoresis identified the right position of the sequence, the DNA sequencing was futher to prove the right position. pcDNA6.2-GW/EmGFP-miR2,pcDNA6.2-GW/EmGFP-miR3 and pcDNA6.2-GW/EmGFP-miR2/3 can successfully down-regulate the proteins respectively which were verified by Western-blot.Conclusions The correctly plasmids were constructed, after transfected into HT22 cells, it can obviously silence the SUMO2/3 gene expression. These plasmids were obstained for the future study.Part II The effection on B35 cell growth after SUMO2/3 gene silencing and the SUMO family protein changes after OGD treatmentObjectives To establish the stably transfected B35 cell line using pcDNA6.2-GW/EmGFP-miR2/3 plasmid and investigation the effection of the SUMO2/3 on B35 cell growth. And the stable B35 cell was treated using OGD which is common used for the ischemia model in vitro, and then we observed the changes of SUMO protein family using Western-blot.Methods The pcDNA6.2-GW/EmGFP-miR2/3 plasmid which has CMV promotor and the control plasmid were used to transfect into B35 cell line, then the stale cell line was selected by blasticidin, the changes of SUMO2/3 protein family was tested by Westen-blot and the cell growth was evaluated using CellTiter-Blue reagent; B35 cell was treated using OGD and then the SUMO protein family were investigated by Western-blot.Results SUMO2/3 protein decreased significantly in B35 cell experssed pcDNA6.2-GW/EmGFP-miR2/3 plasmid comparing to miR-Neg B35 cell. After silencing of the SUMO2/3 expression, B35 growth and proliferation decreased to 64% comparing to miR-Neg B35 cell at 3 days. SUMO1 protein didn’t change after OGD treatment, while the SUMO2/3 in miR-Neg B35 cell decreased at Omin, then increased gradually and reached highest-level at 30 min, after this it decreased little by little and became normal at 3 h. In B35 cell stably expressed the pcDNA6.2-GW/EmGFP-miR2/3 plasmid, SUMO2/3 protein decreased obviously comparing control cell with all the time points because of the silencing of the SUMO2/3 gene.Conclusions Silencing of the SUMO2/3 using miR significantly repressed B35 cell growth and differentiation. SUMO2/3 protein expression in B35 cell stably expressed pcDNA6.2-GW/EmGFP-miR2/3 plasmid was drastically reduced after OGD treatment at all time points comparing to the normal B35 cell and miR-Neg B35 cell.Part III The alteration of SUMO gene expression in spinal cord ischemia injury and investigtion of its mechnismObjectives To development of mice spinal cord ischemia animal model and investigation the SUMO2/3 changes in this model.Methods Male C57B1/6J mice were anesthetized with isoflurane and endotracheally intubated. The middle segment of the thoracic aorta was clamped for 0,8,10 or 12 min via left lateral thoracotomy. Rectal temperature was maintained at 37.0±0.5℃. A laser Doppler probe was used to measure lumbar spinal cord blood flow during thoracic aorta cross-clamping. Open field locomotor function (BBB score)and rotarod performance were evaluated at 1 h and 1,3,5, and 7 days post-injury. Surviving neurons in the lumbar ventral horn were counted at 7 days post-injury using HE staining.Results In the 8 min ischemia group, the BBB score declined to 15 at 1 hour post-injury and then recovered to almost normal by 24 hours. In the groups of 10 or 12 min of ischemia, mice had a more severe decline in BBB scores and a slower recovery, there was significantly differences comparing to 8 min or control group. Similar to the BBB score, Rotarod performance were significantly different in 10 min and 12 min group from control and 8 min group(p<0.01,10 min or 12 min vs.0 min or 8 min). Cross-clamping the middle segment of the thoracic aorta resulted in approximately 90% blood flow reduction in the lumbar spinal cord. Neurological deficit and neuronal cell death were associated with ischemia duration. Another set of mice were subjected to 10 min aortic clamping or sham surgery,4 of 5 mice (80%) in the injured group survived 28 days and had significantly different BBB score and Rotarod performance comparing to control group(p<0.01).Conclusions Cross-clamping of the aorta via left thoracotomy is a simple and reliable method to induce spinal cord ischemia in mice allowing definition of long-term outcome. The SUMO2/3 was significantly increased comparing to the control group, this was similar to the cerebral ischemia.更多还原