论文部分内容阅读
背景:如何抑制或减少神经干细胞凋亡的发生,促进缺氧后神经干细胞的存活,成为脑梗死神经干细胞移植治疗的研究热点。目地:观察在缺氧状态下转染人类persephin基因对神经干细胞凋亡的作用。设计:完全随机分组,对照实验。单位:中山大学附属第二医院神经内科。材料:实验于2006-07/2006-12在中山大学附属第二医院林百欣实验中心完成,重组腺病毒pAd persephin由本实验室构建保存,C17.2神经干细胞由美国哈佛大学医学院Snyder教授惠赠。胰蛋白酶、DMEM/F12购自Gibco公司;胎牛血清来自杭州四季青生物工程公司;多聚赖氨酸购自美国Sigma公司;TUNEL检测试剂盒,阳离子脂质体FuGENE购自瑞士Roche公司;S-P免疫组化试剂盒及DAB显色剂购自福建迈新生物工程公司;鼠抗人Nestin单抗,Persephin兔抗人多抗购自美国Santa Crus公司,persephin反义寡核脱氧苷酸由上海生工合成。方法:①实验干预:将携带人类Persephin基因的重组腺病毒感染C17.2神经干细胞,分为4组,A组:正常对照组,C17.2神经干细胞不作缺氧处理。B组:缺氧处理组,即置于37℃、体积分数0.95N2,体积分数0.05CO2厌氧培养箱培养。C组:缺氧+pAdpersiphin转染组,即:将携带人类Persephin基因的重组腺病毒感染C17.2神经干细胞,pAdpersiphin转染48h后的细胞置同B组条件的厌氧培养箱培养。D组:缺氧+pAdpersiphin转染组+persiphin反义寡核脱氧苷酸,即pAdpersiphin+persiphin反义寡核脱氧苷酸转染。②实验评估:Western-blotting法分析Persephin蛋白的表达;TUNEL法检测凋亡指数;流式细胞术测定细胞凋亡率的变化。主要观察指标:Persephin蛋白的表达、细胞凋亡指数和凋亡率。结果:①Persephin蛋白表达:C组细胞经Western blot检测可见一特异性蛋白条带存在,Mr约为24000,符合预期结果,说明persiphin成功表达,D组亦可见一Mr 24000的条带,但条带较单纯pAdpersiphin感染组明显为淡,说明反义persiphin反义寡核脱氧苷酸可成功抑制pAdCMVpersiphin的表达。②细胞凋亡指数:C组凋亡细胞明显减少,凋亡指数低于B、D组(P<0.01),但高于A组(P<0.01),表明细胞凋亡未完全避免。③细胞凋亡率:B、D组凋亡率明显高于A组(P<0.01),C组明显较B组、D组低(P<0.01)。结论:腺病毒介导的Persephin基因能高效表达Persephin;外源性Persephin对C17.2神经干细胞具有抗凋亡作用,能够提高C17.2神经干细胞对缺氧的耐受性。
Background: How to inhibit or reduce the occurrence of neural stem cells apoptosis and promote the survival of neural stem cells after hypoxia become the research focus of neural stem cell transplantation for cerebral infarction. Objective: To observe the effect of transfection of human persephin gene on the apoptosis of neural stem cells in hypoxia. Design: Complete randomized, controlled experiment. Unit: Second Affiliated Hospital of Sun Yat-sen Neurology. MATERIALS: The experiment was performed at Lin Baxin Experimental Center of the Second Affiliated Hospital of Sun Yat-sen University from July 2006 to December 2006. The recombinant adenovirus pAd persephin was constructed and preserved in our laboratory. C17.2 neural stem cells were kindly donated by Professor Snyder of Harvard Medical School. Trypsin, DMEM / F12 purchased from Gibco; Fetal calf serum from Hangzhou Sijiqing biological engineering company; polylysine from Sigma, USA; TUNEL detection kit, cationic liposome FuGENE purchased from Roche, Switzerland; SP Immunohistochemistry kit and DAB reagent were purchased from Fujian Maixin Bioengineering Co., Ltd. Mouse anti-human Nestin monoclonal antibody and Persephin rabbit anti-human polyclonal antibody were purchased from Santa Clara, USA. Persephin antisense oligodeoxyribonucleotide Synthesis. Methods: ①Experimental intervention: C17.2 neural stem cells were infected with recombinant adenovirus carrying human Persephin gene and divided into four groups. Group A: normal control group, C17.2 neural stem cells without hypoxia treatment. Group B: hypoxic group, which was placed in 37 ℃, the volume fraction of 0.95N2, 0.05CO2 volume fraction anaerobic incubator. Group C: hypoxia + pAdpersiphin transfection group, that is, the recombinant adenovirus carrying human Persephin gene was infected with C17.2 neural stem cells, and cells after 48h transfection with pAdpersiphin were cultured in anaerobic incubator under the condition of group B. Group D: hypoxia + pAdpersiphin transfection group + persiphin antisense oligodeoxyribonucleotide, ie, pAdpersiphin + persiphin antisense oligodeoxyribonucleotide transfection. ② Experimental evaluation: The expression of Persephin protein was analyzed by Western-blotting. The apoptosis index was detected by TUNEL method. The apoptosis rate was determined by flow cytometry. MAIN OUTCOME MEASURES: Persephin protein expression, apoptosis index and apoptosis rate. Results: ①Persephin protein expression: Western blot showed that a specific protein band was present in group C, Mr was about 24000, which was in line with the expected result, indicating that persiphin was successfully expressed, and a Mr 24000 band was also seen in group D, Compared with the simple pAdpersiphin infection group was significantly lighter, indicating antisense persiphin antisense oligonucleotide deoxyribonucleotide can successfully inhibit the expression of pAdCMVpersiphin. ② Apoptosis index: The apoptotic cells in group C were significantly decreased, the apoptotic index was lower than that in group B and D (P <0.01), but higher than that in group A (P <0.01), indicating that apoptosis was not completely avoided. (3) Apoptosis rate: The apoptotic rate in groups B and D was significantly higher than that in group A (P <0.01). The apoptosis rate in group C was significantly lower than that in group B and group D (P <0.01). Conclusion: Adenovirus-mediated Persephin gene can express Persephin efficiently. Exogenous Persephin has anti-apoptotic effect on C17.2 neural stem cells and can improve the tolerance of C17.2 neural stem cells to hypoxia.