【作者】 王晓龙；

【导师】 华育平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2006， 博士

【摘要】 Selenoprotein W(SelW)蛋白在所有含硒蛋白中是发现得较晚的一个,虽有间接证据证明其在代谢过程中的角色与骨骼肌健康密切相关,但是尚未有支持这一论断的直接实验证据。牛、羊、猴、人类和啮齿类组织中的Selenoprotein W都对食物中硒的水平敏感,并主要积聚于骨骼肌;其结构特点与其在缺硒状态肌肉组织中无法检测到的事实都显示其和硒缺乏性骨骼肌病变之间有着密切的关系而值得研究。因此定性、定量地考察Selenoprotein W的作用,有利于分析其对于骨骼肌细胞代谢的重要意义。同时,通过检测Selenoprotein W定性、定量下调后细胞产生的变化,可以准确反应目标蛋白与骨骼肌细胞健康之间的关系,模拟白肌病的临床病理过程。 本研究中Selenoprotein W mRNA和蛋白的下调采用RNA干扰技术,首先根据目的mRNA翻译区序列合成了21bp长的,具有特殊结构要求的双链siRNA,参照文献记载,选取对小鼠骨骼肌细胞转染效率较高的阳离子脂质体Lipofactamine为转染试剂,使其与合成的siRNA形成复合体,利用胞饮和范德华力作用机理,将复合体导入小鼠骨骼肌细胞;当复合体成功进入细胞后,利用细胞所特有的机制,与细胞内特殊蛋白形成沉默复合物,发挥剪切作用,达到降解目标mRNA的目的。随后,利用蛋白印记和荧光实时定量PCR技术对细胞模型中的目标基因及其表达蛋白的动力学变化进行研究,细胞状态的变化由流式细胞术进行测量。转染效率通过转染荧光标记的与目标序列无同源性的siRNA进行报告。本研究平行地在原代细胞和传代细胞进行。 在针对C2C12细胞系中SelW基因的RNA干扰实验中,最高的干扰效率见于转染后24小时,本实验的转染效率为50%至70%。 在阳性小RNA效能比较实验中,选取生物学特性稳定的传代细胞作为实验材料,实验用细胞依转染不同的两个合成的siRNA分为组1,组2,并按转染试剂推荐的常规剂量,分别转染1ug小RNA。24小时和48小时后分别进行流式细胞术检测。 在剂量反应检测实验中,选取实验证明具有良好效果的1号siRNA进行实验,将肌细胞分为6组,分别对应6个梯度进行实验,以便依次梯度降低SelW mRNA水平,考察针对小鼠骨骼肌细胞的合适转染剂量。每组依次转染2ug,1.5ug,1.2ug,1ug,0.5ug,0.25ug siRNA,各组依次按1～6编号。24小时后分别进行检测。 在2个合成siRNA干扰效能比较实验的基础上,在实验确定了小鼠肌细胞最适的siRNA转染剂量后,进行了干扰效率检测实验,实验设立阳性组、阴性组和空白对照组3组,分别转染1号阳性siRNA、阴性siRNA(各1ug)和不转染siRNA(不转染任何外来物质仅进行培养基更换)。 上述所有实验都进行后续检测,包括:流式细胞术、定量PCR和Western blot,分别针对实验用细胞状态、目的基因和蛋白的变化。 结果:更多还原

【Abstract】 Selenoprotein W was one of Selenoproteins which was discovered lately. There are indirect evidences to verify that its role in metabolism has relationship with the healthy of skeletal muscle tissue, but direct evidence still did not seen. The Selenoprotein Ws in the tissues of bovine, ovine, monkey, human and rodents are sensitive to the level of dietetic selenium and mainly accumulate in the skeletal muscle. Its structure and undetectable characteristic in selenium deficient muscle tissue all demonstrate that there is a mysterious relationship between selenoprotein W and selenium deficient muscle diseases. To investigate the role of selenoprotein W quantitatively and qualitatively is helpful to analysis its important meaning to skeletal muscle metabolism. Meanwhile, monitoring changes of cell caused by quantitative and qualitative down-regulating of Selenoprotein W to reflect the relationship between target protein and the healthy of muscle cell, and analogy the clinical pathological course of white muscle disease.The down-regulation of Selenoprotein W mRNA by RNAi in this research, which was performed by introduced synthesized 21bp double strands siRNA into mouse skeletal muscle cell, in which a complex contains lipofactamine. Plus reagents and siRNA will be transfected into cell by pinocytosis and van der waals force;after the complex get into cells successfully, a RNA-induced silencing complex will be generated by the specific mechanism of cell to degenerate Selenoprotein W mRNA. The dynamics of mRNAs and proteins of Selenoprotein W were monitored by real time PCR and western blot;while cell status was measured by Flow cytometry. Transfection effectiveness was reported by a florescent labeled siRNA without homology to the sequence of target mRNA. This research was performed on passage and primary cell parallel.The effectiveness of RNAi in C2C12 cell on selenoprotein W gene was assayed. The highest effectiveness of interfering can be seen 24 hours later after transfection. The transfection effectiveness in this research were from 50% to 70%.The 2 positive siRNAs were compared in passage cell under the frame of the effectiveness to cause cell changing. Cells were separated into 2 groups to transfect 2 different positive siRNAs separately as lug according to the instruction of lipofectamine reagent. The cells were assayed by flow cytometery at 24 and 48 hours after transfection.In dose reaction assay the No. 1 siRNA with superior effectiveness was selected for the experiments and cells were divided into 6 groups to correspondence with 6 dose gradients of the same siRNA so as to down-regulate the level of SelW mRNA step by step. Every groups were transfected with siRNA in the sequence of 2ug, 1.5ug, 1.2ug, lug, 0.5ug, 0.25ug, andwere marked as 1 ~6 . Assay was done 24 hours after transfection.On the basis of the comparing of 2 siRNAs and dose reaction assay, the assay of effectiveness of RNA interfering was performed, in which positive, negative and blank control groups were set to be transfected with the No. 1 positive siRNA, negative siRNA and nothing (just changing the medium).All the cells used in prior experiments were assayed by flow cytometery, real-time PCR and Western blot to report cell status, the changes of target genes and proteins.Results:1. Either of the 2 synthesized siRNAs can trigger RNA interfering;but there was difference of effectiveness between the 2 siRNAs according to the results of flow cytometery;the No. 1 siRNA (31.7%) is better than No.2 (14.5%), so the No. 1 siRNA was selected for later experiments. The most suitable time for assay is 24 hours later after transfection.2.In dose reaction assay in passage cell, except from 3 groups with excessive dose of siRNA transfection with a 50~70% cell layer detached and fail to be assayed, the results in other groups were: apoptosis rate 32.5%, 12.4%, 6.58%;the amounts of SelW mRNA 0.25, 0.39, and 0.524 by standard curve method of Real time PCR;In protein assay, set the value of group 6 as standard, the amounts of proteins were 17.26, 31.08 and 50. All evidences show that lug of siRNA is most suitable dose, which not only can trigger highest effectiveness of interfering, but also sufficient to sustain a balance between cell healthy and interfering.3. In RNAi effectiveness test experiment in passage cell, the result of Flow cytometry showed that positive group can cause a highest apoptosis rate of 37.5%. There were significant difference while comparing the positive group with negative (0.93%)and blank controls(0.7%): P=3.08xl0"9 < 0.05, P=2.95*10"9 < 0.05. Real time PCR result indicated that the mRNA of SelW in positive group(0.275) dropped approximately 72.4 % compare to blank group(0.996), and negative control group(0.9867) dropped 0.93%. In the assay for the changing of proteins, taking blank control as a standard (100), the amounts of proteins in positive, negative group were: 25.07, 98.75;to compare with blank control, the amounts of proteins in positive and negative group dropped 74.93% and 1.25%.One should be noted was that the reactions in primary cell in dose reaction assay, RNAi effectiveness assay were consistent with that in passage cell. But the changing caused by RNA interfering in primary cell was weakness than that in passage cell. In dose reaction assay, overdose siRNA caused cell death;When lug, 0.5ug, 0.25ug were transfected, apoptosis rate were 18.3%, 8.1%, 4.3%;the amount of mRNAs were 0.22, 0.25, 0.32;amounts of protein were 39.5,46.2,and 50. In RNA interfering effectiveness assay, results in positive, negative and blank control group: anontosis rate were 20.0%, 1.03%, 0.4%;the amounts of target gene were 0.534, 0.663, 0.67;the amounts of proteins were 41.2, 48.8, 50.Tests illustrated: 1 .In the course of skeletal muscle metabolism, the depletion of SelW induce to apoptosis, and one function of SelW protein is apoptic inhibitor, which illustrated from degeneration to necrosis were not the only pathological mechanism of selenium deficient muscle disease.2.There is a threshold in RNA interfering, i.e. Better result needs enough dose of siRNA. This is meaningful to anti-virus and gene therapy introduced by RNAi. 3. The effectiveness of RNAi is not influenced by the abundance of target gene. Though SelW is a low abundance gene in muscle cell, experiment demonstrated that a better result of RNAi could be triggered on it. This supported the theory that the resistances to RNAi of different genes are heredity. 4. RNAi in primary cell is more conservative. All results come from every experiments in primary cell showed the effectiveness in primary cell was lower than that in passage cell, and the reason needs further study.更多还原