反义基因抑制PLOD2和Ι型胶原蛋白表达的研究

发布时间：2018-01-04 17:32

本文关键词：反义基因抑制PLOD2和Ι型胶原蛋白表达的研究　出处：《江西医学院》2005年硕士论文　论文类型：学位论文

【摘要】：目的观察不同序列的反义寡脱氧核苷酸(oligodeoxynucleotide,ODN)对小鼠成纤维细胞(NIH/3T3) PLOD2(procollage-lysine 2-oxoglutarate5-dioxygenase 2)mRNA 和Ι型胶原蛋白表达的抑制效果。方法NIH/3T3 成纤维细胞接种于6 孔板后,加入三种不同序列的反义ODN(针对翻译起始部、针对第一第二外显子连接部、针对翻译终止部)和一种公共错义链。经5%CO2 37℃培养后,收集细胞以提取总RNA 和总蛋白,然后用RT-PCR 方法检测PLOD2mRNA、Ι型胶原蛋白α1 链(COLIα1)mRNA 的表达,同时以β-actin 表达为内对照;另以Western blot 方法检测Ι型胶原蛋白的表达量。将所得结果行组间方差统计分析。结果1.对PLOD2mRNA、COLIα1mRNA 的影响:翻译起始部、第一外显子和第二外显子连接部、翻译终止部的反义ODN 作用后的PLOD2 抑制率分别是0.57±0.05、0.22±0.05、0.31±0.06。反义ODN 组对PLOD2mRNA 的表达具有显著的抑制作用(P0.05)。其中,翻译起始部的反义ODN 表现的抑制作用最强。反义及错义ODN 组COLIα1/β-actin 标准值分别是1.08±0.05、1.08±0.04、1.10±0.02、1.13±0.01,空白组为1.14±0.04,反义组与各对照组相比无明显下降,各组间无显著性差异(P㧐0.05)。 2.对Ι型胶原蛋白表达的影响:翻译起始部、第一外显子和第二外显子连接部、终止密码部的反义ODN 作用后的Ι型胶原蛋白抑制率分别是0.44±0.01、0.18±0.04、0.23±0.03。反义ODN 组能显著的抑制Ι型胶原蛋白的表达(P0.05)。其中,翻译起始部位的反义ODN 表现的抑制作用最强。结论反义ODN 对小鼠NIH/3T3 细胞PLOD2mRNA 和Ι型胶原蛋白的表达有明显的抑制作用。不同序列的反义ODN 对PLOD2mRNA 和Ι型胶原蛋白表达
[Abstract]:Objective To observe the effect of different sequence of antisense oligodeoxynucleotides (oligodeoxynucleotide, ODN) on mouse fibroblast (NIH/3T3) PLOD2 (procollage-lysine 2-oxoglutarate5-dioxygenase 2) the inhibitory effect of the expression of mRNA and type icollagen.
Methods NIH/3T3 fibroblasts were seeded in 6 well plate, adding three kinds of antisense sequence of ODN (according to the translation initiation of the first exon junction, the translation termination Department) and a common missense chain. Cultured 5%CO2 to 37 DEG C, cells were collected to extract total RNA and proteins then use the RT-PCR method to detect the PLOD2mRNA, type I collagen alpha 1 chain (COLI alpha 1) the expression of mRNA, while the beta -actin expression as an internal control; the other by Western blot methods to detect the expression of type I collagen. The results were statistical analysis of variance.
The effect of COLI 1. on PLOD2mRNA, alpha 1mRNA: translation initiation of exons and exon junction first, antisense ODN of translation termination after PLOD2 inhibition rates were significantly inhibited with the expression of 0.57 + 0.05,0.22 + 0.05,0.31 + 0.06. antisense ODN group of PLOD2mRNA (P0.05). Among them, the strongest inhibitory effect of antisense ODN expression of translation initiation. Antisense and missense ODN group COLI alpha 1/ beta -actin standard values were 1.08 + 0.05,1.08 + 0.04,1.10 + 0.02,1.13 + 0.01, 1.14 + 0.04 in blank group, antisense group and the control group had no significant decrease compared to no significant differences between the groups (P? 0.05).
Effect of expression of type I collagen 2.: translation initiation of exons and exon junction first, termination type icollagen antisense ODN password Ministry of the inhibition rate were 0.44 + 0.01,0.18 + 0.04,0.23 + 0.03. antisense ODN group can significantly inhibit the expression of type I collagen egg white (P0.05). Among them, the strongest inhibitory effect of antisense ODN expression of translation initiation site.
Conclusion the expression of antisense ODN on PLOD2mRNA and NIH/3T3 cells of mouse type I collagen significantly inhibited collagen. Different sequences of antisense ODN on the expression of PLOD2mRNA and type I

【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R575.2;R346

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