mRNA差异显示技术在皮肤挫伤中的应用

发布时间：2018-02-15 04:01

本文关键词： 法医病理学差异显示技术锚定引物随机引物　出处：《山西医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:运用差异显示技术结合银染初步筛选大鼠皮肤挫伤后皮肤、肌肉的基因表达差异,旨在寻找一种或几种与损伤相关的敏感基因,为法医学病理学实践中推断时间提供科学、有效的参考依据。方法:实验分为正常对照组(n=6)与实验损伤组(n=6)。实验损伤组:用戊巴比妥钠腹腔注射麻醉大鼠,常规手术消毒后,用250克重力锤在150cm高度自由落下,造成右后肢股四头肌处皮肤、肌肉挫伤,于挫伤后4h将大鼠脱颈处死,于挫伤处取皮肤肌肉组织。正常对照组:将大鼠脱颈处死,取相应皮肤肌肉组织。提取对照组与损伤组的总RNA,经质量评价后,无降解的RNA进行反转录-聚合酶链式反应。以4种锚定引物和3种随机引物,共3×4=12种组合的非特异性引物进行扩增,对扩增后的产物进行1.5%的琼脂糖凝胶电泳,初步筛选差异条带。待确定正常组与损伤组有差异条带之后,将扩增产物进行8%的聚丙烯酰胺凝胶电泳,电压400V、时间3h。对聚丙烯酰胺凝胶进行固定、漂洗、染色、显色、终止、漂洗。用无菌刀片切下差异条带,进行2次扩增。经过TIANquick Maxi Purification Kit纯化之后的扩增产物与pMD18-T Vector连接并转染到JM109感受态细胞中,之后在SOC培养基、LB琼脂平板培养基(Amp+)中进行培养。挑选白色单菌落接种到含有Amp+LB液体培养基过夜培养,送菌液进行测序,测序结果提交GenBank进行对比。结果:(1)实验组与对照组皮肤肌肉总RNA的质量符合实验要求,其A260/A280值可达1.913。(2)PCR扩增产物进行1.5%的琼脂糖凝胶电泳后,实验组皮肤与肌肉与对照组比较出现差异条带。(3)PCR扩增产物进行8%的聚丙烯酰胺凝胶电泳,银染后实验组皮肤与肌肉出现差异条带,差异条带大约在150-600bp之间,总计22条差异条带。(4)2次扩增产物与pMD18-T Vector连接并转染到JM109感受态细胞中,LB琼脂平板培养基(Amp+)中进行培养。出现白色菌落与蓝色菌落。(5)菌液进行测序,测序结果提交GenBank进行对比,发现5个差异表达片段与GenBank的大鼠肌钙蛋白、核酸结合蛋白、细胞色素c氧化酶、Rattus norvegicus myxovirus (influenza virus) resistance 2、Mouse DNA sequence from clone RP23-403G13有同源性,其余的差异条带没有同源性。结论:大鼠皮肤损伤过程中有多种基因参与表达。肌钙蛋白与细胞色素c氧化酶在损伤后,它们的mRNA表达均增加,但不清楚对损伤过程是一种保护作用或是损害作用,以及与损伤时间是否存在一定数量关系还有待进一步研究。
[Abstract]:Objective: to screen the difference of gene expression in the muscle of rat skin contusion by differential display technique combined with silver staining in order to search for one or more sensitive genes related to injury. To provide a scientific and effective reference for the practice of forensic pathology. Methods: the experiment was divided into normal control group and experimental injury group. Experimental injury group: rats were anesthetized with pentobarbital sodium intraperitoneally. After routine operation, 250 g gravity hammer was used to drop freely at 150 cm height. The skin and muscle contusion of the quadriceps femoris of the right hind limb were caused, the rats were killed at 4 hours after contusion, and the skin muscle tissue was taken from the contusion. The skin muscle tissue was taken. The total RNAs of the control group and the injured group were extracted. After the quality evaluation, the non-degradable RNA was reverse transcription-polymerase chain reaction. Four kinds of anchoring primers and three kinds of random primers were used. A total of 12 combinations of nonspecific primers were amplified, the amplified products were amplified by 1.5% agarose gel electrophoresis, and the differential bands were preliminarily screened. Polyacrylamide gel electrophoresis of 8%, voltage 400V, time 3 hours. The polyacrylamide gel was fixed, rinsed, dyed, colored, stopped, rinsed. After purification by TIANquick Maxi Purification Kit, the product was ligated with pMD18-T Vector and transfected into JM109 receptive cells. After that, the culture was carried out in the SOC medium, LB Agar plate medium, and the white colony was inoculated into the liquid medium containing Amp LB for overnight culture, and the bacterial solution was sequenced. The sequencing results were submitted to GenBank for comparison. Results 1) the quality of total RNA in skin and muscle of the experimental group and the control group met the requirements of the experiment, and the A260 / A280 value of the A260 / A280 value could reach 1.913.02 / 2 PCR amplification product after 1.5% agarose gel electrophoresis. Compared with the control group, the skin and muscle of the experimental group showed different bands of polyacrylamide gel electrophoresis of 8%, and the difference bands of the skin and muscle of the experimental group were about 150-600 BP after silver staining. A total of 22 differentially amplified products were linked to pMD18-T Vector and transfected into the LB-LB Agar plate medium for culture. The white colony and blue colony were sequenced. The sequencing results were submitted to GenBank for comparison. It was found that the five differentially expressed fragments were homologous to GenBank rat troponin, nucleic acid binding protein and cytochrome c oxidase (Rattus norvegicus myxovirus influenza virus) resistance 2 mouse DNA sequence from clone RP23-403G13, but the other differentially expressed bands had no homology. Conclusion: there are many genes involved in the expression of troponin and cytochrome c oxidase in the process of skin injury in rats. The expression of mRNA is increased after injury, but it is not clear whether the expression of troponin or cytochrome c oxidase is a protective or damaging effect on the process of injury. And whether there is a certain amount of relationship with the time of injury remains to be further studied.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919

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