【作者】 孙俊红；

【导师】 王英元；

【作者基本信息】 山西医科大学 ， 法医学， 2009， 博士

【摘要】 目的运用差异显示技术结合硝酸银染色筛选大鼠肌肉挫伤后与正常肌肉表达差异的基因,并通过Real-time PCR方法研究差异基因与损伤时间的关系,旨在寻找一种或几种与损伤相关的敏感基因,为法医学损伤时间推断提供科学、有效的指标。方法1. 12只健康成年SD大鼠随机分为实验组和正常对照组。实验组大鼠建立肌肉挫伤模型,损伤后4小时处死,提取100mg损伤处肌肉组织。对照组大鼠在规定时间内处死,提取相同部位100mg肌肉组织。两组肌肉组织经SV Total RNA Isolation system提取总RNA,并经Agilent 2100 Bioanalyzer进行质量控制检测, SIN值大于8.0的样本经反转录后用于下游实验。2.采用4种锚定引物和3种随机引物共12种组合对实验组和对照组样本进行PCR扩增,扩增产物经聚丙烯酰胺凝胶电泳分离后硝酸银染色进行差异表达分析。采用煮沸法回收差异条带,并进行二次扩增,扩增产物纯化后与载体连接、克隆,进行序列分析,将测序所得差异序列与GenBank进行同源性比较。3. 4只健康成年SD大鼠随机分为正常对照组和损伤组,提取各组样本的总RNA。在差异序列两端设计特异性引物,采用SYBR?Green I染料法进行Real-time PCR扩增,通过分析扩增曲线及溶解曲线,鉴定差异表达基因的真伪。4. 12只健康成年SD大鼠随机分为正常对照组、损伤后6小时组和损伤后12小时组。采用Real-time PCR检测正常肌肉组织、损伤后6小时肌肉组织和损伤后12小时肌肉组织中β-Actin等九种常用内参基因的表达水平,通过Normfinder、geNorm和BestKeeper三种软件分析九种常用内参基因在肌肉组织损伤后表达的稳定性,寻找合适的内参基因。5. 54只健康成年SD大鼠随机分为正常对照组和损伤组,建立不同损伤时间大鼠肌肉挫伤模型,提取总RNA、反转录后采用差异基因的上下游特异性引物进行Real-time PCR扩增,分析差异基因与损伤时间的关系。结果1.采用Progema公司的SV Total RNA Isolation System提取的总RNA具有较高的RIN值,且没有DNA及蛋白质的污染。各样本RIN值在8.3～8.8之间,均大于8.0。每个样本都可见清晰的18s及28s峰,在18s峰之前基线平坦,没有降解的RNA片段出现。28s峰之后,基线也比较平直,说明没有基因组DNA及蛋白质的污染,可以保证后续验证实验的准确性。2.经过对21条感兴趣的差异条带进行回收及再扩增,共有12条差异条带得到成功回收。DNA重组、转化及蓝白斑筛选重组成功的载体,经序列分析有5条差异条带片段较长,其他条带片段较短,多数为引物二聚体序列。3.经过与GenBank进行同源性比较及Real-time PRC鉴定,5条差异性片段均从总RNA中得到了有效扩增,且溶解曲线均为单一峰型,说明均为阳性差异片段。其中3条为已知基因,与sTnI、Mx2及Cox6c匹配度较高,另外两条可能为新基因,有待于进一步研究。4.通过Real-time PCR的方法测定损伤后不同时间点肌肉组织中常用内参基因的CT值,经geNorm、Normfinder及BestKeeper软件分析,得出RPL13和RPL32在肌肉组织损伤后表达相对稳定,可以作为内参基因对目的基因进行均一化处理。而常用的β-Actin及GAPDH在不同损伤时间肌肉组织中表达不稳定。5、经Real-time PCR分析,sTnI mRNA在损伤后表达逐渐降低,在0.5 h、1 h、6 h sTnI mRNA表达量分别为正常组的78.17%、41.58%、32.13%,且差异具有统计学意义(P<0.05),6 h~36 h sTnI mRNA表达量与对照组相比无统计学差异(P>0.05)。Mx2 mRNA在损伤后0.5小时表达骤然升高达到正常对照组的84.3倍,随着损伤时间的延长,其表达量逐渐下降,损伤后18小时降低到正常对照组的0.65倍,在损伤后24小时又出现一个表达高峰,达正常对照组的37.1倍,随后又逐渐下降。Frag-3 mRNA在损伤早期随着时间延长表达量逐渐增高,损伤后6小时其表达量高达正常对照组的243.5倍,损伤后12小时恢复到接近正常表达量水平,损伤后18小时,又出现一个小的表达高峰,为正常对照组的11.38倍,随后又下降至正常组水平。肌肉挫伤后Cox6c mRNA表达量的变化幅度较小,在正常组表达量的0.24~1.57倍之间。损伤后0.5小时,Cox6c mRNA表达量最高,为正常对照组的1.57倍。随着损伤时间的延长,其表达量逐渐减少,在1小时和6小时分别为正常对照组的1.29倍和1.19倍。损伤后18小时,其表达量已降至正常组水平以下,30小时表达量最低,为正常对照组的0.24倍。Frag-5 mRNA在肌肉损伤后36小时内均呈现相对高表达的状态。损伤的初始阶段其表达量迅速升高, 0.5小时达到正常对照组的5.7倍,1小时达到一个高峰,为正常对照组的6.19倍。随后开始下降,12小时达到最低表达量,为正常对照组的1.2倍。在18小时达到又一个高峰,为正常对照组的8.6倍。结论通过DDRT-PCR技术共发现5条差异性片段,其中三条与sTnI、Mx2及Cox6c的基因序列匹配度较高,另外两条可能为新基因。经实时荧光定量分析,sTnI、Cox6c mRNA相对表达量与损伤时间有关,随肌肉挫伤后时间的延长呈现规律性变化,且其变化幅度较小,速度较平稳,可以考虑作为损伤时间推断的标记。更多还原

【Abstract】 Objective: Differential display was used to isolate early time-related genes from the contused skeletal muscle of rats in order to find one or several sensitive gene, and the expression levels of the differential display genes mRNA in skeletal muscle were examined by real-time PCR to determine whether it can be used as a marker for wound age estimation.Methods: 1. A total of 12 Sprague-Dawley male rats, around 10 to 12 weeks old, weighing between 250 and 300 g, were used in this study. The rats were divided into 2 parts: control group (n=6) and contusion group (n=6). Animals were kept under a 12 h light-dark cycle with free access to food and water. After the rats were anesthetized with ethylether and the right posterior limb was shaved, a depilatory agent was applied to remove residual hair. Subsequently, the rats were placed on a foam bed, and a 250 g counterpoise was raised and allowed to fall freely 150 cm through a clear Lucite guide tube onto the right posterior limb of rats. After injury was made, the rats were reared in a cage and fed commercial rat food and tap water ad libitum. At 4 h after contusion, the rats were sacrificed with a lethal dose of pentobarbital (350 mg/kg of body weight intraperitoneal injection). Approximate 100 mg muscle sample was dissected from the right posterior limb, cut into two parts and immediately frozen in liquid nitrogen. Total RNA was isolated from muscle specimens (weighing approximately 50 mg) using the SV Total RNA Isolation System following the instructions provided with the kit. The concentration (ng/μL) and total RNA integrity was assessed using an Agilent 2100 Bioanalyser by loading samples onto the Eukaryote total RNA nano-chip. And only those RNA with a RNA Integrity Number (RIN) above 8.0 and with clearly visible 28/18s peaks was used for the following experiment.2. A total of 12 combinations of anchor primers and random primers were used to amplify the cDNA from the control group and contused group skeletal muscle. The PCR products were analyzed by the PAGE with silver staining. The differential display bands were purified with the vector, cloned, sequence analyzed and homologated with GenBank.3. The specific primers to the differential dispaly fragments were used to amplify the specific PCR products from the total RNA by the Real-time PCR, in order to indentify the authenticity of differential display fragments.4. In order to identify the endogenous reference genes for normalization of the gene expression in the contused skeletal muscle, a panel of nine candidate reference genes, such asβ-Actin and GAPDH, were tested in the control group, 6 h and 12 h group after contusion by the Real-time PCR. Then the potential endogenous reference genes were tested by three different software packages, Normfinder, geNorm and BestKeeper.5. A total of 54 Sprague-Dawley male rats, which were divided into 2 parts : control group (n=6) and 0.5, 1, 6, 12, 18, 24, 30 and 36 h (n=6) contusion group, were used to examine the expression levels of the differential display genes mRNA in skeletal muscle by real-time PCR.Results: 1.All samples were performed on Agilent 2100 Bioanalyzer using the RNA 6000 LabChips Kit. Approximate 0.1-0.4μg of total RNA was extracted from each milligram wet muscle using the SV Total RNA Isolation System. And only those RNA with a RNA Integrity Number (RIN) above 8.0 and with clearly visible 28/18s peaks was used for the following experiment.2. A total of 21 differential display bands were cut from the PAGE, and 12 of them were successfully amplified. Then the DNA was recombined to the vector, and cloned in the E.coli. The sequence was analyzed by the Takara. As a result, 5 fragments were long enough to be homologated with GenBank.3. Each of 5 fragments was successfully amplified from the total RNA using the special primers. And the dissociation curve of them all showed singal cusps, which suggested that there were no other PCR productions besides our target gene, and there was no primer dimmer.4. The results, which were analyzed by the Normfinder, geNorm and BestKeeper, showed that the RPL13 and RPL32 were more stable than other endogenous reference genes in normalizing the genes expression of skeletal muscle.5. The expression level of sTnI mRNA decreased gradually after contusion, at 0.5 h , 1 h and 6 h after contusion, the expression level decreased to 78.17% (P<0.05), 41.58% (P<0.05) and 32.13% of that in the control group. The expression level of Mx2 mRNA upgraded to 84.3 times (P<0.05) of that in the control group at 0.5 h after contusion, then it decreased gradually, at 18 h the expression level was 0.65 times of that in the control group. At 24 h after injury there was another peak of expression which reached 37.1 times of that in the normal control group. The expression level of Frag-3 mRNA upgraded to 243.5 times of that in the normal group at 6 h after contusion, then it decreasd to normal expression level. At 18 h after injury, there was another peak of expression which was 6.3 times of that in the normal group, and then it again decreased to normal expression level. The expression level of Cox6c mRNA had a lesser change. It varied from 0.24 to 1.57 times of that in the normal group. The highest expression level was 1.57 times of that in the control group at 0.5 h after contusion, and the minimum expression level was 0.24 times of that in the control group at 30 h after contusion. The expression level of Frag-5 mRNA was high during 36 h after contusion. At 0.5 h , 1 h and 6 h after contusion, the expression level upgraded to 5.7, 6.1 and 4.27 times of that in the control group . The highest expression level was 8.6 times to that in the control group at 18 h after contusion. At 24 h, 30 h, and 36 h after injury, the expression level decreased to 2.9, 2.6 and 1.8 times of that in the control group.Conclusion: Five fragments were found by DDRT-PCR. Three of them had a high homology with the gene sequence of sTnI, Mx2 and Cox6c, and the other two fragments may be new genes. sTnI mRNA and Cox6c mRNA changed regularly along with the injury time, and could be considered as new markers of wound time estimation.更多还原