大鼠皮肤挫伤后ICAM-1和NF-κB mRNA表达与损伤时间的研究
发布时间：2019-01-15 07:59
【摘要】： 目的:应用实时荧光定量RT-PCR技术检测大鼠皮肤挫伤后皮肤和肌肉组织中两种基因(ICAM—1和NF-κB)mRNA的时序性表达规律,探讨这两种基因mRNA表达在损伤时间推断中的可行性,旨为法医实践中推断早期皮肤损伤时间提供科学的实验依据。 方法:选取健康成年SD大鼠48只,随机分为对照组(6只)和实验组(42只)。均实施麻醉、褪毛后,实验组大鼠采用自由落体打击法于右后肢股四头肌部位造成皮肤挫伤模型,损伤后0.5h、1h、6h、12h、18h、24h、30h七个时间点(每个时间点6只)取挫伤处皮肤及肌肉组织各30mg,置于液氮中保存备用;对照组大鼠未打击造伤,其余处理与实验组相同。以膜吸附技术为基础的SV Total RNA提取皮肤和肌肉组织中的总RNA,逆转录合成cDNA第一条链,梯度浓度稀释cDNA原液分别作每个基因与内参基因(mL32)相对定量的标准曲线;SYBR GreenⅠ嵌合荧光法实时荧光定量PCR检测ICAM-1和NF-κB mRNA逆转录合成第一条cDNA链的相对表达量,分别与损伤时间进行统计学分析。 结果:(1)紫外-可见光分光光度计和Agilent 2100芯片生物分析仪检测RNA纯度、浓度及完整性较好,均可满足下一步实验的要求;(2)ICAM-1和NF-κB分别与内参基因rpL32扩增效率一致,说明内参基因选择正确,引物设计特异性强、反应性能良好:(3)大鼠皮肤挫伤后ICAM-1 mRNA在皮肤组织中的表达0.5h达到峰值,24h下降至正常对照组的7倍随后继续升高;在肌肉组织中于损伤后0.5h表达显著增强(P＜0.001),6h达到峰值,18h降至最低后再次升高:(4)NF-κB mRNA在皮肤组织中于损伤后0.5h表达显著增强(P＜0.05),6h出现高峰,18h降至正常对照组以下,随后略有回升;肌肉组织中损伤后0.5h表达显著增强(P＜0.001),1h达到峰值,此后逐渐降低并于18h出现第二次表达高峰。 结论:大鼠皮肤挫伤后皮肤和肌肉组织中ICAM-1 mRNA和NF-κB mRNA表达随着损伤时间呈规律性变化,可望用于法医学鉴定及早期法医学损伤时间推断。实时荧光定量RT-PCR技术检测分子水平的变化敏感而且准确,适合法医学研究及检案的需要。
[Abstract]:Objective: to detect the temporal expression of two genes (ICAM-1 and NF- 魏 B) mRNA) in the skin and muscle tissues of rats after skin contusion by real-time fluorescence quantitative RT-PCR. To explore the feasibility of mRNA expression of these two genes in the estimation of injury time in order to provide scientific experimental basis for estimating the time of early skin injury in forensic practice. Methods: 48 healthy adult SD rats were randomly divided into control group (n = 6) and experimental group (n = 42). All the rats were anesthetized, and the rats in the experimental group were exposed to the skin contusion in the quadriceps femoris of the right hind limb by free falling body attack method, and the skin contusion model was established at 0.5 h, 1 h, 12 h and 18 h, 24 h after the injury. At 30 h, the skin and muscle tissue of the contusion were 30 mg at each time point (6 rats per time point) and stored in liquid nitrogen. The rats in the control group had no injury, and the other treatments were the same as those in the experimental group. SV Total RNA based on membrane adsorption technique was used to extract total RNA, from skin and muscle tissue to synthesize the first strand of cDNA. Gradient concentration dilution of cDNA solution was used as the relative quantitative standard curve of each gene and internal reference gene (mL32). SYBR Green 鈪，
本文编号：2409022
[Abstract]:Objective: to detect the temporal expression of two genes (ICAM-1 and NF- 魏 B) mRNA) in the skin and muscle tissues of rats after skin contusion by real-time fluorescence quantitative RT-PCR. To explore the feasibility of mRNA expression of these two genes in the estimation of injury time in order to provide scientific experimental basis for estimating the time of early skin injury in forensic practice. Methods: 48 healthy adult SD rats were randomly divided into control group (n = 6) and experimental group (n = 42). All the rats were anesthetized, and the rats in the experimental group were exposed to the skin contusion in the quadriceps femoris of the right hind limb by free falling body attack method, and the skin contusion model was established at 0.5 h, 1 h, 12 h and 18 h, 24 h after the injury. At 30 h, the skin and muscle tissue of the contusion were 30 mg at each time point (6 rats per time point) and stored in liquid nitrogen. The rats in the control group had no injury, and the other treatments were the same as those in the experimental group. SV Total RNA based on membrane adsorption technique was used to extract total RNA, from skin and muscle tissue to synthesize the first strand of cDNA. Gradient concentration dilution of cDNA solution was used as the relative quantitative standard curve of each gene and internal reference gene (mL32). SYBR Green 鈪，
本文编号：2409022
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