【摘要】： 目的:應(yīng)用實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測(cè)大鼠皮膚挫傷后皮膚和肌肉組織中兩種基因(ICAM—1和NF-κB)mRNA的時(shí)序性表達(dá)規(guī)律,探討這兩種基因mRNA表達(dá)在損傷時(shí)間推斷中的可行性,旨為法醫(yī)實(shí)踐中推斷早期皮膚損傷時(shí)間提供科學(xué)的實(shí)驗(yàn)依據(jù)。 方法:選取健康成年SD大鼠48只,隨機(jī)分為對(duì)照組(6只)和實(shí)驗(yàn)組(42只)。均實(shí)施麻醉、褪毛后,實(shí)驗(yàn)組大鼠采用自由落體打擊法于右后肢股四頭肌部位造成皮膚挫傷模型,損傷后0.5h、1h、6h、12h、18h、24h、30h七個(gè)時(shí)間點(diǎn)(每個(gè)時(shí)間點(diǎn)6只)取挫傷處皮膚及肌肉組織各30mg,置于液氮中保存?zhèn)溆?對(duì)照組大鼠未打擊造傷,其余處理與實(shí)驗(yàn)組相同。以膜吸附技術(shù)為基礎(chǔ)的SV Total RNA提取皮膚和肌肉組織中的總RNA,逆轉(zhuǎn)錄合成cDNA第一條鏈,梯度濃度稀釋cDNA原液分別作每個(gè)基因與內(nèi)參基因(mL32)相對(duì)定量的標(biāo)準(zhǔn)曲線;SYBR GreenⅠ嵌合熒光法實(shí)時(shí)熒光定量PCR檢測(cè)ICAM-1和NF-κB mRNA逆轉(zhuǎn)錄合成第一條cDNA鏈的相對(duì)表達(dá)量,分別與損傷時(shí)間進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果:(1)紫外-可見(jiàn)光分光光度計(jì)和Agilent 2100芯片生物分析儀檢測(cè)RNA純度、濃度及完整性較好,均可滿足下一步實(shí)驗(yàn)的要求;(2)ICAM-1和NF-κB分別與內(nèi)參基因rpL32擴(kuò)增效率一致,說(shuō)明內(nèi)參基因選擇正確,引物設(shè)計(jì)特異性強(qiáng)、反應(yīng)性能良好:(3)大鼠皮膚挫傷后ICAM-1 mRNA在皮膚組織中的表達(dá)0.5h達(dá)到峰值,24h下降至正常對(duì)照組的7倍隨后繼續(xù)升高;在肌肉組織中于損傷后0.5h表達(dá)顯著增強(qiáng)(P＜0.001),6h達(dá)到峰值,18h降至最低后再次升高:(4)NF-κB mRNA在皮膚組織中于損傷后0.5h表達(dá)顯著增強(qiáng)(P＜0.05),6h出現(xiàn)高峰,18h降至正常對(duì)照組以下,隨后略有回升;肌肉組織中損傷后0.5h表達(dá)顯著增強(qiáng)(P＜0.001),1h達(dá)到峰值,此后逐漸降低并于18h出現(xiàn)第二次表達(dá)高峰。 結(jié)論:大鼠皮膚挫傷后皮膚和肌肉組織中ICAM-1 mRNA和NF-κB mRNA表達(dá)隨著損傷時(shí)間呈規(guī)律性變化,可望用于法醫(yī)學(xué)鑒定及早期法醫(yī)學(xué)損傷時(shí)間推斷。實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測(cè)分子水平的變化敏感而且準(zhǔn)確,適合法醫(yī)學(xué)研究及檢案的需要。
[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，

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