本文選題：臨床法醫(yī)學(xué) + 損傷時(shí)間推斷��； 參考：《山西醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的：應(yīng)用免疫組化(SABC法)、免疫印跡(Western blot)法及實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測(cè)大鼠腦挫傷后腦組織中NF-κB的表達(dá)規(guī)律,探討NF-κB表達(dá)與腦損傷經(jīng)過時(shí)間的關(guān)系,以期為法醫(yī)學(xué)實(shí)踐中早期推斷腦損傷時(shí)間提供科學(xué)的實(shí)驗(yàn)依據(jù)。 方法：選取64只健康成年SD大鼠,雌雄不限,體重250±10g(由山西醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供),隨機(jī)分為實(shí)驗(yàn)組(n=56)及對(duì)照組(n=8),實(shí)驗(yàn)組依據(jù)損傷時(shí)間不同再分為7個(gè)亞組。對(duì)大鼠實(shí)施麻醉后,實(shí)驗(yàn)組采用自由落體打擊法于顱骨中線旁3mm處造成腦挫傷模型,于損傷后1h、4h、12h、48h、72h、7d、14d七個(gè)時(shí)間點(diǎn)(每個(gè)時(shí)間點(diǎn)8只)取挫傷處腦組織用常規(guī)蘇木素-伊紅染色技術(shù)(hematoxylin and eosin,HE);對(duì)大鼠腦挫傷后14d內(nèi)繼發(fā)性腦損傷的病理學(xué)改變、NF-κB的分布特征進(jìn)行光鏡觀察,同時(shí)另取100mg,置于-80℃液氮中保存?zhèn)溆�；�?duì)照組大鼠僅鉆孔處理,但不受打擊,其余處理與實(shí)驗(yàn)組相同。應(yīng)用免疫組化(SABC)法、免疫印跡(Western blot)法及實(shí)時(shí)熒光定量RT-PCR技術(shù)結(jié)合圖像分析技術(shù)半定量檢測(cè)大鼠腦組織NF-κB含量的變化,并與對(duì)照組作比較。將所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果：(1)紫外-可見光分光光度計(jì)檢測(cè)RNA純度、濃度及完整性較好,可以滿足下一步實(shí)驗(yàn)的要求;(2) NF-κB分別與內(nèi)參基因rpL32擴(kuò)增效率一致,說明內(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)免疫組化(SABC)法、大鼠腦挫傷后在腦組織中于傷后1h即有少量NF-κB陽性細(xì)胞,48h陽性細(xì)胞明顯增加并達(dá)到峰值,72h隨后逐漸減少,7d后偶見陽性細(xì)胞,14d后未見陽性細(xì)胞。(4)免疫印跡(Western blot)法,大鼠腦挫傷后在腦組織中于傷后1h NF-κB蛋白增加,12-48達(dá)高峰,72h后下降,14d基本恢復(fù)到正常水平(5)實(shí)時(shí)熒光定量RT-PCR法,大鼠腦挫傷后在腦組織中于傷后1h NF-κBmRNA表達(dá)即快速增加,4h后逐漸下降,在到48h達(dá)低谷,而在72h增加并達(dá)高峰,7d和14d時(shí)表達(dá)弱。 結(jié)論：大鼠腦挫傷后腦組織中NF-κB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。免疫組化技術(shù)操作簡(jiǎn)單且易行,免疫印跡(Western blot)法、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精確,均適合法醫(yī)學(xué)研究及檢案的需要。
[Abstract]:Objective: to detect the expression of NF- 魏 B in brain tissue of rats after brain contusion by immunohistochemistry (SABC method), Western blotting (blot) and real-time fluorescence quantitative RT-PCR (RT-PCR), and to explore the relationship between NF- 魏 B expression and brain injury duration. In order to provide scientific experimental basis for early estimation of brain injury time in forensic practice. Methods: 64 healthy adult SD rats, weighing 250 鹵10g (provided by Experimental Animal Center, Shanxi Medical University), were randomly divided into two groups: experimental group (n = 56) and control group (n = 8). The experimental group was subdivided into 7 subgroups according to the time of injury. After anaesthesia, the brain contusion model was established in the experimental group at 3mm near the midline of skull by free falling body attack method. The pathological changes of NF- 魏 B in rat brain contusion were observed under light microscope by routine hematoxylin-eosin staining technique (hematoxylin and eosinhe), and the pathological changes of NF- 魏 B were observed under light microscope at 14 days after injury, and the brain tissue of contusion was taken from 7 time points (8 rats at each time point), and the pathological changes of NF- 魏 B in rats were observed by light microscope, and the pathological changes of NF- 魏 B in brain contusion were observed by light microscope. At the same time, 100 mg was taken and stored in liquid nitrogen at -80 鈩，

本文編號(hào)：2063429