【作者】 陈国蕾；

【导师】 焦炎；

【作者基本信息】 山西医科大学 ， 法医学， 2010， 硕士

【摘要】 目的：应用免疫组化SABC法、免疫印迹Western Bloting法和实时荧光定量RT-PCR技术检测大鼠脑挫伤后HIF-1α蛋白和HIF-1αmRNA的时序性表达规律,探讨HIF-1α推断脑挫伤的可行性,旨在为法医实践中推断脑挫伤经过时间提供依据。方法：选取健康成年SD大鼠64只,随机分为对照组和实验组,每组8只。参照Feeney法建立大鼠闭合性脑挫伤模型,实验组按损伤时间不同分为伤后1h、6h、12h、48h、72h、7d、14d共7组,对照组动物仅切开头皮,有顶开骨窗,不致脑损伤,在损伤后依规定时间内处死动物,对照组动物于钻孔后1h处死,取出脑组织,以脑挫伤灶为中心旁开1mm行大鼠脑冠状切面取材,置于4%多聚甲醛PBS液中固定,用于免疫组织化学染色；于脑挫伤灶取脑组织-80℃液氮保存用于蛋白和总RNA的提取。应用免疫组化SABC法和免疫印迹Western Bloting法检测各组HIF-1α蛋白的表达,利用图像分析技术定量测定免疫组化染色切片的阳性细胞数及以条带积分光密度值,所得数据用用SPSS13.0软件对所得数据进行统计学分析。将提取的脑组织中的总RNA,逆转录合成cDNA第一条链,采用实时荧光定量PCR检测H1F-1amRNA逆转录合成第一条cDNA链的相对表达量,所得数据用用SPSS13.0软件对所得数据进行统计学分析。结果：(1)HE结果：对照组神经元细胞形态结构正常,核染色较淡、圆形,核仁清楚。损伤组可见脑实质散在出血,蛛网膜下腔、侧脑室出血。所选切面各部位均可见程度不一的神经元细胞核深染、固缩,神经元细胞水肿,周围出现空隙,逐渐发展为胞核破碎溶解,神经元坏死消失,周围胶质细胞增生；(2)免疫组织组化学SABC法染色结果：对照组神经细胞偶见HIF-1α表达,阳性产物呈棕黄色,主要位于神经细胞胞核及胞浆内：伤后1h挫伤灶周围即可观察到阳性反应细胞,呈散在少量分布；伤后12h可见表达HIF-1α的神经细胞数增多,表达强度增强,差异有显著性(P<0.05)；48h后达高峰,阳性细胞聚集成簇,周围可见大量棕黄色产物。随后阳性反应细胞逐渐减少,7d后仍见少量表达,14d后基本恢复正常；(3)免疫印迹Western Bloting结果：脑挫伤后1h即可见条带,12h时条带明显增宽,48h的条带最宽,随后开始减少,到14d时条带几乎不可见。除14d组外,各实验组与对照组比较,差异有统计学意义(P<0.05)；实验组间两两比较,除12h组和72h组之间无统计学意义(P>0.05)外,其余各组间差异均有统计学意义(P<0.05)；(4)实时荧光定量RT-PCR检测结果：HIF-1αmRNA于脑挫伤后1h表达显著增强(P<0.01),达到高峰,为正常组的75.58倍,随后逐渐下降,至48h时又出现一次高峰,随后又开始下降,14d时降至正常水平。结论：大鼠脑挫伤后脑组织中HIF-1α蛋白和HIF-1αmRNA的表达随着损伤时间的延长都呈时序性规律变化,但变化规律有所不同,HIF-1αmRNA的表达高峰出现较早,可用于较早损伤时间的推断,两者综合分析,可望为法医学颅脑损伤时间的推断提供客观、科学的依据。更多还原

【Abstract】 Objective:To examine the sequential expression trend of HIF-la protein and HIF-lamRNA in the brain after cerebral contusion of rats by immunohistochemical SABC, Western Bloting and real-time fluorescent quantitative RT-PCR,evaluate the application for estimating the early time of cerebral contusion in forensic medicine aspect.Methods:The 64 SD rats were divided into control group and experimental group randomly,(n=8). Based on Feeney’s method, the rat model of cerebral contusion was established. Animals were sacrifice at lh,6h,12h,48h,72h,7d,14d after cerebral contusion.Rats in control group were cut scalp and opend window in right parietal bone without cerebral contusion,were sacrifice after 1h.The brain tissue was cut in coronal section and fixed in 4% paraform for immunohistochemical.The brain tissue were cut respectively in each experimental group and then the samples were stored in-80°liquid nitrogen for the isolation of protein and total RNA. the expression of HIF-1a protein was detected by immunohistochemical SABC and Western Bloting.Quantitative Determination of immunohistochemical staining sections of positive cellsand band integral optical density value were detected by image analysis technique.Total RNA was isolated from each sample and reverse transcript to cDNA,then their reaction outcome were quantitated by real-time fluorescent quantitative PCR.The results were assessed by statistic analysis.Results:(1) The result of HE:The morphology of neuronal cell is normol, cell nuclear is round and nuclear staining is light with clear nucleolus in control group.experimental group can be seen scattered intraparenchymal hemorrhage, subarachnoid space and lateral ventricle hemorrhage.Deeply stained nuclei and condensation of neuron cells, edema appears around the gap to varying degrees are visible in different parts of neurons of the selected aspect, and gradually developed into nucleus broken and dissolved, neuronal necrosis disappeared, the surrounding glial cell proliferation;(2) The result of immunohistochemical SABC:Only few HIF-la-positive cells at control group,The positive product can be seen in cytoplasm and nucleus The expression of HIF-1αprotein could be detected at 1h after injury,The quantity of HIF-la-positive cells increased remarkably after 12h after injury(P<0.05);peaked at 48h, Little expression was found at 7d after injury.The 14d group was similar as the control group.(3) The result of Western Bloting:Bands can see at 1h after Cerebral contusion, widened significantly at12h, widest bands at 48h,then began to decrease,the band is almost invisible at 14d.Each experimental group compara to control group except 14d groups, the difference had statistical significance(P<0.05).Significant statistic difference was found in multiple comparison (P<0.05)except groups between 12h and 48h(P>0.05).(4) The result of real-time fluorescent quantitative PCR:The expression of HIF-1αmRNA was obviously increased at 1h(P<0.01)after cerebral contusion and arrived the peak value.then fell but another peak appear at 48h.the control group and the 14d group showed negative staining.Conclusion:The time-dependent expression of HIF-1αprotein and HIF-1αmRNA in the brain tissue after cerebral contusion is different,the peak of.HIF-1αmRNA more early than HIF-1αprotein,they may be used for the estimation of posttraumatic intervals after cerebral contusion in forensic practice.更多还原