大鼠溺死内参基因筛选和水通道蛋白5的表达

发布时间：2018-04-16 19:07

本文选题：溺死 + ACTB　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的探讨溺死大鼠死后不同时间点mRNA的变化及稳定性,筛选稳定的内参基因,并定量水通道蛋白5(Aquaporin,AQP-5)mRNA的表达。方法选取30只无特定病原体(Specific pathogen Free,SPF)级健康成年SD(Sprague Dawley)大鼠,并按照随机单位组设计分组法,以每组6只,随机均匀分为对照组(脊椎脱臼法处死)、溺死15min组(溺死尸体置于水中15min),溺死4h组(溺死尸体置于水中4h),溺死16h组(溺死尸体置于水中16h),溺死48h组(溺死尸体置于水中48h)。取溺死尸体及对照组尸体解剖,观察肺脏变化,并分取肺组织,一部分用4%多聚甲醛(polyformaldehyde,PFA)中性PBS(phosphate buffer saline)液固定,用于形态学检测;一部分液氮速冻,提取RNA,采用qPCR(Real Time PCR,实时荧光定量聚合酶链反应)筛选稳定的内参基因以及对AQP-5进行定量检测。结果与对照组比较,各溺死组大鼠肺部均呈严重水肿,一定程度破裂出血,并可感觉到明显溺液存于肺脏。通过提取RNA,并行q PCR,geNorm和NormFinder排序筛选发现ACTB(β-Actin,β-肌动蛋白)为最稳定的内参基因;检测AQP-5的表达我们发现溺死大鼠较对照组大鼠AQP-5相对表达量下降。结论筛选出ACTB为最稳定的内参基因,各溺死时间点大鼠AQP-5 mRNA的表达均显著下调。本研究中AQP-5 mRNA检测在大鼠溺死表达有一定阳性意义,有助于法医溺死鉴定。
[Abstract]:Objective to investigate the changes and stability of mRNA at different time points after drowning in rats, to screen stable internal reference genes and to quantify the expression of aquaporin aquaporin AQP-5 mRNA.Methods Thirty healthy adult SD(Sprague Dawley rats without specific pathogen were selected, and 6 rats in each group were divided into two groups according to random unit group.They were randomly divided into control group (spinal dislocated method, 15min group, drowning dead body in water for 15 minutes, drowning dead body in water for 4 hours, drowning dead body in water for 16 hours, drowning dead body in water for 16 hours and drowning dead body in water for 48 hours.The dead body of drowning and the control group were dissected to observe the changes of lung, and the lung tissue was divided into two groups. One part was fixed with 4% paraformaldehyde polyformalin polyformaldehydede (PFA-PFA-neutral PBS(phosphate buffer salineine) solution for morphological examination, and one part of liquid nitrogen was frozen quickly.RNAs were extracted, qPCR(Real Time PCR and real-time fluorescence quantitative polymerase chain reaction (PCR) were used to screen stable internal reference genes and to detect AQP-5 quantitatively.Results compared with the control group, the lungs of the rats in each drowning group showed severe edema, rupture and hemorrhage to some extent, and obviously drowned fluid could be found in the lungs.The expression of ACTB (尾 -Actinin, 尾 -actin) was found to be the most stable internal reference gene, and the relative expression of AQP-5 in drowning rats was lower than that in control rats.Conclusion ACTB is the most stable internal reference gene, and the expression of AQP-5 mRNA is down-regulated at all drowning time points in rats.In this study, the expression of AQP-5 mRNA was positive in the drowning of rats, which was helpful for forensic drowning identification.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：D919

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