發(fā)布時(shí)間：2018-04-16 19:07

  本文選題：溺死 + ACTB�。� 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的探討溺死大鼠死后不同時(shí)間點(diǎn)mRNA的變化及穩(wěn)定性,篩選穩(wěn)定的內(nèi)參基因,并定量水通道蛋白5(Aquaporin,AQP-5)mRNA的表達(dá)。方法選取30只無特定病原體(Specific pathogen Free,SPF)級(jí)健康成年SD(Sprague Dawley)大鼠,并按照隨機(jī)單位組設(shè)計(jì)分組法,以每組6只,隨機(jī)均勻分為對(duì)照組(脊椎脫臼法處死)、溺死15min組(溺死尸體置于水中15min),溺死4h組(溺死尸體置于水中4h),溺死16h組(溺死尸體置于水中16h),溺死48h組(溺死尸體置于水中48h)。取溺死尸體及對(duì)照組尸體解剖,觀察肺臟變化,并分取肺組織,一部分用4%多聚甲醛(polyformaldehyde,PFA)中性PBS(phosphate buffer saline)液固定,用于形態(tài)學(xué)檢測(cè);一部分液氮速凍,提取RNA,采用qPCR(Real Time PCR,實(shí)時(shí)熒光定量聚合酶鏈反應(yīng))篩選穩(wěn)定的內(nèi)參基因以及對(duì)AQP-5進(jìn)行定量檢測(cè)。結(jié)果與對(duì)照組比較,各溺死組大鼠肺部均呈嚴(yán)重水腫,一定程度破裂出血,并可感覺到明顯溺液存于肺臟。通過提取RNA,并行q PCR,geNorm和NormFinder排序篩選發(fā)現(xiàn)ACTB(β-Actin,β-肌動(dòng)蛋白)為最穩(wěn)定的內(nèi)參基因;檢測(cè)AQP-5的表達(dá)我們發(fā)現(xiàn)溺死大鼠較對(duì)照組大鼠AQP-5相對(duì)表達(dá)量下降。結(jié)論篩選出ACTB為最穩(wěn)定的內(nèi)參基因,各溺死時(shí)間點(diǎn)大鼠AQP-5 mRNA的表達(dá)均顯著下調(diào)。本研究中AQP-5 mRNA檢測(cè)在大鼠溺死表達(dá)有一定陽性意義,有助于法醫(yī)溺死鑒定。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：D919

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