【摘要】： 目的：本研究從基因庫中篩選出兩種穩(wěn)定表達(dá)于不同個體與組織細(xì)胞中的看家基因GAPDH和β-actin，采用實時熒光定量RT-PCR技術(shù)研究大鼠死后不同時間和不同臟器中兩種看家基因的mRNA降解規(guī)律，建立mRNA降解的程度與死亡時間關(guān)系的回歸方程，探索用于晚期死亡時間推斷較為理想的組織，并試圖建立標(biāo)準(zhǔn)化的RNA提取與定量檢測方法，，希望能最終應(yīng)用于法醫(yī)學(xué)實踐中。 方法：(1)選取SD大鼠28只，按不同死后經(jīng)過時間分對照組(死后即刻)和實驗組1～6(死后1天、3天、5天、7天、9天、12天)，每組4只，對照組脊椎脫臼法處死后快速取出心、肝、脾、肺、腎、腦，稱量多個小塊(1g)組織置于液氮中保存?zhèn)溆�。實驗組脊椎脫臼法處死后置于人工氣候箱內(nèi)，設(shè)定條件為溫度20℃，濕度50％，白天、黑夜各12小時。分別于1天、3天、5天、7天、9天、12天后各取出4只大鼠的心、肝、脾、肺、腎、腦，稱量多個小塊(1g)組織置于液氮中保存?zhèn)溆谩?2)采用以膜提取技術(shù)(不含苯酚)為基礎(chǔ)和以異硫氰酸胍／苯酚法為原理的兩種商品化試劑盒對大鼠心、肝、脾、肺、腎、腦組織進(jìn)行總RNA的提取，并進(jìn)行總RNA的評價與鑒定，以分析評價兩種提取方法的優(yōu)劣及適用范圍。(3)對組織中GAPDH mRNA和β-actin mRNA進(jìn)行一步法RT-PCR檢測，應(yīng)用凝膠圖像分析系統(tǒng)分別對各組織中GAPDH mRNA和β-actin mRNA擴(kuò)增產(chǎn)物電泳圖目標(biāo)條帶進(jìn)行灰度掃描和光密度分析，計算出各條帶相對灰度值和光密度值，結(jié)果以相對灰度(Relative grayscale)和積分光密度值(Integral optical density，IOD)表示，并篩選出在死后較長時間段仍能檢出的組織臟器。(4)根據(jù)一步法RT-PCR擴(kuò)增后產(chǎn)物凝膠電泳的實驗結(jié)果，本文選取了在死后5天內(nèi)仍能檢測出擴(kuò)增產(chǎn)物的腦組織和脾臟，使用SYBR GreenⅠ嵌合熒光法實時熒光定量RT-PCR技術(shù)，根據(jù)熒光信號的累積情況實時監(jiān)測GAPDH mRNA和β-actin mRNA不同時間點的RT-PCR進(jìn)程，并將PMI延長至12天，結(jié)果用循環(huán)閾值(簡稱C_t值)表示，分析死后經(jīng)過時間與C_t值的線性關(guān)系，并最終建立死亡時間推斷回歸方程。(5)為探討同一種看家基因上的不同位點，其降解速率是否也有一定規(guī)律性，本文選取了在死后較長時間內(nèi)仍能檢測出擴(kuò)增產(chǎn)物的腦組織，對GAPDH mRNA的6個不同位點進(jìn)行不同時間點的實時熒光定量RT-PCR監(jiān)測，這些位點依次分布于GAPDH mRNA的5'～3'端。并將PMI時間延長、間隔增加(死后即刻、1天、5天、9天、12天)，結(jié)果用循環(huán)閾值(簡稱C_t值)表示。通過檢測大鼠死后不同時間腦組織中各位點GAPDH mRNA的降解，分析不同位點的降解規(guī)律及其與死后經(jīng)過時間的相關(guān)性，試圖尋找單一看家基因的降解規(guī)律。 結(jié)果：(1)總RNA評價與鑒定均以對照組為例作比較，采用TRIzol試劑盒與SVTotal RNA Isolation System提取的總RNA純度與得率均可滿足下一步實驗要求。各組織OD_(260／280)均在1．9～2．1之間，結(jié)果無顯著性差異(P0．01)。但在SV Total RNA Isolation System中加入了DNaseⅠ消化，有效的去除了DNA污染，因此提取成功率明顯高于TRIzol試劑盒。除SV Total RNA Isolation System對組織提取上限量要求較低外，不同提取方法獲得的各組織臟器總RNA得率無顯著性差異。各組織臟器中總RNA得率依次為肝臟脾臟心臟大腦腎臟肺臟。均可滿足下一步實驗要求。(2)GAPDH mRNA、β-actin mRNA進(jìn)行RT-PCR反應(yīng)后，擴(kuò)增產(chǎn)物的相對灰度與積分光密度值隨死后經(jīng)過時間的延長而逐漸減小，且與死亡時間顯著相關(guān)，從擴(kuò)增產(chǎn)物電泳圖中可見大鼠脾臟和腦組織GAPDH mRNA和β-actin mRNA在死后5天內(nèi)可被檢出，心臟和腎臟在死后3天內(nèi)可被檢出，而肝臟和肺臟GAPDH mRNA和β-actin mRNA降解較快，僅在死后1天內(nèi)被檢出。以脾臟和腦組織檢出時間最長。(3)使用SYBR GreenⅠ嵌合法實時熒光定量RT-PCR，根據(jù)熒光信號的累積實時監(jiān)測腦組織和脾臟GAPDH mRNA和β-actin mRNA不同時間點的RT-PCR進(jìn)程，經(jīng)線性回歸分析，GAPDH mRNA和β-actin mRNA的C_t值均與PMI之間存在顯著的相關(guān)性，并得出死亡時間推斷的回歸方程(腦組織GAPDH：Y=15．312+1．531X，R~2=0．943；腦組織β-actin：Y=15．609+1．750X，R~2=0．953；脾臟GAPDH：Y=19．571+1．453X，R~2=0．852；脾臟β-actin：Y=21．769+1．274X，R~2=0．808)。(4)經(jīng)線性回歸分析，腦組織中各位點GAPDH mRNA均與死后經(jīng)過時間有相關(guān)性，但不同位點的mRNA降解速率不同。各位點線性回歸方程如下：GAPDH mRNA1：Y=15．501+1．577X R~2=0．968，斜率為1．577GAPDH mRNA2：Y=15．717+1．596X R~2=0．980，斜率為1．596GAPDH mRNA3：Y=15．772+1．553X R~2=0．973，斜率為1．553GAPDH mRNA4：Y=15．487+0．936X R~2=0．965，斜率為0．936GAPDH mRNA5：Y=15．580+0．892X R~2=0．949，斜率為0．892GAPDH mRNA6：Y=15．570+0．829X R~2=0．956，斜率為0．829各個線性回歸方程中，GAPDH mRNA1～GAPDH mRNA3的斜率為1．577、1．596、1．553，這三個位點的降解速率基本相同。GAPDH mRNA4～GAPDH mRNA6的斜率為0．936、0．892、0．829，這三個位點的降解速率基本相同。但與前三個位點相比，斜率減小，說明后三個位點mRNA的降解速率要慢于前三個位點。以GAPDH mRNA 6作為外標(biāo)，GAPDHmRNA1～GAPDH mRNA3與其的比值同PMI之間存在顯著的相關(guān)性(回歸方程分別為Y=1．015+0．029X，R~2=0．898；Y=1．028+0．030X，R~2=0．871；Y=1．031+0．028X，R~2=0．879)。而GAPDH mRNA4和GAPDH mRNA 5與作為外標(biāo)的GAPDH mRNA 6的比值同PMI之間無相關(guān)性。 結(jié)論：(1)在研究機(jī)體死后mRNA降解規(guī)律的實驗中，總RNA的提取是實驗成功與否的關(guān)鍵，而不同商品化提取試劑均可滿足實驗要求，但從法醫(yī)學(xué)實驗室質(zhì)量控制及檢驗結(jié)果是否具有可比性的角度出發(fā)，SV Total RNA Isolation System效果更好。(2)與一步法RT-PCR技術(shù)相比，SYBR GreenⅠ嵌合法實時熒光定量RT-PCR在定量分析mRNA降解的研究中是一個更理想的技術(shù)手段。選用看家基因作為PMI推斷的研究對象，可在法醫(yī)檢案中消除其它基因因為個體差異帶來的誤差，更具實用性。C_t值作為動態(tài)監(jiān)測機(jī)體死后不同時間點的客觀指標(biāo)，與死后不同時間點的線性關(guān)系良好，推斷死后經(jīng)過時間尤其是晚期死亡時間較為理想，(3)死后mRNA特別是看家基因mRNA的組織差異性研究表明，腦組織和脾臟中mRNA穩(wěn)定性較好，適用于PMI特別是晚期PMI的推斷。除環(huán)境溫度外，環(huán)境濕度也是死亡時間推斷研究中的重要影響因素，在今后的研究中應(yīng)加以考慮。(4)同一種組織中，同一種看家基因的不同位點存在降解速率的差異性。造成這種差異性的原因可能是機(jī)體死亡后，在保證RNA分子完整性的前提下，mRNA的降解可能是從5'端向3'端進(jìn)行的。因此選取看家基因作為引物時，應(yīng)盡量選取靠近3'端的位點，這些位點降解速率慢，可能更適于研究晚期死亡時間的推斷。同時在實驗室結(jié)果比較中，也應(yīng)將引物序列標(biāo)準(zhǔn)化，便于不同實驗室的結(jié)果比較與評價。(5)采用RNA尤其是mRNA作為研究目標(biāo)進(jìn)行死亡時間推斷為此研究領(lǐng)域提供了一個新的思路。
[Abstract]:AIM: To screen two kinds of housekeeping genes GAPDH and beta-actin stably expressed in different individuals and tissues and cells from the gene pool. Real-time fluorescence quantitative RT-PCR was used to study the mRNA degradation of two kinds of housekeeping genes in different organs and different time after death in rats, and to establish the relationship between the degree of mRNA degradation and the time of death. Regression equation is used to explore the ideal tissue for estimating the time of late death, and to establish a standardized method for RNA extraction and quantitative detection, hoping to be eventually applied in forensic practice.
Methods: (1) Twenty-eight SD rats were divided into control group (immediately after death) and experimental group (1-6 days, 3 days, 5 days, 7 days, 9 days, 12 days) according to different postmortem passage time. Four rats in each group were selected. In control group, the heart, liver, spleen, lung, kidney, brain were quickly removed after vertebral dislocation. After death, the rats were placed in an artificial climate chamber under the following conditions: temperature 20 C, humidity 50%, day and night 12 hours. The hearts, livers, spleens, lungs, kidneys, and brains of four rats were taken out after 1 day, 3 days, 5 days, 7 days, 9 days, and 12 days respectively, and several small pieces (1g) of tissue were weighed and stored in liquid nitrogen. Two commercial kits based on guanidine isothiocyanate/phenol method were used to extract total RNA from heart, liver, spleen, lung, kidney and brain of rats, and the total RNA was evaluated and identified in order to analyze the advantages and disadvantages of the two methods and evaluate their applicability. (3) GAPDH mRNA and beta-actin mRNA in tissues were detected by one-step RT-PCR, and gel was used. The image analysis system scans the target bands of GAPDH mRNA and beta-actin mRNA amplification products electrophoresis map in each tissue by gray scale scanning and optical density analysis, and calculates the relative gray value and optical density value of each band. The results are expressed by relative gray scale (Relative gray scale) and integral optical density (IOD). (4) According to the results of one-step RT-PCR amplified product gel electrophoresis, brain tissues and spleens which could detect the amplified products within 5 days after death were selected. The real-time fluorescence quantitative RT-PCR technique of SYBR Green I chimeric fluorescence was used to detect the amplified products according to the accumulation of fluorescent signals. The RT-PCR process of GAPDH mRNA and beta-actin mRNA at different time points was monitored and the PMI was prolonged to 12 days. Results The linear relationship between postmortem passage time and C_t value was analyzed by cyclic threshold value, and the regression equation was established to deduce the postmortem time. (5) To explore the different sites in the same housekeeping gene, the degradation rate was as follows. In this study, six different sites of GAPDH mRNA were monitored by real-time fluorescence quantitative RT-PCR at different time points. These sites were distributed in the 5'-3'end of GAPDH mRNA in turn. The PMI time was prolonged and the interval was increased (immediately after death, 1 day, 5 days). By detecting the degradation of GAPDH mRNA at different sites in brain tissues at different time points after death, we analyzed the degradation regularity of different sites and their correlation with postmortem passage time, and tried to find the degradation regularity of single housekeeper gene.
Results: (1) Total RNA evaluation and identification were compared with the control group. The purity and yield of total RNA extracted by TRIzol kit and SVTotal RNA Isolation System could meet the next experimental requirements. OD_ (260/280) of all tissues ranged from 1.9 to 2.1, and the results showed no significant difference (P 0.01). DNA contamination was effectively removed by adding DNase I digestion, so the success rate of extraction was significantly higher than that of TRIzol kit. Except for SV Total RNA Isolation System, which requires a lower upper limit of tissue extraction, there was no significant difference in the total RNA yield of different tissues obtained by different methods. The relative gray and integral optical density of the amplified products decreased with the prolongation of postmortem time, and were significantly correlated with the time of death. GAPDH in the spleen and brain tissues of rats could be seen from the electrophoresis of the amplified products. MRNA and beta-actin mRNA could be detected within 5 days after death, heart and kidney could be detected within 3 days after death, while GAPDH mRNA and beta-actin mRNA in liver and lung degraded more rapidly, only detected within 1 day after death. Spleen and brain tissues were detected for the longest time. (3) SYBR Green I embedded real-time fluorescent quantitative RT-PCR was used according to the accumulation of fluorescent signals. Real-time RT-PCR of GAPDH mRNA and beta-actin mRNA in brain and spleen was monitored. Linear regression analysis showed that there was a significant correlation between C_t values of GAPDH mRNA and beta-actin mRNA and PMI. The regression equation for estimating the time of death (GAPDH: Y = 15.312 + 1.531X, R~2 = 0.943; beta-actin in brain tissue) was obtained. Y = 15.609 + 1.750X, R~2 = 0.953; spleen GAPDH: Y = 19.571 + 1.453X, R~2 = 0.852; spleen beta-actin: Y = 21.769 + 1.274X, R~2 = 0.808). (4) By linear regression analysis, the GAPDH mRNA in brain tissues were correlated with postmortem time, but the mRNA degradation rates at different sites were different. GAPDH mRNA1:Y = 15.501+15.501+1.577X R~2 = 0.968, with a slope of 1.577 GAPDH mRNA2:Y = 15.717 + 1.596X R~2 = 0.980, with a slope of 1.596 GAPDH mRNA3:Y = 15.772 + 15.772 + 1.772 + 1.553X R~2 = 0.973, and 1.553GGAPDH mRNA4:Y = 4:Y = 15.487 + 0.936X R~2 = 15.487 + 0.936X R~2 = 0.965, and 1.577 GAPDH mRNA4:Y = 15.487 + 0.487 + 0.936X R~2 = 0.965, and 0.2X R~2=0.949 The slope of GAPDH mRNA 6:Y=15.570+0.829XR~2=0.956, and the slope of GAPDH mRNA 1-GAPDH mRNA 3 was 1.577,1.596,1.553 in each linear regression equation of 0.829. The degradation rates of the three sites were basically the same. The slope of GAPDH mRNA 4-GAPDH mRNA 6 was 0.936,0.892,0.829, and the degradation rates of the three sites were 1.577,1.596,1.553. Compared with the first three sites, the slope decreased, indicating that the degradation rate of mRNA in the latter three sites was slower than that in the former three sites. R~2=0.871; Y=1.031+0.028X, R~2=0.879). However, the ratio of GAPDH mRNA 4 and GAPDH mRNA 5 to GAPDH mRNA 6 as an external standard had no correlation with PMI.
Conclusion: (1) The extraction of total RNA is the key to the success of the experiment in studying the degradation of postmortem mRNA. Different commercial extracting reagents can meet the experimental requirements, but SV Total RNA Isolation System is more effective from the point of view of quality control and comparability of test results in forensic laboratory. Compared with one-step RT-PCR, SYBR Green I real-time fluorescence quantitative RT-PCR is a more ideal technique for quantitative analysis of mRNA degradation. Choosing the housekeeping gene as the research object of PMI inference can eliminate the errors caused by individual differences in forensic medical records and is more practical. The objective indexes of postmortem monitoring at different time points have a good linear relationship with postmortem time points, and the postmortem passage time, especially the late postmortem time, is more ideal. (3) The study on the tissue differences of postmortem mRNA, especially the home-care gene mRNA, shows that the mRNA in brain tissue and spleen is stable and suitable for PMI, especially in the late stage. PMI inference. In addition to ambient temperature, ambient humidity is also an important factor in the study of time-to-death inference, which should be taken into account in future studies. (4) In the same tissue, there are differences in degradation rates at different sites of the same housekeeping gene. The reason for this difference may be that RNA molecules are guaranteed after death. In the premise of integrity, the degradation of mRNA may occur from the 5'end to the 3'end. Therefore, when selecting the watcher gene as a primer, we should try to select sites close to the 3'end. The degradation rate of these sites is slow, which may be more suitable for studying the inference of late-stage mortality. Comparing and evaluating the results with the laboratory. (5) Using RNA, especially mRNA, as the research target to infer the time of death provides a new idea for this research field.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：D919;Q953

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王穎希;RNA用于體液鑒別的方法學(xué)建立及其法醫(yī)學(xué)應(yīng)用研究[D];山西醫(yī)科大學(xué);2012年

本文編號：2217492