改良彗星試驗檢測兔死后肝細胞核DNA含量變化及其法醫(yī)學意義
發(fā)布時間：2018-11-10 22:58
【摘要】： 目的: 應用改良彗星試驗方法檢測兔死后肝細胞核DNA的降解情況,研究死后細胞核DNA含量變化與死亡時間的關系,從而探討死后細胞核DNA含量改變在早期死亡時間推斷中的應用價值。 方法: 健康成年大白兔經(jīng)斷頸錯位處死后,置于室溫下,分別在處死后0h、3h、6h、9h、12h、15h、18h、21h、24h、27h、30h共11個時間點進行在體肝臟組織取材,并按相應時間段分組共有11個組別。應用改良彗星試驗方法技術對各組樣本進行電泳,染色,在熒光顯微鏡下對電泳圖像進行觀察、攝像,再用CASP圖像分析軟件對彗星圖像尾部情況進行分析,采用尾長(TL)、尾矩(TM)兩個參數(shù)作為主要的分析指標,最后應用SAS 9.0統(tǒng)計分析軟件對圖像分析所得數(shù)據(jù)進行統(tǒng)計學相關回歸分析。 結(jié)果: 應用改良彗星試驗共制得凝膠體528塊,無一例損壞脫落,試驗的繁瑣程度得到明顯的減輕、實驗時間明顯縮短。彗星試驗顯示:兔死后肝細胞核DNA經(jīng)過單細胞凝膠電泳出現(xiàn)了明顯的彗星圖像,并隨死亡時間的推移彗星圖像變得越清晰明顯,而后又逐漸變得模糊不清;最早出現(xiàn)的時間是在死后3h,其拖尾率達到98%,而在死后30h彗星圖像就變得模糊不清,不能進行準確辨認。圖像分析顯示:在死后0-18h隨死亡時間的延長,彗星尾部逐漸增長、增寬,表現(xiàn)尾部情況的TL、TM參數(shù)值逐漸增大,18h時達到高峰,而后在21-27h有所下降并在一定范圍內(nèi)波動,二參數(shù)值表現(xiàn)出相對的穩(wěn)定性,而在30h則不能檢測到彗星圖像相關數(shù)據(jù),表明彗星圖像不存在。統(tǒng)計分析顯示:在死后0-18h各組間TL、TM值存在顯著性差異,而在21-27h各組間TL、TM值無顯著性差別;TL、TM在死后3-18h與死亡時間緊密相關,相關系數(shù)分別達到0.99614、0.99077,p㩳0.01,回歸分析二者與死亡時間在此時間段幾乎成直線關系。 結(jié)論: 1.改良彗星試驗使得實驗更為成功,更能夠快速、簡便、靈敏、準確檢測死后細胞核DNA的降解,結(jié)合圖像分析軟件大大提高了結(jié)果分析的精確性和敏感性,在早期死亡時間推斷中有較高的應用價值。 2.兔在死后3h肝細胞核DNA就已經(jīng)開始發(fā)生降解,死后9-18h降解明顯加速,而后出現(xiàn)一段相對的穩(wěn)定期,最后溶解消失。 3.尾長和尾矩良好地反映了DNA降解情況,是彗星試驗研究死后DNA含量變化與死亡時間關系的很好的指標。 4.死后細胞核DNA含量變化與死亡時間存在一定的線性關系,研究二者關系將在早期死亡時間推斷中有重要的意義。 5.應用改良彗星試驗測定死后肝細胞核DNA含量變化可望成為早期死亡時間推斷的更簡便方法。 6.圖像分析軟件為彗星試驗結(jié)果分析提供了便利。
[Abstract]:Objective: to detect the degradation of DNA in the liver nucleus of rabbits after death by modified comet assay, and to study the relationship between the changes of nuclear DNA content and the time of death after death. To explore the application value of nuclear DNA content change in early death time estimation. Methods: the healthy adult white rabbits were killed by cervical dislocations and placed at room temperature. The liver tissue samples were collected from the liver in vivo at 30 hours after the death at 1h, 3h, 6h, 9h, 12h, 15h, 18h, 21h, 21h, 24h, 27h, 30h, respectively. According to the corresponding time groups, there are 11 groups. The modified comet assay technique was applied to the electrophoresis and staining of each group of samples. The electrophoretic images were observed under fluorescence microscope, the image was photographed, and the tail of comet image was analyzed by CASP image analysis software, and the tail length (TL), was used. The two parameters of tail moment (TM) are used as the main analysis indexes. Finally, the statistical correlation regression analysis of the image analysis data is carried out by using the SAS 9.0 statistical analysis software. Results: a total of 528 gel bodies were prepared by modified comet assay, none of them was damaged and shedding, the tedious degree of the experiment was obviously reduced, and the experimental time was shortened obviously. Comet assay showed that the DNA of the liver nucleus appeared obvious comet image by single cell gel electrophoresis after death, and the comet image became clearer and more obvious with the passage of death time, and then became blurred gradually. The earliest time was 3 hours after death, and the tail trailing rate reached 98%, but the comet image became blurred and could not be accurately identified 30 hours after death. Image analysis showed that the tail of comet gradually increased and widened with the prolongation of death time from 0-18 h after death, the TL,TM parameter of tail increased gradually, and reached the peak at 18 h. Then it decreased at 21-27 h and fluctuated in a certain range. The two parameter values showed relative stability, but the relative data of comet images could not be detected at 30 h, indicating that the comet images did not exist. Statistical analysis showed that there was significant difference in TL,TM between groups at 0-18 h after death, but no significant difference in TL,TM between groups at 21-27 h. TL,TM was closely related to the time of death from 3 to 18 hours after death, and the correlation coefficient was 0.99614 ~ 0.9907p0. 01, respectively. The regression analysis showed that there was almost a linear relationship between the time of death and the time of death. Conclusion: 1. The improved comet assay makes the experiment more successful, and it is more rapid, simple, sensitive and accurate in detecting the degradation of nuclear DNA in postmortem nuclei, and the accuracy and sensitivity of the result analysis are greatly improved by combining with image analysis software. It has high application value in early death time inference. 2. The degradation of DNA in the liver nuclei of rabbits began to occur 3 hours after death, and accelerated obviously from 9 to 18 hours after death, followed by a relatively stable period, and finally dissolved and disappeared. 3. The tail length and tail moment well reflect the degradation of DNA, which is a good index to study the relationship between the changes of DNA content and the time of death in comet assay. 4. There is a linear relationship between the changes of nuclear DNA content and the time of death after death, and the study of the relationship between them will be of great significance in the estimation of early death time. 5. The modified comet assay is expected to be a simple method for estimating the early death time of liver nuclei. 6. Image analysis software provides convenience for the analysis of comet test results.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：D919
本文編號：2323806
[Abstract]:Objective: to detect the degradation of DNA in the liver nucleus of rabbits after death by modified comet assay, and to study the relationship between the changes of nuclear DNA content and the time of death after death. To explore the application value of nuclear DNA content change in early death time estimation. Methods: the healthy adult white rabbits were killed by cervical dislocations and placed at room temperature. The liver tissue samples were collected from the liver in vivo at 30 hours after the death at 1h, 3h, 6h, 9h, 12h, 15h, 18h, 21h, 21h, 24h, 27h, 30h, respectively. According to the corresponding time groups, there are 11 groups. The modified comet assay technique was applied to the electrophoresis and staining of each group of samples. The electrophoretic images were observed under fluorescence microscope, the image was photographed, and the tail of comet image was analyzed by CASP image analysis software, and the tail length (TL), was used. The two parameters of tail moment (TM) are used as the main analysis indexes. Finally, the statistical correlation regression analysis of the image analysis data is carried out by using the SAS 9.0 statistical analysis software. Results: a total of 528 gel bodies were prepared by modified comet assay, none of them was damaged and shedding, the tedious degree of the experiment was obviously reduced, and the experimental time was shortened obviously. Comet assay showed that the DNA of the liver nucleus appeared obvious comet image by single cell gel electrophoresis after death, and the comet image became clearer and more obvious with the passage of death time, and then became blurred gradually. The earliest time was 3 hours after death, and the tail trailing rate reached 98%, but the comet image became blurred and could not be accurately identified 30 hours after death. Image analysis showed that the tail of comet gradually increased and widened with the prolongation of death time from 0-18 h after death, the TL,TM parameter of tail increased gradually, and reached the peak at 18 h. Then it decreased at 21-27 h and fluctuated in a certain range. The two parameter values showed relative stability, but the relative data of comet images could not be detected at 30 h, indicating that the comet images did not exist. Statistical analysis showed that there was significant difference in TL,TM between groups at 0-18 h after death, but no significant difference in TL,TM between groups at 21-27 h. TL,TM was closely related to the time of death from 3 to 18 hours after death, and the correlation coefficient was 0.99614 ~ 0.9907p0. 01, respectively. The regression analysis showed that there was almost a linear relationship between the time of death and the time of death. Conclusion: 1. The improved comet assay makes the experiment more successful, and it is more rapid, simple, sensitive and accurate in detecting the degradation of nuclear DNA in postmortem nuclei, and the accuracy and sensitivity of the result analysis are greatly improved by combining with image analysis software. It has high application value in early death time inference. 2. The degradation of DNA in the liver nuclei of rabbits began to occur 3 hours after death, and accelerated obviously from 9 to 18 hours after death, followed by a relatively stable period, and finally dissolved and disappeared. 3. The tail length and tail moment well reflect the degradation of DNA, which is a good index to study the relationship between the changes of DNA content and the time of death in comet assay. 4. There is a linear relationship between the changes of nuclear DNA content and the time of death after death, and the study of the relationship between them will be of great significance in the estimation of early death time. 5. The modified comet assay is expected to be a simple method for estimating the early death time of liver nuclei. 6. Image analysis software provides convenience for the analysis of comet test results.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：D919
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