死后間隔時間推斷的新研究
發(fā)布時間：2018-07-31 18:17
【摘要】： 死后間隔時間(postmortem interval，PMI)或稱死亡時間推斷(estimation of time since death，ETSD)是法醫(yī)病理學(xué)實際檢案及研究工作的重點和難點之一。長期以來，國內(nèi)外的學(xué)者們已采用多種技術(shù)與方法嘗試PMI的推斷，尤其是早期PMI的推斷，如利用死后各種尸體現(xiàn)象、肌肉超生反應(yīng)、玻璃體液鉀離子濃度、死后血液的生化變化等，但其準確性常受到外界各種因素的影響，往往誤差較大。至目前為止，尚無公認的簡易實用、準確可靠的推斷方法，尤其是中晚期PMI的推斷方法更不盡人意。因此，本課題研究大鼠死后皮下肌肉組織電阻抗幅值和相位角的變化規(guī)律，以探討生物電阻抗推斷中晚期PMI的價值，試圖發(fā)展一種簡單方便、準確可靠的推斷中晚期PMI的新方法；選擇前期研究尚未用過的分析指標——headDNA％，應(yīng)用單細胞凝膠電泳技術(shù)檢測大鼠死后腦、骨髓細胞核DNA降解隨PMI的變化規(guī)律，以探索單細胞凝膠電泳技術(shù)推斷早期PMI的新參數(shù)。 第一部分大鼠死后皮下肌肉組織電阻抗幅值和相位角變化推斷死后間隔時間的實驗研究 【目的】研究大鼠死后皮下肌肉組織電阻抗幅值和相位角的變化規(guī)律，探討生物電阻抗技術(shù)推斷中晚期PMI的價值，試圖發(fā)展一種簡單方便、準確可靠的推斷中晚期PMI的新方法。同時從肌肉組織形態(tài)學(xué)的角度初步分析了電阻抗幅值變化趨勢產(chǎn)生的機制。 【方法】取Wistar健康成年大鼠20只，分成4個實驗組，每組5只，分別對應(yīng)于四個不同的測量溫度(9±1℃、14±1℃、16±1℃、19±1℃)。用頸椎脫臼的方式處死動物，將電極插入大腿部肌肉并固定。迅速將動物置于設(shè)定溫度的恒溫培養(yǎng)箱或冰箱中，利用四電極阻抗測量系統(tǒng)測量大鼠死后皮下肌肉組織電阻抗幅值和相位角。自死后0h開始，每隔3h記錄一次電阻抗數(shù)據(jù)(夜間關(guān)機時除外)和環(huán)境溫度。當(dāng)實驗時間足夠長時(阻抗下降到原始阻抗的70％左右或15天以上)便可停止實驗。再用Excel對所得的離散阻抗數(shù)據(jù)進行簡單的均值、方差分析，用MATLAB對阻抗數(shù)據(jù)進行相關(guān)分析和直線擬合并作圖、計算相關(guān)系數(shù)。 取前述4只實驗大鼠(1只為死前對照組，3只為死后實驗組)，分別于死后12h、24h和72h在大鼠電阻抗測量部位皮下肌肉組織取材、常規(guī)制片、HE染色后，在光鏡下觀察組織形態(tài)學(xué)變化，并與其電阻抗的變化進行對比分析。 【結(jié)果】 1．所有死亡大鼠皮下肌肉組織的電阻抗幅值都呈現(xiàn)出相似的先升后降的變化趨勢，且中、長期的下降部分有一定的線性變化趨勢。死亡初期(PMI=1～4天)電阻抗幅值迅速上升，達峰值后隨著時間的推移，呈線性下降趨勢(PMI=4～15天)，最終趨于平坦。 2．所有死亡大鼠皮下肌肉組織的電阻抗相位角沒有相似的變化規(guī)律，其變化雜亂無章。 3．相對電阻抗可以一定程度上消去不同大鼠由于初值的差異造成的特異性，而且同一溫度組不同大鼠之間的離散性較絕對電阻抗小。 4．溫度越高絕對電阻抗或相對電阻抗上升得越快，下降得亦更快；溫度越低絕對電阻抗或相對電阻抗上升得越緩慢，下降得更緩慢。絕對電阻抗或相對電阻抗下降段斜率亦與溫度的變化呈正相關(guān)關(guān)系。 5．各溫度組下降段斜率與PMI的相關(guān)系數(shù)均大于0.97，表明各溫度組下降段與PMI之間的相關(guān)性很高，因此，有可能利用這一線性關(guān)系推斷中、晚期PMI。 6．大鼠死前核未見改變，肌束橫紋清晰；死后12h，表層少數(shù)肌束腫脹，肌絲多數(shù)呈細顆粒狀或勻質(zhì)狀，橫紋消失；死后72h，較多見肌纖維腫脹、顆粒樣變、橫紋消失。隨著PMI推移逐步產(chǎn)生細胞膜自溶裂解過程，組織的導(dǎo)電性增加，因而表現(xiàn)為電阻抗不斷下降的結(jié)果。 【結(jié)論】 1．大鼠死后皮下肌肉組織電阻抗幅值隨時間的推移呈現(xiàn)先上升后下降的變化趨勢，并且下降部分的電阻抗幅值變化和PMI之間存在線性關(guān)系。在測量環(huán)境相似的條件下，電阻抗變化也相似。 2．環(huán)境溫度是影響電阻抗幅值最主要的參數(shù)，且環(huán)境溫度越高其變化速度越快。因此，電阻抗的測量還要注意其適用的溫度范圍，溫度過高和過低都不適合使用。 3．測量電阻抗幅值時，將絕對電阻抗轉(zhuǎn)換成相對電阻抗，更能反映電阻抗變化的客觀規(guī)律。 4．電阻抗下降段斜率與溫度的變化呈正相關(guān)關(guān)系，且在不同環(huán)境溫度時，下降段斜率與PMI的相關(guān)性都較高。如果找出下降段起點、峰值、斜率這三者之間的確定關(guān)系，就可利用下降段的線性關(guān)系為推斷中晚期PMI提供一定的判斷依據(jù)。 5．由于電阻抗相位角受環(huán)境因素的影響很大，目前還不能用于PMI的推斷。 6．通過對電阻抗變化趨勢產(chǎn)生的機制從組織形態(tài)變化方面進行的初步探討，得出了和細胞生物學(xué)理論解釋相一致的結(jié)果。 7．相對于其它PMI推斷技術(shù)而言，電阻抗法具有測量簡單方便、準確可靠的優(yōu)點，但是鑒于目前的檢測水平和環(huán)境因素的限制，能否應(yīng)用于實際情況，發(fā)展成為一種新型的PMI推斷技術(shù)，還需要做進一步的研究和探討。 第二部分單細胞凝膠電泳檢測大鼠死后腦、骨髓細胞核DNA降解推斷死后間隔時間的研究 【目的】應(yīng)用單細胞凝膠電泳技術(shù)(Single-cell gel electrophoresis，SCGE)直接檢測大鼠死后不同溫度下腦、骨髓細胞核DNA降解的情況，選擇既往研究尚未用過的參數(shù)headDNA％分析不同溫度下DNA降解的程度隨PMI變化的規(guī)律，建立兩者的回歸方程，并與標準指標尾長(TL)、Oliver尾距(TM)進行對比，以期為該方法推斷早期PMI探索新的參數(shù)。 【方法】以頸髓離斷法處死大鼠，保存在溫度分別控制在10℃±1℃或20℃±1℃的實驗環(huán)境下，在大鼠死后0～40h內(nèi)，，每隔4h，分別對腦組織和骨髓取材，并制作單細胞懸液；然后進行單細胞凝膠電泳；在熒光顯微鏡下觀察并拍照。所攝圖像用CASP0.22彗星圖像分析軟件進行檢測，選擇既往研究尚未用過的參數(shù)headDNA％分析不同溫度下DNA降解的程度隨PMI變化的規(guī)律，用SAS8.0和SPSS12.0分析軟件進行統(tǒng)計學(xué)分析，建立兩者的回歸方程，并與標準指標尾長(TL)、Oliver尾距(TM)的分析結(jié)果進行對比。同時，依據(jù)建立的方程和headDNA％估計值95％可信區(qū)間計算公式反推PMI范圍的計算公式。 【結(jié)果】 1．大鼠死后，腦細胞、骨髓細胞在電泳圖上出現(xiàn)了不同程度的彗星形拖尾； 2．大鼠死后早期PMI內(nèi)，不論在10℃還是20℃下，腦、骨髓細胞核HeadDNA％的下降與PMI之間存在著良好的線性關(guān)系，建立了四個線性回歸方程，其確定系數(shù)均較高，可用于早期PMI推斷； 3．死后細胞核HeadDNA％的下降受環(huán)境溫度、組織種類的影響； 4．建立了由已知HeadDNA％值反推PMI范圍的計算公式：如腦組織在20℃下，據(jù)某已知HeadDNA％值反推PMI范圍的計算公式： timel=(86.83-DNA_(已知))／1.9634 time2=(99.27-DNA_(已知))／1.9634 5．選用的參數(shù)HeadDNA％較前期研究慣用的標準指標TL、TM與PMI的線性回歸方程擬合較理想； 6．腦組織、骨髓組織分別用三種參數(shù)與PMI擬合的線性回歸分析，前者的確定系數(shù)較后者高。 【結(jié)論】 1．單細胞凝膠電泳技術(shù)可應(yīng)用與早期PMI的推斷； 2．環(huán)境溫度和組織差異明顯影響死后核DNA的降解； 3．新參數(shù)HeadDNA％較前期研究慣用的標準指標TL、TM推斷PMI的價值更高，但其可靠性有待于今后進一步的研究，尤其是人體材料研究的驗證； 4．由已知HeadDNA％值可反推PMI的范圍； 5．腦組織是較骨髓組織更為理想的備選檢材。
[Abstract]:The postmortem interval time (postmortem interval, PMI) or death time inference (estimation of time since death, ETSD) is one of the key and difficult points in the actual forensic pathology examination and research work. For a long time, scholars at home and abroad have adopted a variety of techniques and methods to try the inference of PMI, especially early PMI inference, such as the use of death. All kinds of postmortem phenomena, muscle excess reaction, concentration of potassium ion in vitreous fluid, biochemical changes in blood after death, but their accuracy is often influenced by various external factors and often has great errors. Up to now, there is no recognized simple practical, accurate and reliable inference method, especially in the middle and late PMI. This study studies the variation of the amplitude and phase angle of the electrical impedance of the subcutaneous muscle tissue after the death of the rat, in order to explore the value of the advanced PMI in the early stage of the bioelectrical impedance inference, and to try to develop a simple, accurate and reliable new method to infer the middle and late PMI, and choose the unused analysis index of the previous study, headDNA%, to apply the single fine. Cell gel electrophoresis was used to detect the rat's dead brain, and the DNA degradation of the nucleus of bone marrow was changed with PMI, and the new parameters of early PMI were deduced by single cell gel electrophoresis.
Part one: inferring postmortem interval from the changes of electrical impedance and phase angle in rat subcutaneous muscle tissue after death
[Objective] to study the changes of the amplitude and phase angle of the electrical impedance of the subcutaneous muscle tissue of the rat after death, and to explore the value of the advanced PMI by bioelectrical impedance technique, and try to develop a simple, accurate and reliable new method to infer the middle and late PMI. The mechanism of the trend.
[Methods] 20 healthy adult rats were divided into 4 experimental groups, which were divided into 4 experimental groups, with 5 rats in each group, corresponding to four different measuring temperatures (9 + 1, 14 + 1, 16 + 1, 19 + 1). The animals were killed by the dislocated cervical vertebra, and the electrodes were inserted into the thigh muscles and fixed. The animals were quickly placed in a thermostat incubator or refrigerator at setting temperature. A four electrode impedance measurement system was used to measure the amplitude and phase angle of the electrical impedance of the subcutaneously muscle tissue of the rat after death. From 0h after death, the electrical impedance data was recorded every 3H (except at night) and the ambient temperature. When the experiment time was long enough (the impedance dropped to about 70% or more of the original impedance), the experiment could be stopped. Then Exce could be stopped. The discrete impedance data obtained by L are simple mean, variance analysis, correlation analysis of impedance data by MATLAB and the drawing of straight line, and the correlation coefficient is calculated.
The 4 experimental rats (1 for pre death control group and 3 for the postmortem group) were obtained after death, 12h, 24h and 72h were harvested subcutaneously in the electrical impedance measurement site of rats. The changes of histomorphology were observed under the light microscope, and the changes of electrical impedance were compared and analyzed after HE staining.
[results]
1. the amplitudes of the electrical impedance of the subcutaneous muscle tissues of all the dead rats showed a similar trend in the first rise and then descending. In the meantime, the long-term descending part had a certain linear trend. The amplitude of the electrical impedance increased rapidly in the early period of death (PMI=1 to 4 days), and then a linear descending trend (PMI=4 to 15 days) with the passage of time. It tends to be flat.
2. the electrical impedance phase angle of subcutaneous muscle tissue of all the dead rats did not change in a similar way.
3. relative electrical impedance can eliminate the specificity of different rats due to the difference of initial values, and the dispersion of the rats in the same temperature group is smaller than that of the absolute impedance.
4. the higher the temperature is, the faster the absolute electrical impedance and relative electrical impedance rise and the faster the decrease. The lower the temperature is, the slower the absolute electrical impedance or relative electrical impedance rises, and the decrease is more slow. The slope of absolute electrical impedance or relative impedance is also positively related to the change of temperature.
5. the correlation coefficient of the slope of each temperature group and the PMI is more than 0.97, indicating that the correlation between the decrease segments of each temperature group and the PMI is very high. Therefore, it is possible to use this linear relationship to deduce the late PMI..
In the 6. rats, the nucleus of the 6. rats was not changed and the transverse lines were clear. After death, a few muscle bundles were swollen. Most of the muscle fibers were fine grainy or homogeneous, and the transverse lines disappeared. After death, the muscle fibers swelled, granulated, and the transverse lines disappeared. As PMI progressively produced the process of cell membrane self dissolution and cracking, the electrical conductivity of tissue increased and thus manifested as 72h. The result of constant decrease in electrical impedance.
[Conclusion]
After the death of the 1. rats, the amplitude of the electrical impedance of the subcutaneous muscles increased first and then decreased with time, and there was a linear relationship between the amplitude of the electrical impedance of the descending part and the PMI. The electrical impedance changes were similar under the condition of similar measurement environment.
2. environment temperature is the most important parameter that affects the amplitude of electrical impedance, and the higher the environment temperature is, the faster the change speed. Therefore, the measurement of electrical impedance should pay attention to its applicable temperature range, both too high temperature and too low are not suitable for use.
3. when measuring the magnitude of the electrical impedance, the absolute impedance will be converted to the relative impedance, which can better reflect the objective law of the change of electrical impedance.
4. the slope of the descending section of electrical impedance is positively correlated with the change of temperature, and the correlation between the slope of the descending section and the PMI is higher at different ambient temperatures. If we find the relationship between the starting point, the peak and the slope of the descending section, the linear relation of the descending section can be used to provide a certain basis for the inference of the late PMI.
5. because electrical impedance phase angle is greatly influenced by environmental factors, it can not be used for PMI inference at present.
6. through the preliminary discussion of the mechanism of the change of electrical impedance from the change of tissue morphology, the result is consistent with the theoretical explanation of cell biology.
7. compared with other PMI inference techniques, electrical impedance method has the advantages of simple and convenient measurement and accurate and reliable. However, in view of the current level of detection and the restriction of environmental factors, it is necessary to study and discuss whether it can be applied to the actual situation and develop into a new type of PMI inference technology.
In the second part, single cell gel electrophoresis was used to detect the postmortem interval of DNA in rats.
[Objective] to use Single-cell gel electrophoresis (SCGE) to directly detect the DNA degradation of brain and bone marrow nuclei at different temperatures after death in rats, and select the unused parameter headDNA% to analyze the regularity of the degree of DNA degradation with PMI at different temperatures at different temperatures and establish the regression equations. It is compared with the standard index tail length (TL) and Oliver trailing distance (TM), in order to infer new parameters for early PMI by this method.
[Methods] the rats were killed by the cervical spinal cord disconnection, and stored in the experimental environment under the temperature of 10, 1 or 20 and 20 C, respectively, in 0 ~ 40H after the death of the rat, every 4h, respectively, of the brain tissue and bone marrow, and the single cell suspension was made, then the single cell gel electrophoresis was carried out, and the photo was observed and photographed under the fluorescence microscope. CASP0.22 comet image analysis software was used to detect the changes of the degree of DNA degradation at different temperatures at different temperatures. The regression equation was established by SAS8.0 and SPSS12.0 analysis software, and the analysis of the tail length of the standard index (TL) and the tail distance of Oliver (TM) was analyzed with the SAS8.0 and SPSS12.0 analysis software. At the same time, the calculation formula of PMI range is deduced from the established equation and the 95% confidence interval formula of headDNA% estimation.
[results]
1. after the death of the rats, the brain cells and bone marrow cells showed different comet shaped tails in the electrophoregram.
In the early postmortem PMI of 2. rats, there was a good linear relationship between the decrease of HeadDNA% in the brain and the nucleus of bone marrow, no matter at 10 or 20 C, and four linear regression equations were established. The coefficients were all high, which could be used for early PMI inference.
3. the decrease of HeadDNA% in nuclei after death was influenced by environmental temperature and tissue types.
4. Establish a formula for estimating the PMI range from the known head DNA% value. For example, the formula for estimating the PMI range from the known head DNA% value in brain tissue at 20 C is established.
Timel= (86.83-DNA_ (known)) / 1.9634
Time2= (99.27-DNA_ (known)) / 1.9634
5. the parameter HeadDNA% selected is better than the standard regression equation TL used in previous studies, and the linear regression equation between TM and PMI.
6. the linear regression analysis of brain tissue and bone marrow tissue was fitted with three parameters and PMI. The former had higher coefficient of determination than the latter.
[Conclusion]
1. single cell gel electrophoresis technique can be used to deduce early PMI.
2. the difference of environmental temperature and tissue significantly affected the degradation of DNA after death.
3. the new parameter HeadDNA% is compared with the standard index TL, which is used in the previous study, and TM deduce that the value of PMI is higher, but its reliability needs to be further studied in the future, especially in the research of human material.
4. the range of PMI can be pushed back by the known HeadDNA% value.
5. brain tissue is an ideal alternative material for bone marrow tissue.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：D919
本文編號：2156427
[Abstract]:The postmortem interval time (postmortem interval, PMI) or death time inference (estimation of time since death, ETSD) is one of the key and difficult points in the actual forensic pathology examination and research work. For a long time, scholars at home and abroad have adopted a variety of techniques and methods to try the inference of PMI, especially early PMI inference, such as the use of death. All kinds of postmortem phenomena, muscle excess reaction, concentration of potassium ion in vitreous fluid, biochemical changes in blood after death, but their accuracy is often influenced by various external factors and often has great errors. Up to now, there is no recognized simple practical, accurate and reliable inference method, especially in the middle and late PMI. This study studies the variation of the amplitude and phase angle of the electrical impedance of the subcutaneous muscle tissue after the death of the rat, in order to explore the value of the advanced PMI in the early stage of the bioelectrical impedance inference, and to try to develop a simple, accurate and reliable new method to infer the middle and late PMI, and choose the unused analysis index of the previous study, headDNA%, to apply the single fine. Cell gel electrophoresis was used to detect the rat's dead brain, and the DNA degradation of the nucleus of bone marrow was changed with PMI, and the new parameters of early PMI were deduced by single cell gel electrophoresis.
Part one: inferring postmortem interval from the changes of electrical impedance and phase angle in rat subcutaneous muscle tissue after death
[Objective] to study the changes of the amplitude and phase angle of the electrical impedance of the subcutaneous muscle tissue of the rat after death, and to explore the value of the advanced PMI by bioelectrical impedance technique, and try to develop a simple, accurate and reliable new method to infer the middle and late PMI. The mechanism of the trend.
[Methods] 20 healthy adult rats were divided into 4 experimental groups, which were divided into 4 experimental groups, with 5 rats in each group, corresponding to four different measuring temperatures (9 + 1, 14 + 1, 16 + 1, 19 + 1). The animals were killed by the dislocated cervical vertebra, and the electrodes were inserted into the thigh muscles and fixed. The animals were quickly placed in a thermostat incubator or refrigerator at setting temperature. A four electrode impedance measurement system was used to measure the amplitude and phase angle of the electrical impedance of the subcutaneously muscle tissue of the rat after death. From 0h after death, the electrical impedance data was recorded every 3H (except at night) and the ambient temperature. When the experiment time was long enough (the impedance dropped to about 70% or more of the original impedance), the experiment could be stopped. Then Exce could be stopped. The discrete impedance data obtained by L are simple mean, variance analysis, correlation analysis of impedance data by MATLAB and the drawing of straight line, and the correlation coefficient is calculated.
The 4 experimental rats (1 for pre death control group and 3 for the postmortem group) were obtained after death, 12h, 24h and 72h were harvested subcutaneously in the electrical impedance measurement site of rats. The changes of histomorphology were observed under the light microscope, and the changes of electrical impedance were compared and analyzed after HE staining.
[results]
1. the amplitudes of the electrical impedance of the subcutaneous muscle tissues of all the dead rats showed a similar trend in the first rise and then descending. In the meantime, the long-term descending part had a certain linear trend. The amplitude of the electrical impedance increased rapidly in the early period of death (PMI=1 to 4 days), and then a linear descending trend (PMI=4 to 15 days) with the passage of time. It tends to be flat.
2. the electrical impedance phase angle of subcutaneous muscle tissue of all the dead rats did not change in a similar way.
3. relative electrical impedance can eliminate the specificity of different rats due to the difference of initial values, and the dispersion of the rats in the same temperature group is smaller than that of the absolute impedance.
4. the higher the temperature is, the faster the absolute electrical impedance and relative electrical impedance rise and the faster the decrease. The lower the temperature is, the slower the absolute electrical impedance or relative electrical impedance rises, and the decrease is more slow. The slope of absolute electrical impedance or relative impedance is also positively related to the change of temperature.
5. the correlation coefficient of the slope of each temperature group and the PMI is more than 0.97, indicating that the correlation between the decrease segments of each temperature group and the PMI is very high. Therefore, it is possible to use this linear relationship to deduce the late PMI..
In the 6. rats, the nucleus of the 6. rats was not changed and the transverse lines were clear. After death, a few muscle bundles were swollen. Most of the muscle fibers were fine grainy or homogeneous, and the transverse lines disappeared. After death, the muscle fibers swelled, granulated, and the transverse lines disappeared. As PMI progressively produced the process of cell membrane self dissolution and cracking, the electrical conductivity of tissue increased and thus manifested as 72h. The result of constant decrease in electrical impedance.
[Conclusion]
After the death of the 1. rats, the amplitude of the electrical impedance of the subcutaneous muscles increased first and then decreased with time, and there was a linear relationship between the amplitude of the electrical impedance of the descending part and the PMI. The electrical impedance changes were similar under the condition of similar measurement environment.
2. environment temperature is the most important parameter that affects the amplitude of electrical impedance, and the higher the environment temperature is, the faster the change speed. Therefore, the measurement of electrical impedance should pay attention to its applicable temperature range, both too high temperature and too low are not suitable for use.
3. when measuring the magnitude of the electrical impedance, the absolute impedance will be converted to the relative impedance, which can better reflect the objective law of the change of electrical impedance.
4. the slope of the descending section of electrical impedance is positively correlated with the change of temperature, and the correlation between the slope of the descending section and the PMI is higher at different ambient temperatures. If we find the relationship between the starting point, the peak and the slope of the descending section, the linear relation of the descending section can be used to provide a certain basis for the inference of the late PMI.
5. because electrical impedance phase angle is greatly influenced by environmental factors, it can not be used for PMI inference at present.
6. through the preliminary discussion of the mechanism of the change of electrical impedance from the change of tissue morphology, the result is consistent with the theoretical explanation of cell biology.
7. compared with other PMI inference techniques, electrical impedance method has the advantages of simple and convenient measurement and accurate and reliable. However, in view of the current level of detection and the restriction of environmental factors, it is necessary to study and discuss whether it can be applied to the actual situation and develop into a new type of PMI inference technology.
In the second part, single cell gel electrophoresis was used to detect the postmortem interval of DNA in rats.
[Objective] to use Single-cell gel electrophoresis (SCGE) to directly detect the DNA degradation of brain and bone marrow nuclei at different temperatures after death in rats, and select the unused parameter headDNA% to analyze the regularity of the degree of DNA degradation with PMI at different temperatures at different temperatures and establish the regression equations. It is compared with the standard index tail length (TL) and Oliver trailing distance (TM), in order to infer new parameters for early PMI by this method.
[Methods] the rats were killed by the cervical spinal cord disconnection, and stored in the experimental environment under the temperature of 10, 1 or 20 and 20 C, respectively, in 0 ~ 40H after the death of the rat, every 4h, respectively, of the brain tissue and bone marrow, and the single cell suspension was made, then the single cell gel electrophoresis was carried out, and the photo was observed and photographed under the fluorescence microscope. CASP0.22 comet image analysis software was used to detect the changes of the degree of DNA degradation at different temperatures at different temperatures. The regression equation was established by SAS8.0 and SPSS12.0 analysis software, and the analysis of the tail length of the standard index (TL) and the tail distance of Oliver (TM) was analyzed with the SAS8.0 and SPSS12.0 analysis software. At the same time, the calculation formula of PMI range is deduced from the established equation and the 95% confidence interval formula of headDNA% estimation.
[results]
1. after the death of the rats, the brain cells and bone marrow cells showed different comet shaped tails in the electrophoregram.
In the early postmortem PMI of 2. rats, there was a good linear relationship between the decrease of HeadDNA% in the brain and the nucleus of bone marrow, no matter at 10 or 20 C, and four linear regression equations were established. The coefficients were all high, which could be used for early PMI inference.
3. the decrease of HeadDNA% in nuclei after death was influenced by environmental temperature and tissue types.
4. Establish a formula for estimating the PMI range from the known head DNA% value. For example, the formula for estimating the PMI range from the known head DNA% value in brain tissue at 20 C is established.
Timel= (86.83-DNA_ (known)) / 1.9634
Time2= (99.27-DNA_ (known)) / 1.9634
5. the parameter HeadDNA% selected is better than the standard regression equation TL used in previous studies, and the linear regression equation between TM and PMI.
6. the linear regression analysis of brain tissue and bone marrow tissue was fitted with three parameters and PMI. The former had higher coefficient of determination than the latter.
[Conclusion]
1. single cell gel electrophoresis technique can be used to deduce early PMI.
2. the difference of environmental temperature and tissue significantly affected the degradation of DNA after death.
3. the new parameter HeadDNA% is compared with the standard index TL, which is used in the previous study, and TM deduce that the value of PMI is higher, but its reliability needs to be further studied in the future, especially in the research of human material.
4. the range of PMI can be pushed back by the known HeadDNA% value.
5. brain tissue is an ideal alternative material for bone marrow tissue.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：D919
【引證文獻】
相關(guān)期刊論文 前1條
1 文星;梁志宏;張根偉;黃嵐;王忠義;;基于穩(wěn)態(tài)空間分辨光譜的豬肉新鮮度檢測方法[J];農(nóng)業(yè)工程學(xué)報;2010年09期
本文編號：2156427
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