The Study On Relationship Between Degradation Of Retinal Nucleic Acid And PMI In Rat After Death

Part â… :Image analysis for degradation of DNA in retinal nuclei of rat after death[Background]The estimation of Postmortem interval (PMI), especially the early elapsed time since death has been one of the difficult fields in forensic pathology. Accurate estimation for PMI could provide clues for criminal investigation and evidences for trial or judicial proceedings. Number of previous studies for PMI focused on the postmortem phenomenon. But the methods of observation and inspection for postmortem phenomenon were affected larger by external environment factors and the subjective experience of observers; they can only estimate the rough scope of PMI. Enzymohistochemistry and postmortem chemistry, especially the change of Vitrea potassium since death, also were the hot spots of forensic pathology studies. As reported in the study, the different environmental temperature, testing methods led to the larger difference of the results. So there are certain academic controversies about the value of these methods in the practice of forensic medicine. Thus far, there is no recognized accurate and reliable method for the estimation of PMI. In recent years, along with the development of molecular biology and image analysis technology, domestic and foreign researchers has already begun to study the change of nuclear DNA since death and use it for research of estimation of PMI.Somatic cell nucleus DNA is the genetic material with remarkable biochemical stability. For the same species, the content of nuclear DNA in different individuals and different organizations was on the same average level. Sine death, various factors may damage the structure of cell wall and produced damage to the cytolysosomes in cytoplasm, lysomal enzymes were released and activated, lead to cell-digesting, structural collapse. Degradation of cell nucleus DNA also gradually took place. So after dying, the number of cell nucleus DNA will reduce until to disappear completely.Domestic and foreign scholars had done research for degradation of DNA since death using Flow cytometric analysis; found that after dying, the content of DNA gradually reduced with the extension of PMI. But because of expensive equipment and higher laboratory techniques required, fewer morphological parameters, flow cytometry was not easily used extensively for estimation of PMI. Along with the rapid development and the wide use of image analysis and computer technology, precision of image analysis techniques (IAT) has reached a maximum level of Flow cytometric analysis for quantitative analysis. Domestic and foreign scholars had applied image analysis systems to study degradation of cellular nuclear DNA since death; the results were consistent with the result of Flow cytometric analysis system. As a result of the impact of internal and external environment, the rule of DNA degradation was different in different tissue. Use of the traditional organizations which were little affected by environment could enhance the reliability of the results of research for the relevance between DNA degradation and PMI. Retina in the relatively airtight eyeball, is affected slightly the external environment factors. Therefore, the use of IAT analysis for degradation of retinal nuclear DNA will be possible to infer PMI.[Purposes] On the basis of early experimental research, this experiment is designed to use the Compute Image Analyze Technique(CIAT) to detect degradation of retinal cellular nuclear DNA sine death and analysis the relationship between degradation of retinal nuclear DNA and the time since death.[Methods] 90 healthy adult SD rats, only ordinary class, female, weighting 250g+10g, according to different PMI, randomly divided into 15 groups (one group each 2h after dying), the temperature 20 degrees. Every two hours within 28h since death, Withdraw the rat retina as experimental object, to make cytology smear, fixed with cold Carnoy fixation, stained by Feulgen staining. Then to measure three parameters of DNA by the color image analyze instrument, which are Integral Optical Density (IOD), Average Optical Density (AOD), and Index of Density (ID). The data was analyzed with software of Excel 2003 and SPSS 12.0 software for linear regression statistical analysis.[Results] the average optical density and the integral optical density decline along with the time since death, the index of density reflects an upward trend. To better reflect the relevance of retina cell nucleus DNA content and PMI, a simple linear regression model about PMI and every experimental parameter was established. Within 28h since death the regression equations as follows: (1) Within 28h since death linear fitting regression equation about the relationship between IOD and PMI:Y_IOD = -0.097 X_IOD +18.903 (R2=0.968)(2) Within 28h since death linear fitting regression equation about the relationship between AOD and PMI:Y_AOD =-0.009 X_AOD+0.590 (R2=0.949)(3) Within 28h since death linear fitting regression equation about the relationship between IOD and PMI:Y_ID =0.122X_ID +2.246 (R2 = 0.951)The determination coefficient R² of three regression equations is greater than 0.9, which indicated that the linear fitting degree of three regression equations about the relationship between PMI and DNA degradation is better. The return model has the remarkable statistics significance after the variance examination (P<0.001). There is linear correlation relation between degradation of DNA and PMI. The determination coefficient R² of regression equation about the relationship between AOD and PMI is the biggest, which indicated that the correlation dependence between PMI and AOD is the best one of three.[Conclusions] (1) The first experiment using 1AT study retina cell nucleus DNA degradation after dying conclude that the content of retina cell nucleus DNA degraded gradually with the extension of the time since death and the degradation of DNA has good relevance with PMI. Application of computer image analysis techniques for measuring the change of DNA content since death will become an accurate, objective method of estimation of PMI.(2) The linear fitting degree of three regression equations about the relationship between PMI and retinal nuclear DNA degradation is better. The degradation of retinal nuclear DNA was little affected by environmental factors in the death 28h. Retina organization is the ideal experimental subject of research for the early death time.(3) Index of density, average optical density and integral optical density are the good parameters of IAT used for research the relevance between the cell nucleus DNA degradation and post mortem interval.(4) IAT is an objective, effective experimental method for measurement of DNA content since death. The linear relationship between the three parameters of IAT and PMI reflect the good relevance between retina cell nucleus DNA content and PMI within 28h since death, and its reliability need to be certificated in human body or in practice further in the future. If conjoined with other assessment methods in forensic practice, it can infer the time since death more accurately. Part â…¡Compound fluorescence RT-PCR technique measure mRNA degradation in retina of rat after death[Background] Along with the development of molecular biology and the renewal of examination method, time dependent nucleic acid degradation gradually become the focus of forensic attention. Somatic cell nucleus DNA is the genetic material with remarkable biochemical stability, and it is stable in a longer time after dying. The domestic and foreign forensic medicine scholars utilizing the different method have done the massive research to the relation of cell nucleus DNA degradation in different organizations and postmortem interval (PMI), and thought DNA degradation of the organism cell nucleus has relevance with PMI after death. Compared to DNA, the general view is that in the living and dead body mRNA will rapidly degrade as a result of the active role of prevail RNA enzymes. However, many studies reported that in certain organism organization organs, such as the brain organization, RNA has the quite high stability, may in a longer time not degrade after dying. The relative stability of the mRNA makes it possible to study the relationship of mRNA degradation after dying and PMI. Only a few literatures reported the relation of mRNA degradation and PMI, thinking that degradation of the organism cell mRNA also has relevance with PMI and using appropriate methods to exam mRNA degradation is the important premise for studying the relationship of mRNA degradation and PMI.[Purpose] To measure quantitatively degradation of retinal cell mRNA in rat after dying, and to further study relationship between mRNA degradation and PMI, providing the basic research for using the mRNA degradation to infer PMI.[Method] 60 healthy adult SD rats, only ordinary class, female, weighting 250g+10g, according to different PMI, randomly divided into 15 groups (one group each 2h after dying). Withdraw the rat retinas of 0²8h groups as a experimental object. Trizol withdraw method organizations extracts total RNA of organization sample, the spectrophotometric method surveys the absorbance value of total RNA to attract the luminosity value, housekeeping genes, β-actin, Pgk1, Rpl4 mRNA, were reverse transcribed and amplified by one-stage process compound fluorescence RT-PCR system. Capillary electrophoresis and fluorescence detection analysis for the PCR products was done by ABI 3,100 DNA auto analyzer, GeneMapper v3.2 software survey the result and output the data automatically. The utilization of regression analysis and the variance examination using SPSS 12.0 statistic software carried on to analysis the relation of each group of measurement data and the PMI, comparing with degeneration degree of three mRNA along with the PMI. Degradation degree (result of Part â… ) of DNA and mRNA after dying was also compared to infer the reliability of relation between mRNA degradation and PMI.[Result] Within 28h after dies, absorbance value of total RNA reduced along with the extension of PMI. RT-PCR products of Housekeeping genes, β-actin, Pgk1, and Rpl4 mRNA decreased along with the extension of PMI, and there degradation had the similar degree with the total RNA degeneration. 28h after dying three were only few RT-PCR products of mRNA. Within 28h after dying, the equation of linear regression fitting the relationship between PMI and mRNA degradation as follows:(1) Linear fitting regression equation about the relationship between PMI and beta -actin gene mRNA:Y_β-actin=-4436.205X_β-actin+127581.7 (R2=0.976)(2) Linear fitting regression equation about the relationship between PMI and pgkl gene mRNA:Y_pgk1 =-1993.884X_pgk1+57651.54 (R2=0.973)(3) Linear fitting regression equation about the relationship between PMI and rpl4 gene mRNA:Y_rpl4=-1189.791X_rpl4+34533.46 (R2=0.955)The determination coefficient R² of three regression equations is greater than 0.9, which indicated that the Linear fitting degree of tree regression equations about the relationship between PMI and mRNA degradation is better. The return model has the remarkable statistics significance after the variance examination (P<0.001). There is linear correlation relation between degradation of mRNA and PMI. Compared with mRNA degeneration and PMI existence linear correlation relations, their determination coefficients are in the high level, the correlation dependence between PMI and mRNA degradation even must surpass with the correlation dependence between PMI with degradation.[Conclusion] (1) For the first time, The experiment using compound fluorescence RT-PCR and capillary electrophoresis analysis technique researched the degradation of retinal cell mRNA within 28 hours after rat dying, and concludes that after dying retinal cell mRNA will degrade gradually along with the extension of PMI and has good relevance with PMI. Application of appropriate quantitative methods to measure somatic cell mRNA content after dying may be a new direction of PMI inference research.(2) The retina locating in the relatively airtight eyeball, is affected slightly the external environment factor, degeneration of mRNA in 28h after dying and PMI has better linear relation. Compared with other organization, the degradation speed of retinal mRNA is quicker. Thus the retina organization is good selection for studying the relationship between the degeneration of mRNA and the early death time.(3) Three housekeeping genes, β-actin, Pgk1 and Rpl4 mRNA, have the consistent speed of degradation, which is also similar with the total RNA degeneration speed. The result prompts housekeeping gene mRNAs possibly have the similar rule of degradation after dying.(4) Within 28h after dying, the linear return model fitting the relationship of the degradation of three housekeeping gene mRNAs and PMI is good comparatively, which prompts that the degradation of housekeeping gene mRNAs may be made to infer the early death time.(5) The better linear relation between PMI and the degradation of mRNA in retina, the reliability of which need to be confirmed further more in other animal tissues, human body, before it be applied for forensic examination. (6) The linear relation mRNA degradation is similar to the linear relation DNA degradation. The mRNA degradation to infer PMI has the reliability. Jointly applies degradation of DNA and RNA for the inference of PMI will be able to have the very great degree to enhance the result the reliability and the precision.