【作者】 王祖峰；

【导师】 王英元； 高彩荣；

【作者基本信息】 山西医科大学 ， 法医病理学， 2003， 硕士

【摘要】 目的 应用Southern杂交技术，对皮肤、肌肉端粒DNA片断长度(mean length of telomere restriction fragment，简称：TRF)进行观察，测定不同年龄段人群的端粒DNA片断长度值，以期初步探明不同年龄段人群的端粒DNA片断长度的变化规律，绘制出端粒DNA片断长度值随年龄变化的标准曲线，比较性别、籍贯对端粒DNA片断长度的影响，以期为法医实践工作中对无名尸体年龄推断提供理论依据。 方法 采用Southern杂交的方法。随机取60名无关个体的皮肤、71名无关个体的肌肉，用蛋白酶K消化样本，酚、氯仿法抽提出DNA，测出DNA的浓度，用地高辛标记探针并确定探针最适工作浓度，Hinf Ⅰ酶消化DNA，琼脂糖凝焦电泳分离DNA片断后，行真空转膜，洗膜，进行Southern杂交，BCIP／NBT法染色显色后，进行图像分析，得出不同年龄段供体的端粒DNA片断长度值。 结果 对皮肤、肌肉组织端粒DNA片断长度值与年龄的依存关系进行回归分析，得到的回归系数分别为：b_皮=53.302、b_肌=54.147；回归方程模型为： Y皮=53.302·l／X+6.069 Y肌=54.147·l／X+6.047 [Y：代表年龄(岁)；X：代表端粒DNA片断长度值(Kb)] 用得到的端粒DNA片断长度值，检验性别、籍贯对皮肤、肌肉的TRF值的影响关系，发现：(1)在不同性别间，端粒DNA片断长度值有显著性差别(p＜0.05)，同年龄组女性较男性的TRF值大。(2)对比山西和北京两地人群的TRF值，未发现显著性差别(P＞0.05)。(3)比较同年龄组的皮肤和肌肉之间的TRF值，也未发现显著性差别(P＞0.05)。 11}西医科丸学2()()3届硕1学位论文 结论皮肤、肌肉的端粒DNA片断长度随着年龄的增加而逐渐缩短，其随年龄缩短的速率在各个年龄段不同，青少年时期缩短的速率较快，成年后缩短速率相对减慢。在不同性别之间，1司年龄组女性较男胜的端粒D肋片断长度值大。对比同年龄组d_l西人群和北京人群的端粒I)NA片断一长度，没有发现显著性差别。皮肤和肌肉两种组织的端粒DN八片一断用工年龄推断时，没有优劣之分。更多还原

【Abstract】 Objective Through measure telomere length ( mean length of telomere restriction fragment, TRF) of dermal and intramuscular , and study it’s length correlating with the different human ’ s age. The TRF was examined by Southern blotting. The formula to age estimating was obtained by regression analysis between the TRF and the age. Compared different effecting of gender and territory to estimate age of human. Using of our method can give a rough estimate of age of subject in forensic samples that carry no morphological information.Methods DNA was extracted from sample of intramuscular and dermal, using proteinase K digest the sample, each sample was extracted twice with the phenol/chloroform, the oligonucleotide (CCCTAA)3 probe was labeled with digoxin(DlG) and decided it’s best working concentration, DNA was digested with the restriction enzyme Hinf 1 , then DNA fragment was separated by agrose gel electrophoresis and transferred to a nylon membrane by vacuum blotter, the membrane was hybridized with the telomeric probe, stained with BCIP/NBT, analyzed by using a densitometric imaging system and mean length of TRF calculated.Results The relation of TRF and age was anal yzed by regression analysis, and the regression equation can be included in the following formula:Y(1)=53.302.1/X+6.069 Y(2)=54.147.1/X+6.047[Y: age (year),X: TRF(kb). (1) dermal, (2) intramuscular.] T-test result: There is a significant difference existed (p<0.05) in different gender, the TRF of female is longer than that of male. To the different territory, TRF is not a significant difference (P>0. 05) between the human of SHANXI and that of BEIJING. There is not a significant difference (P>0. 05) for estimating of age between the TRF of intramuscular and that of dermal.Conclusion The gradual shortening of TRF in dermal and intramuscular cells of human has a obvious correlation with the age, it has a fast shorten in the adolescent, and then it has a relative slower shorten in the adult. The TRF of female is longer than that of male to the same age. To the human of SHANGXI and BFIJING, there is not an obvious difference to the TRF. When the TRF of intramuscular is used for estimating age of human, the result is as good as that of dermal.更多还原