皮肤、肌肉端粒DNA推断年龄的研究
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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。
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.
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。
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.
引文
1 张继宗，闵建雄，陈世贤，等．男性青少年X线片的骨骺特征及年龄推断[J]。中国法医学杂志，2001，16(2)：91
2 Tanner JM, Gibbons RD. A computerized image analysis system for estimating Tanner-Whitehouse 2 bone age[J]. Horm Res, 1994, 42(6): 282
3 宋宏伟，贾静涛．根据维吾尔族牙磨耗度推断年龄的研究[J]，中国法医学杂志，2000，15(1)：27
4 陈世贤．法医人类学[M]．北京：人民卫生出版社，1998．20
5 Harley CB, Futcher AB, Greider CW. Telomeres shorten during aging of human fibroblasts [J]. Nature, 1990, 345(62740): 458
6 Hastie ND, Dempster M, Dunlop MG, et al. Telomere reduction in human colorectal carcinoma with aging [J]. Nature, 1990,346(6287): 866
7 Lindsey J, McGill-NI, Lindsey-LA, et al. In vivo loss of telomeric repeats with age in humans[J]. Mutat Res, 1991, 256:45
8 Decary S, Mouly, Hamida CB, et al. Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy [J]. Hum Gene Ther, 1997, 8(12): 1429
9 Bodnar AG, Kim NW, Effros RB, et al. Mechanism of telomerase induction during T cell activation [J]. Exp cell Res, 1996, 228:58
10 许丽艳，李恩民，沈忠英．非同位素标记检测人染色体端粒长度[J]．癌变·畸变·突变[J]，2001，13(1)：1
11 F．奥斯伯，R．布伦特等著，颜子颖等译．精编分子生物学试验指南[M]．北京：科学出版社(第一版)，2001．341
12 Blackburn EH, Gall JG. A tandemly repented sequence at the termini of
the extra chromosomal ribosomal RNA Genes in Tetrahymena[J]. J Mol Bio, 1978, 120: 33
13 Zakian VA. Telomeres, Beginning to understand the end[J]. Science, 1995, 270:1601
14 Blackburn EH. Structure and function of telomeres[J]. Nature, 1991, 350:569
15 Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence (TTAGGG)n Present at the telomeres of human chromosomes [J]. Proc Natl Acad Sci USA, 1988,85:6622
16 Allshire RC, Gosden JR, Cross SH, et al. Telomeric repeat from T thermophila cross hybridizes with human telomeres [J]. Nature, 1988, 332(6165): 656
17 任建国，周军，戴尧仁．端粒及端粒酶的研究进展[J]．生物化学与生物物理进展，1999，26(5)：415
18 Allsopp BC, Vaziri H, Patterson C, et al. Telomere length predicts replicative capacity of human fibroblasts [J]. Proc Natl Acad Sci USA, 1992, 89:10114
19 杨仕明，房殿春，罗元辉，等．不同年龄供体正常粘膜端粒状念分析[J]．第三军医大学学报，1999，21(2)：126
20 Melk A, Ramassar V, Helms LM. Telomere shortening in kidneys with age[J]. J AM Soc Nephrol, 2000,11(3):444
21 Aikata H, Takaishi H, Kawakami Y, et al. Telomere reduction in human liver tissues with age and chronic inflammation [J]. Exp Cell Res, 2000, 256(2): 578
22 Jackie BT, Andrew PK, Fiona W, et al. Telomere length shortening in
chronic myelogenous leukemia is associated with reduced time to accelerated phase [J]. Blood, 2000,96(1): 358
23 Yang L, Suwa T, Wright WE, et al. Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells [J]. Mech Ageing Dev, 2001, 122(15): 1685
24 Jane DR, Rosemary EG, Robert FW, et al. Dynamics of telomere shortening in neutrophils and T Lymphocytes during ageing and the ralationship to skewed X chromosome inactivation patterns [J]. British Journal of Haematology, 2000, 109:272
25 Nakamura TM, Cech TR. Reversing time, Origin of telomerase[J]. Cell, 1998,92(5): 587
26 Bodnar AG, Ouellette M, Frolkis M, et al. Extension of lifespan by introduction of telomerase into normal human cells [J]. Science, 1998, 279(5349): 349
27 Aviv A, Aviv H. Telomeres,hidden mosaicism,loss of heterozygosity, and complex genetic traits [J]. Hum Genet, 1998,103(1):2
28 Harley CB, Vaziri H, Counter CM, et al. The telomere hypothesis of cellular aging [J]. Exp Geronatol,1992, 27:375
29 葛璐璐，刘超，王穗保．端粒DNA与年龄相关性的研究进展[J]．中国法医学杂志，2001，16(2)：121
30 张黎明，贺茜，陈志龙．端粒、端粒酶与细胞寿命及癌症的关系[J]．自然杂志，2001，23(3)：139
31 王镜岩，朱圣庾，徐长法．生物化学[M]．北京：高等教育出版社(第三版)，2002．426
32 杨庆恩．DNA在法庭科学中的应用[M]．北京：中国人民公安大学出
版社，1994．39
33 李玉林．分子病理学[M]．北京：人民卫生出版社，2002．287
34 Blackburn EH. Switching and Signaling at the Telomere[J]. Cell, 2001,106:661
35 王曦．端粒酶及其在肿瘤诊治方面的研究[J]．国外医学肿瘤学分册，1998，25(2)：80
36 Robert PL, Jose BC, Catherine B, et al. Extension of Cell Life-Span and Telomere Length in Animals Cloned from Senescent Somatic Cells[J]. Science, 2000, 288:665
37 卢圣栋．现代分子生物学实验技术[M]．北京：中国协和医科大学出版社(第二版)，1999．195
38 王怀安，顾明，林准．中华人民共和国民法通则[S]．中华人民共和国法律全书(2)，长春：吉林人民出版社，1999．127
39 王怀安，顾明，林准．中华人民共和国刑法[S]．中华人民共和国法律全书(1)，长春：吉林人民出版社，1999．58
2 Tanner JM, Gibbons RD. A computerized image analysis system for estimating Tanner-Whitehouse 2 bone age[J]. Horm Res, 1994, 42(6): 282
3 宋宏伟，贾静涛．根据维吾尔族牙磨耗度推断年龄的研究[J]，中国法医学杂志，2000，15(1)：27
4 陈世贤．法医人类学[M]．北京：人民卫生出版社，1998．20
5 Harley CB, Futcher AB, Greider CW. Telomeres shorten during aging of human fibroblasts [J]. Nature, 1990, 345(62740): 458
6 Hastie ND, Dempster M, Dunlop MG, et al. Telomere reduction in human colorectal carcinoma with aging [J]. Nature, 1990,346(6287): 866
7 Lindsey J, McGill-NI, Lindsey-LA, et al. In vivo loss of telomeric repeats with age in humans[J]. Mutat Res, 1991, 256:45
8 Decary S, Mouly, Hamida CB, et al. Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy [J]. Hum Gene Ther, 1997, 8(12): 1429
9 Bodnar AG, Kim NW, Effros RB, et al. Mechanism of telomerase induction during T cell activation [J]. Exp cell Res, 1996, 228:58
10 许丽艳，李恩民，沈忠英．非同位素标记检测人染色体端粒长度[J]．癌变·畸变·突变[J]，2001，13(1)：1
11 F．奥斯伯，R．布伦特等著，颜子颖等译．精编分子生物学试验指南[M]．北京：科学出版社(第一版)，2001．341
12 Blackburn EH, Gall JG. A tandemly repented sequence at the termini of
the extra chromosomal ribosomal RNA Genes in Tetrahymena[J]. J Mol Bio, 1978, 120: 33
13 Zakian VA. Telomeres, Beginning to understand the end[J]. Science, 1995, 270:1601
14 Blackburn EH. Structure and function of telomeres[J]. Nature, 1991, 350:569
15 Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence (TTAGGG)n Present at the telomeres of human chromosomes [J]. Proc Natl Acad Sci USA, 1988,85:6622
16 Allshire RC, Gosden JR, Cross SH, et al. Telomeric repeat from T thermophila cross hybridizes with human telomeres [J]. Nature, 1988, 332(6165): 656
17 任建国，周军，戴尧仁．端粒及端粒酶的研究进展[J]．生物化学与生物物理进展，1999，26(5)：415
18 Allsopp BC, Vaziri H, Patterson C, et al. Telomere length predicts replicative capacity of human fibroblasts [J]. Proc Natl Acad Sci USA, 1992, 89:10114
19 杨仕明，房殿春，罗元辉，等．不同年龄供体正常粘膜端粒状念分析[J]．第三军医大学学报，1999，21(2)：126
20 Melk A, Ramassar V, Helms LM. Telomere shortening in kidneys with age[J]. J AM Soc Nephrol, 2000,11(3):444
21 Aikata H, Takaishi H, Kawakami Y, et al. Telomere reduction in human liver tissues with age and chronic inflammation [J]. Exp Cell Res, 2000, 256(2): 578
22 Jackie BT, Andrew PK, Fiona W, et al. Telomere length shortening in
chronic myelogenous leukemia is associated with reduced time to accelerated phase [J]. Blood, 2000,96(1): 358
23 Yang L, Suwa T, Wright WE, et al. Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells [J]. Mech Ageing Dev, 2001, 122(15): 1685
24 Jane DR, Rosemary EG, Robert FW, et al. Dynamics of telomere shortening in neutrophils and T Lymphocytes during ageing and the ralationship to skewed X chromosome inactivation patterns [J]. British Journal of Haematology, 2000, 109:272
25 Nakamura TM, Cech TR. Reversing time, Origin of telomerase[J]. Cell, 1998,92(5): 587
26 Bodnar AG, Ouellette M, Frolkis M, et al. Extension of lifespan by introduction of telomerase into normal human cells [J]. Science, 1998, 279(5349): 349
27 Aviv A, Aviv H. Telomeres,hidden mosaicism,loss of heterozygosity, and complex genetic traits [J]. Hum Genet, 1998,103(1):2
28 Harley CB, Vaziri H, Counter CM, et al. The telomere hypothesis of cellular aging [J]. Exp Geronatol,1992, 27:375
29 葛璐璐，刘超，王穗保．端粒DNA与年龄相关性的研究进展[J]．中国法医学杂志，2001，16(2)：121
30 张黎明，贺茜，陈志龙．端粒、端粒酶与细胞寿命及癌症的关系[J]．自然杂志，2001，23(3)：139
31 王镜岩，朱圣庾，徐长法．生物化学[M]．北京：高等教育出版社(第三版)，2002．426
32 杨庆恩．DNA在法庭科学中的应用[M]．北京：中国人民公安大学出
版社，1994．39
33 李玉林．分子病理学[M]．北京：人民卫生出版社，2002．287
34 Blackburn EH. Switching and Signaling at the Telomere[J]. Cell, 2001,106:661
35 王曦．端粒酶及其在肿瘤诊治方面的研究[J]．国外医学肿瘤学分册，1998，25(2)：80
36 Robert PL, Jose BC, Catherine B, et al. Extension of Cell Life-Span and Telomere Length in Animals Cloned from Senescent Somatic Cells[J]. Science, 2000, 288:665
37 卢圣栋．现代分子生物学实验技术[M]．北京：中国协和医科大学出版社(第二版)，1999．195
38 王怀安，顾明，林准．中华人民共和国民法通则[S]．中华人民共和国法律全书(2)，长春：吉林人民出版社，1999．127
39 王怀安，顾明，林准．中华人民共和国刑法[S]．中华人民共和国法律全书(1)，长春：吉林人民出版社，1999．58
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