利用微卫星标记对中国恒河猴MHC基因遗传同质性分群的研究
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摘要
恒河猴是医学生物学研究中重要的非人灵长类模式动物,广泛应用于传染性疾病模型制作、疫苗开发、干细胞研究以及器官移植,尤其在模拟艾滋病感染人体的实验中,是不可代替的猴艾滋病模型。由于恒河猴地理分布广泛,而且驯化程度有限、无法建立近交系,所以在同一个实验分组中参与实验的恒河猴个体往往是遗传背景异质性,但是医学实验中需要参与实验的动物尽量保持遗传背景的同质性从而使实验结果便于分析,这样的客观矛盾导致实验中往往会出现个体差异。
相同地理来源的恒河猴往往由于地理隔离,个体差异比较小,而主要组织相容性复合物分子是免疫遗传学中最重要的基因,所以选取相同地域来源和相同主要组织相容性复合物分子基因型的恒河猴实验动物参与实验将有利于缩小个体差异。
本文试图根据已知地域性微卫星遗传标记与特定MHC基因高度连锁的微卫星遗传标记,建立一套恒河猴免疫遗传学同质性分类方法。实验结果表明,中国恒河猴的遗传背景复杂,地理来源遗传标记需要进一步完善。特定MHC基因高度连锁的微卫星遗传标记则可以替代连锁的MHC基因,将恒河猴区分为若干个不同的群体。
本方法的建立和进一步完善,在对中国恒河猴遗传背景缺乏详细了解的情况下,将为中国恒河猴参与实验分组提供参考,也可以指导恒河猴繁育管理,长远可用于对相对封闭地区中国恒河猴进行遗传调查并逐步建立遗传同质性群体。
Macaca mulatta is an important non-human primate model in biomedical research. They are widely used in numbers of infectious diseases, vaccine development, stem cell research, organ transplants and especially as an essential AIDS model in a simulated human body of HIV infection experiments. Due to its wide geographical distribution and domestication of a limited degree, it is very difficult to establish the inbred strains. Researcher made great efforts to maintain the homogeneity of genetic background of experimental results to decrease the results error in medical experiments. However, macaca mulatta participated in the same experiment are almost with heterogeneous genetic background. So, the individual differences results in the actual experimental more complicated.
Macaca mulatta with the same geographical origin are close in genetic relationship and relatively small individual differences. The major histocompatibility complex molecular (MHC) genes are the most important immunogenetic genes. So animals with the same geographical origin and the same MHC genotype will be beneficial to reduce individual differences in the experiment.
In this paper, we established a macaca mulatta immunogenetic homogeneity classification based on the known regional micro-satellite genetic markers and micro-satellite linked definitude MHC genes. Our results indicated that the genetic background of Chinese macaca mulatta was complicated. So the genetic marker of demographic origin should be further studied. We could replace the linked MHC genes with micro-satellite linked definitude MHC genes to divide the animals into numbers of subgroups.
As the lack of detailed understanding about the genetic background of Chinese macaca mulatta, our methods will provide the basic research reference for the macaca mulatta medical experiments and the management for the animals' propagation. It could be applied in genetic investigation on macaca mulatta from closed region to established the homogeneous genetic background subgroups in the future.
相同地理来源的恒河猴往往由于地理隔离,个体差异比较小,而主要组织相容性复合物分子是免疫遗传学中最重要的基因,所以选取相同地域来源和相同主要组织相容性复合物分子基因型的恒河猴实验动物参与实验将有利于缩小个体差异。
本文试图根据已知地域性微卫星遗传标记与特定MHC基因高度连锁的微卫星遗传标记,建立一套恒河猴免疫遗传学同质性分类方法。实验结果表明,中国恒河猴的遗传背景复杂,地理来源遗传标记需要进一步完善。特定MHC基因高度连锁的微卫星遗传标记则可以替代连锁的MHC基因,将恒河猴区分为若干个不同的群体。
本方法的建立和进一步完善,在对中国恒河猴遗传背景缺乏详细了解的情况下,将为中国恒河猴参与实验分组提供参考,也可以指导恒河猴繁育管理,长远可用于对相对封闭地区中国恒河猴进行遗传调查并逐步建立遗传同质性群体。
Macaca mulatta is an important non-human primate model in biomedical research. They are widely used in numbers of infectious diseases, vaccine development, stem cell research, organ transplants and especially as an essential AIDS model in a simulated human body of HIV infection experiments. Due to its wide geographical distribution and domestication of a limited degree, it is very difficult to establish the inbred strains. Researcher made great efforts to maintain the homogeneity of genetic background of experimental results to decrease the results error in medical experiments. However, macaca mulatta participated in the same experiment are almost with heterogeneous genetic background. So, the individual differences results in the actual experimental more complicated.
Macaca mulatta with the same geographical origin are close in genetic relationship and relatively small individual differences. The major histocompatibility complex molecular (MHC) genes are the most important immunogenetic genes. So animals with the same geographical origin and the same MHC genotype will be beneficial to reduce individual differences in the experiment.
In this paper, we established a macaca mulatta immunogenetic homogeneity classification based on the known regional micro-satellite genetic markers and micro-satellite linked definitude MHC genes. Our results indicated that the genetic background of Chinese macaca mulatta was complicated. So the genetic marker of demographic origin should be further studied. We could replace the linked MHC genes with micro-satellite linked definitude MHC genes to divide the animals into numbers of subgroups.
As the lack of detailed understanding about the genetic background of Chinese macaca mulatta, our methods will provide the basic research reference for the macaca mulatta medical experiments and the management for the animals' propagation. It could be applied in genetic investigation on macaca mulatta from closed region to established the homogeneous genetic background subgroups in the future.
引文
[1] Parker RA, Regan MM, Reimann KA (2001) Variability of viral load in plasma of rhesus monkeys inoculated with simian immunodeficiency virus or simian-human immunodeficiency virus: implications for using nonhuman primate AIDS models to test vaccines and therapeutics. J Virol 75, 11234-11238.
[2] Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S, Wolf DP, Mitalipov SM (2007) Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450,497-502.
[3] Davis M, Antoniadis EA, Amaral DG, Winslow JT (2008) Acoustic startle reflex in rhesus monkeys: areview. Rev Neurosci 19, 171-185.
[4] Fechner JH, Jr., Dong Y, Hong X, Brunner KG, Tsuchida M, Neville D, Scharff J, Lee F,Oberley TD, Peters D, Schultz JM, Manthei ER, Hamawy MM, Knechtle SJ (2001) Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids. Transplantation 72, 581-587.
[5] Kanthaswamy S, von Dollen A, Kurushima JD, Alminas O, Rogers J, Ferguson B, Lerche NW, Allen PC, Smith DG (2006) Microsatellite markers for standardized genetic management of captive colonies of rhesus macaques (Macaca mulatta). Am J Primatol 68,73-95.
[6] Hendel H, Caillat-Zucman S, Lebuanec H, Carrington M, O'Brien S, Andrieu JM, Schachter F, Zagury D, Rappaport J, Winkler C, Nelson GW, Zagury JF (1999) New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J Immunol 162,6942-6946.
[7] Kaslow RA, Rivers C, Tang J, Bender TJ, Goepfert PA, El Habib R, Weinhold K, Mulligan MJ (2001) Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines. J Virol 75, 8681-8689.
[8] Carrington M, Bontrop RE (2002) Effects of MHC class I on HIV/SIV disease in primates.Aids 16 Suppl 4, S105-114.
[9] Evans DT, Jing P, Allen TM, O'Connor DH, Horton H, Venham JE, Piekarczyk M, Dzuris J,Dykhuzen M, Mitchen J, Rudersdorf RA, Pauza CD, Sette A, Bontrop RE, DeMars R,Watkins DI (2000) Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression. J Virol 74, 7400-7410.
[10] Chen HY, Di Mascio M, Perelson AS, Ho DD, Zhang L (2007) Determination of virus burst size in vivo using a single-cycle SIV in rhesus macaques. Proc Natl Acad Sci U S A 104,19079-19084.
[11] Liu J, O'Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, Abbink P, Coffey RT,Grandpre LE, Seaman MS, Landucci G, Forthal DN, Montefiori DC, Carville A, Mansfield KG, Havenga MJ, Pau MG, Goudsmit J, Barouch DH (2009) Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457, 87-91.
[12]Daza-Vamenta R,Glusman G,Rowen L,Guthrie B,Geraghty DE(2004) Genetic divergence of the rhesus macaque major histocompatibility complex.Genome Res 14,1501-1515.
[13]Urvater JA,Otting N,Loehrke JH,Rudersdorf R,Slukvin,Ⅱ,Piekarczyk MS,Golos TG,Hughes AL,Bontrop RE,Watkins DI(2000) Mamu-I:a novel primate MHC class I B-related locus with unusually low variability.J Immunol 164,1386-1398.
[14]Doxiadis GG,de Groot N,Claas FH,Doxiadis,Ⅱ,van Rood JJ,Bontrop RE(2007) A highly divergent microsatellite facilitating fast and accurate DRB haplotyping in humans and rhesus macaques.Proc Natl Acad Sci U S A 104,8907-8912.
[15]Otting N,de Vos-Rouweler AJ,Heijmans CM,de Groot NG,Doxiadis GG,Bontrop RE(2007)MHC class I A region diversity and polymorphism in macaque species.Immunogenetics 59,367-375.
[16]Karl JA,Wiseman RW,Campbell KJ,Blasky AJ,Hughes AL,Ferguson B,Read DS,O'Connor DH(2008) Identification of MHC class I sequences in Chinese-origin rhesus macaques.Immunogenetics 60,37-46.
[17]Ouyang D,Xu L,Dai Z,Shi H,Zhang G,Zheng Y,He X(2008) Identification of major histocompatibility complex class I alleles in Chinese rhesus macaques.Acta Biochim Biophys Sin(Shanghai) 40,919-927.
[18]Ouyang DY,Xu LH,Shi HJ,Zheng YT,He XH(2009) Eight novel MHC class I alleles identified in Chinese-origin rhesus macaques.Tissue Antigens 73,285-287.
[19]Hernandez RD,Hubisz MJ,Wheeler DA,Smith DG,Ferguson B,Rogers J,Nazareth L,Indap A,Bourquin T,McPherson J,Muzny D,Gibbs R,Nielsen R,Bustamante CD(2007)Demographic histories and patterns of linkage disequilibrium in Chinese and Indian rhesus macaques.Science 316,240-243.
[20]Smith DG,George D,Kanthaswamy S,(2006) Identification of Country of Origin and Admixture Between Indian and Chinese Rhesus Macaques.International Journal of Primatology 27,3.
[21]Yumiko TT,Michio Y,Taeko N,(2007) Reference strand-mediated conformation analysis-based typing of multiple alleles in the rhesus macaque MHC class I Mamu-A and Mamu-Bloci.Electrophoresis 28,918-924.
[22]Ferguson B,Street SL,Wright H,Pearson C,Jia Y,Thompson SL,Allibone P,Dubay CJ,Spindel E,Norgren RB,Jr.(2007) Single nucleotide polymorphisms(SNPs) distinguish Indian-origin and Chinese-origin rhesus macaques(Macaca mulatta).BMC Genomics 8,43.
[23]李元,用PCR-SSP方法对中国恒河猴MHC-Ⅰ分子分型的初步研究.学位论文,2003
[24]宿兵,王文,兰宏,张亚平,中国猴类的蛋白多态性及其遗传分化关系。1997,遗传学报24(2):109
[25]Penedo MC,Bontrop RE,Heijmans CM,Otting N,Noort R,Rouweler AJ,de Groot N,de Groot NG,Ward T,Doxiadis GG(2005) Microsatellite typing of the rhesus macaque MHC region.Immunogenetics 57,198-209.
[26]Wojcechowskyj JA,Yant LJ,Wiseman RW,O'Connor SL,O'Connor DH(2007) Control of simian immunodeficiency virus SIVmac239 is not predicted by inheritance of Mamu-B~*17-containing haplotypes.J Virol 81,406-410.
[27]Toshihiko Kikuchi,Masayuki Hara,Keiji Terao,Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys(Macaca fascicularis)。Primates(2007) 48:140-146
[28]Raveendran M,Harris RA,Milosavljevic A,Johnson Z,Shelledy W,Cameron J,Rogers J (2006) Designing new microsatellite markers for linkage and population genetic analyses in rhesus macaques and other nonhuman primates.Genomics 88,706-710.
[29]Andrade MC,Penedo MC,Ward T,Silva VF,Bertolini LR,Roberts JA,Leite JR Cabello PH (2004) Determination of genetic status in a closed colony of rhesus monkeys(Macaca mulatta).Primates 45,183-186.
[30]Wiseman RW,Wojcechowskyj JA,Greene JM,Blasky A J,Gopon T,Soma T,Friedrich TC,O'Connor SL,O'Connor DH(2007) Simian immunodeficiency virus SIVmac239 infection of major histocompatibility complex-identical cynomolgus macaques from Mauritius.J Virol 81,349-361.
[31]Doxiadis GG,Otting N,de Groot NG,Bontrop RE(2001) Differential evolutionary MHC class Ⅱ strategies in humans and rhesus macaques:relevance for biomedical studies.Immunol Rev 183,76-85.
[32]Doxiadis GG,Otting N,de Groot NG,de Groot N,Rouweler AJ,Noort R,Verschoor EJ,Bontjer I,Bontrop RE(2003) Evolutionary stability of MHC class Ⅱ haplotypes in diverse rhesus macaque populations.Immunogenetics 55,540-551.
[33]徐来祥,张知彬,宋铭晶,福尔马林保存的动物标本基因组DNA的提取方法。2002动物学报,48(2),264-269
[34]谭卓毅,丁梅,王保捷,福尔马林固定石蜡包埋组织中DNA提取。2006法医学杂志22,455-458
[35]Gilbert MT,Haselkorn T,Bunce M,Sanchez JJ,Lucas SB,Jewell LD,Van Marck E,Worobey M(2007) The isolation of nucleic acids from fixed,paraffin-embedded tissues-which methods are useful when? PLoS ONE 2,e537.
[36]田子强,刘俊峰,张少为,普通甲醛固定石蜡包埋组织提取方法的探讨。2004 chinese journal of cancer 23,342-345
[37]徐敏,纪岩文,张理涛,石蜡包埋皮肤组织DNA提取方法的改进。2007,天津医科大学学报03,440-444
[38]朱丽华,李琳,章广玲,周天戟.石蜡包埋组织PCR检测中DNA提取方法的比较及改进.临床与实验病理学杂志J Clin Exp Pathol2006(4):495-496.
[2] Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S, Wolf DP, Mitalipov SM (2007) Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450,497-502.
[3] Davis M, Antoniadis EA, Amaral DG, Winslow JT (2008) Acoustic startle reflex in rhesus monkeys: areview. Rev Neurosci 19, 171-185.
[4] Fechner JH, Jr., Dong Y, Hong X, Brunner KG, Tsuchida M, Neville D, Scharff J, Lee F,Oberley TD, Peters D, Schultz JM, Manthei ER, Hamawy MM, Knechtle SJ (2001) Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids. Transplantation 72, 581-587.
[5] Kanthaswamy S, von Dollen A, Kurushima JD, Alminas O, Rogers J, Ferguson B, Lerche NW, Allen PC, Smith DG (2006) Microsatellite markers for standardized genetic management of captive colonies of rhesus macaques (Macaca mulatta). Am J Primatol 68,73-95.
[6] Hendel H, Caillat-Zucman S, Lebuanec H, Carrington M, O'Brien S, Andrieu JM, Schachter F, Zagury D, Rappaport J, Winkler C, Nelson GW, Zagury JF (1999) New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J Immunol 162,6942-6946.
[7] Kaslow RA, Rivers C, Tang J, Bender TJ, Goepfert PA, El Habib R, Weinhold K, Mulligan MJ (2001) Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines. J Virol 75, 8681-8689.
[8] Carrington M, Bontrop RE (2002) Effects of MHC class I on HIV/SIV disease in primates.Aids 16 Suppl 4, S105-114.
[9] Evans DT, Jing P, Allen TM, O'Connor DH, Horton H, Venham JE, Piekarczyk M, Dzuris J,Dykhuzen M, Mitchen J, Rudersdorf RA, Pauza CD, Sette A, Bontrop RE, DeMars R,Watkins DI (2000) Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression. J Virol 74, 7400-7410.
[10] Chen HY, Di Mascio M, Perelson AS, Ho DD, Zhang L (2007) Determination of virus burst size in vivo using a single-cycle SIV in rhesus macaques. Proc Natl Acad Sci U S A 104,19079-19084.
[11] Liu J, O'Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, Abbink P, Coffey RT,Grandpre LE, Seaman MS, Landucci G, Forthal DN, Montefiori DC, Carville A, Mansfield KG, Havenga MJ, Pau MG, Goudsmit J, Barouch DH (2009) Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457, 87-91.
[12]Daza-Vamenta R,Glusman G,Rowen L,Guthrie B,Geraghty DE(2004) Genetic divergence of the rhesus macaque major histocompatibility complex.Genome Res 14,1501-1515.
[13]Urvater JA,Otting N,Loehrke JH,Rudersdorf R,Slukvin,Ⅱ,Piekarczyk MS,Golos TG,Hughes AL,Bontrop RE,Watkins DI(2000) Mamu-I:a novel primate MHC class I B-related locus with unusually low variability.J Immunol 164,1386-1398.
[14]Doxiadis GG,de Groot N,Claas FH,Doxiadis,Ⅱ,van Rood JJ,Bontrop RE(2007) A highly divergent microsatellite facilitating fast and accurate DRB haplotyping in humans and rhesus macaques.Proc Natl Acad Sci U S A 104,8907-8912.
[15]Otting N,de Vos-Rouweler AJ,Heijmans CM,de Groot NG,Doxiadis GG,Bontrop RE(2007)MHC class I A region diversity and polymorphism in macaque species.Immunogenetics 59,367-375.
[16]Karl JA,Wiseman RW,Campbell KJ,Blasky AJ,Hughes AL,Ferguson B,Read DS,O'Connor DH(2008) Identification of MHC class I sequences in Chinese-origin rhesus macaques.Immunogenetics 60,37-46.
[17]Ouyang D,Xu L,Dai Z,Shi H,Zhang G,Zheng Y,He X(2008) Identification of major histocompatibility complex class I alleles in Chinese rhesus macaques.Acta Biochim Biophys Sin(Shanghai) 40,919-927.
[18]Ouyang DY,Xu LH,Shi HJ,Zheng YT,He XH(2009) Eight novel MHC class I alleles identified in Chinese-origin rhesus macaques.Tissue Antigens 73,285-287.
[19]Hernandez RD,Hubisz MJ,Wheeler DA,Smith DG,Ferguson B,Rogers J,Nazareth L,Indap A,Bourquin T,McPherson J,Muzny D,Gibbs R,Nielsen R,Bustamante CD(2007)Demographic histories and patterns of linkage disequilibrium in Chinese and Indian rhesus macaques.Science 316,240-243.
[20]Smith DG,George D,Kanthaswamy S,(2006) Identification of Country of Origin and Admixture Between Indian and Chinese Rhesus Macaques.International Journal of Primatology 27,3.
[21]Yumiko TT,Michio Y,Taeko N,(2007) Reference strand-mediated conformation analysis-based typing of multiple alleles in the rhesus macaque MHC class I Mamu-A and Mamu-Bloci.Electrophoresis 28,918-924.
[22]Ferguson B,Street SL,Wright H,Pearson C,Jia Y,Thompson SL,Allibone P,Dubay CJ,Spindel E,Norgren RB,Jr.(2007) Single nucleotide polymorphisms(SNPs) distinguish Indian-origin and Chinese-origin rhesus macaques(Macaca mulatta).BMC Genomics 8,43.
[23]李元,用PCR-SSP方法对中国恒河猴MHC-Ⅰ分子分型的初步研究.学位论文,2003
[24]宿兵,王文,兰宏,张亚平,中国猴类的蛋白多态性及其遗传分化关系。1997,遗传学报24(2):109
[25]Penedo MC,Bontrop RE,Heijmans CM,Otting N,Noort R,Rouweler AJ,de Groot N,de Groot NG,Ward T,Doxiadis GG(2005) Microsatellite typing of the rhesus macaque MHC region.Immunogenetics 57,198-209.
[26]Wojcechowskyj JA,Yant LJ,Wiseman RW,O'Connor SL,O'Connor DH(2007) Control of simian immunodeficiency virus SIVmac239 is not predicted by inheritance of Mamu-B~*17-containing haplotypes.J Virol 81,406-410.
[27]Toshihiko Kikuchi,Masayuki Hara,Keiji Terao,Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys(Macaca fascicularis)。Primates(2007) 48:140-146
[28]Raveendran M,Harris RA,Milosavljevic A,Johnson Z,Shelledy W,Cameron J,Rogers J (2006) Designing new microsatellite markers for linkage and population genetic analyses in rhesus macaques and other nonhuman primates.Genomics 88,706-710.
[29]Andrade MC,Penedo MC,Ward T,Silva VF,Bertolini LR,Roberts JA,Leite JR Cabello PH (2004) Determination of genetic status in a closed colony of rhesus monkeys(Macaca mulatta).Primates 45,183-186.
[30]Wiseman RW,Wojcechowskyj JA,Greene JM,Blasky A J,Gopon T,Soma T,Friedrich TC,O'Connor SL,O'Connor DH(2007) Simian immunodeficiency virus SIVmac239 infection of major histocompatibility complex-identical cynomolgus macaques from Mauritius.J Virol 81,349-361.
[31]Doxiadis GG,Otting N,de Groot NG,Bontrop RE(2001) Differential evolutionary MHC class Ⅱ strategies in humans and rhesus macaques:relevance for biomedical studies.Immunol Rev 183,76-85.
[32]Doxiadis GG,Otting N,de Groot NG,de Groot N,Rouweler AJ,Noort R,Verschoor EJ,Bontjer I,Bontrop RE(2003) Evolutionary stability of MHC class Ⅱ haplotypes in diverse rhesus macaque populations.Immunogenetics 55,540-551.
[33]徐来祥,张知彬,宋铭晶,福尔马林保存的动物标本基因组DNA的提取方法。2002动物学报,48(2),264-269
[34]谭卓毅,丁梅,王保捷,福尔马林固定石蜡包埋组织中DNA提取。2006法医学杂志22,455-458
[35]Gilbert MT,Haselkorn T,Bunce M,Sanchez JJ,Lucas SB,Jewell LD,Van Marck E,Worobey M(2007) The isolation of nucleic acids from fixed,paraffin-embedded tissues-which methods are useful when? PLoS ONE 2,e537.
[36]田子强,刘俊峰,张少为,普通甲醛固定石蜡包埋组织提取方法的探讨。2004 chinese journal of cancer 23,342-345
[37]徐敏,纪岩文,张理涛,石蜡包埋皮肤组织DNA提取方法的改进。2007,天津医科大学学报03,440-444
[38]朱丽华,李琳,章广玲,周天戟.石蜡包埋组织PCR检测中DNA提取方法的比较及改进.临床与实验病理学杂志J Clin Exp Pathol2006(4):495-496.