先天性牙根发育不良致病相关基因的克隆研究
|
摘要
牙根发育不良疾病是牙齿根部生理性发育出现障碍的疾病，可分为：牙根牙本质发育缺陷；牙根牙骨质发育缺陷；牙根形态异常(不全、缺如或短锥状)；牙根附着器官发育异常等类型。国外研究报道乳、恒牙列均可发生，其结果可导致牙根缺如或短根，严重者可造成牙齿过早脱落。国外学者统计本病发病率在1％～10％之间。它不仅严重阻碍牙列功能的正常发挥，而且影响患者的容貌，给患者的生理和心理造成极大的危害，从而成为口腔科学工作者亟为关注的课题。但到目前为止，国内外有关牙根发育不良致病相关基因的研究未见报导，至今牙根的发育机制仍不甚明了，是否存在牙根发育的特异性调控基因还有待进一步研究。本研究目的是初步筛选和克隆导致牙根发育不良疾病的相关基因。采用临床就诊的3种不同类型的牙根发育不良病例，通过临床、实验室和病理检查及分析，通过分子遗传学研究的功能克隆策略初步筛选致病候选基因，并且进行基因突变检测确定基因的遗传学改变，进而再通过差减克隆方法从牙根发育不良疾病患儿与健康对照儿童的发育差异表达文库中筛选出致病相关基因，并通过牙齿发育的组织时空定位以及真核表达载体转染细胞，初步探讨其在牙根生理发育中的分子调控机制，以期阐明导致牙根发育不良疾病的分子遗传学基础，有望为牙根生理发育的分子机制提供新的理论基础，为该疾病的临床诊断、治疗和预防提供实验和理论依据。
第四军医大学博士学位论文
本研究主要研究内容及结果如下。
第一部分牙根发育不良患者的检查与分析研究
本部分研究是对3种不同临床表型的牙根发育不良患儿采
用致病候选基因功能性克隆的策略，通过详细的临床、实验室
检查与分析，以及患儿脱落牙齿的病理学检测，探索患儿的异
常生理功能(检测指标)，明确其临床和病理学诊断，从而初
步筛选出可疑的致病候选基因。临床、实验室检查与分析结果
发现本研究选择的3种不同临床表型的牙根发育不良疾病可能
是基因突变导致的遗传性疾病或发育异常疾病。通过分析研究，
初步分别诊断为:病例l是一种新表型的牙根发育缺陷的低碱
J胜磷酸醋酶症;病例2是儿童型低碱性磷酸醋酶症;病例3是
一种新表型的综合征(遗传性牙本质发育不全、牙根发育不良、
少汗型外胚叶发育不全)
病理学检测结果表明:病例1脱落牙齿的牙冠发育良好，
牙根结构发育不全，牙本质矿化不足，根端有异常牙质团块堆
积，并伴有牙质吸收现象，符合牙根发育不良疾病的病理表现。
病例2牙齿髓室以及根管扩大呈“贝壳”或“气泡”样结构改
变，牙骨质结构缺失，生长间歇线异常增多，牙本质小管粗细、
排列不均，牙本质的矿化不良，符合牙齿型低碱性磷酸酷酶症
疾病的病理表现。病例3牙齿的釉牙本质界呈线性结构且间隙
增宽，牙本质结构杂乱无章，并堆积髓腔，无牙根结构，是一
种复杂的新型综合征。
第二部分牙根发育不良患者致病候选基因的突变检测
根据上述的牙根发育不良疾病患儿的检查与分析，初步挑
选ALPL基因、EDA基因作为牙根发育不良疾病致病候选基因，
通过PCR及单链构象多态性(SSCP)分析、基因测序等方法进行
突变基因的检测。结果发现:
病例1的致病候选基因ALPL塞因Exons出现异常，测序
第四军医大学博士学位论文
发现583位点出现基因突变G一A，并且为杂合子结构，密码子
CGU一CAu可能部分引起第1 36个氨基酸的改变，精氨酸变成组
氨酸。该突变型为583G>A，R136H。因此可以确定致病候选基
因ALPL是本病例的致病相关基因。
病例2的致病候选基因ALPL基因的ExonlZ存在一个C一G
变化，该基因为纯合子结构，该基因1581位点出现基因突变。
密码子CCU一GCU引起第446个氨基酸的改变，脯氨酸( Pro，P)
变成丙氨酸(Gly，G)。该突变型为1581C>G，P446G。因此结合
临床症状可以确定ALPL是病例2的致病基因。
病例3的致病候选基因EDA基因的Ex。ng存在一个T一C
变化，该基因为杂合子结构，来自父系或母系染色体该基因
1231位点可能出现基因突变。密码子CUG一CCU可能部分引起
第330个氨基酸的改变，亮氨酸(Leu，L)变成脯氨酸(Pr。，P)。
该突变型为1231T>C，L33OP。结果说明EDA基因是该患儿的
一个致病相关基因。
经过数据库和文献检索，本实验发现3种突变型均未见相
同报道，初步认为是与牙根发育不良表型相关的新的突变型。
第三部分牙根发育不良致病相关基因的克隆及筛选研究
由于病例1具有较为典型的牙根发育缺陷症状，与以往牙
齿型低碱性磷酸酩酶症的症状不完全相符。牙根发育不良的临
床表型是否为低碱性磷酸醋酶症的新口腔表现形式，还是作为
一种独立疾病与低碱性磷酸酷酶症同时在患者身上发病，仍难
以解释。本研究通过差减克隆策略的改良消减杂交技术筛选该
患儿与健康兄弟对照之间发育表达差异基因，探寻牙根发育的
致病相关基因。
首先构建了牙根发育不良疾病差异表达基因文库，文库总
浓度为4.0x1O2。随机挑取100个白色克隆，ECoR工酶切后，
含有插段的克隆90个，占90%。56obp以上的片段有72个。
Hypoplasia of teeth root (HTR) disease is a disorder with physiological development of teeth root. According to clinical manifestation, it was classified into many subgroup: Dentinogenesis defect of root; cementinogenesis defect of root; shape anomaly; defect of teeth root attachment organ and so on.. It was reported that HTR could occur with an incidence of 1-10% in deciduous and permanent dentition, which resulted in non-root or short root. HTR could affect not only normal dentition function but also appearance of face, which brings severe harms to physiological and psychological activities. So it was a interested topic for stomatological workers. However up to now, the causative genes of HTR weren't reported, and the mechanism of teeth root formation were unknown. It needed further research if it existed specific genes initiating root development. So Aims of my research is to screen and clone causative genes of HTR. Firstly patients with 3 kind of HTR phenotype were collected to screen candidate gene by c
linic, laboratory, pathologic examination, then by fuctional cloning strategy of molecular genetic research. Furtherly candidate genes were inspected to find mutation sites for identifying genetic change of genes. Then from differentially expressed auxo-genes libarary between HTR children and normal control children causative genes were defined by subtractive cloning method, finally which are studied to explore molecular mechanism regulating
teeth root formation by location of timing and tissue expessing, moreover cell
transfection of Eukaryotic expression vector. So it is wished to clarify molecular genetic backgroud of HTR disease, and provide new theory about molecular mechanism of teeth root biological development, and obtain practicable and theoretical results for clinical diagnosis, therapeutics and prevention of HTR disease. The following experiments were carried out.
Section one : The inspection and analysis of HTR patients In this experiment, three children with various HTR phenotype were inspected clinically and by laboratory test, then their shedding teeth were examined by pathologic test. Finally abnormal results were found to diagnose the entity of disease, furthermore to pick up the suspected candidate gene by functional clone strategy. The results of clinic and laboratory test were analyzed to show that 3 kind of HTR diseases are probably the inherited disease with mutated gene or the dysplasia disease. They were separately and preliminarily assessed as a novel hypophosphatasia with HTR phenotype, a typical children hypophosphatasia, a novel syndrome (including Dentinogenesis imperfecta DGI, hypohidrotic ectodermal dysplasia HED, HTR). Pathologic test of patient one showed that teeth crown grew well but without teeth root structure, with sub-mineralized dentin, resorption, and abnormal dentin stacking in apical area, so it was HTR. Pathologic test of patient two showed that pulp chamber and root canal enlarged as "shell or bubble"structure, cementin disappeared with sub-mineralized dentin, incremental line were increasing, Caliber and arrangement of dentinal tubule weren't average. So it was diagnosed as odontophosphatasia. Pathologic test of patient three showed that enamel-dentinal junction teeth arranged as line structure with broad interspace, dentin formed in a great mess, and pulp chamber was filled without root structure. So it was diagnosed as a complex
syndrome.
Section two : Mutation screening of candidate genes for HTR patient
Based on the results of section one, ALPL and EDA gene were picked up as candidate genes for HTR patient by functional clone strategy. Gene mutation were screened by PCR-SSCP technique and gene sequencing. Results of Patient one showed that Exon5 of ALPL gene were abnormal by electrophoresis methods, then sequencing revealed a G--A transition of 583rd site and heterozygote structure. Codon CCU-GCU was predicted to result in a substitution of Arginine by Histidine at 136th ammo acid. Mutation type was 583G>A, R136H. Results of Patient two revealed a C-G
第四军医大学博士学位论文
本研究主要研究内容及结果如下。
第一部分牙根发育不良患者的检查与分析研究
本部分研究是对3种不同临床表型的牙根发育不良患儿采
用致病候选基因功能性克隆的策略，通过详细的临床、实验室
检查与分析，以及患儿脱落牙齿的病理学检测，探索患儿的异
常生理功能(检测指标)，明确其临床和病理学诊断，从而初
步筛选出可疑的致病候选基因。临床、实验室检查与分析结果
发现本研究选择的3种不同临床表型的牙根发育不良疾病可能
是基因突变导致的遗传性疾病或发育异常疾病。通过分析研究，
初步分别诊断为:病例l是一种新表型的牙根发育缺陷的低碱
J胜磷酸醋酶症;病例2是儿童型低碱性磷酸醋酶症;病例3是
一种新表型的综合征(遗传性牙本质发育不全、牙根发育不良、
少汗型外胚叶发育不全)
病理学检测结果表明:病例1脱落牙齿的牙冠发育良好，
牙根结构发育不全，牙本质矿化不足，根端有异常牙质团块堆
积，并伴有牙质吸收现象，符合牙根发育不良疾病的病理表现。
病例2牙齿髓室以及根管扩大呈“贝壳”或“气泡”样结构改
变，牙骨质结构缺失，生长间歇线异常增多，牙本质小管粗细、
排列不均，牙本质的矿化不良，符合牙齿型低碱性磷酸酷酶症
疾病的病理表现。病例3牙齿的釉牙本质界呈线性结构且间隙
增宽，牙本质结构杂乱无章，并堆积髓腔，无牙根结构，是一
种复杂的新型综合征。
第二部分牙根发育不良患者致病候选基因的突变检测
根据上述的牙根发育不良疾病患儿的检查与分析，初步挑
选ALPL基因、EDA基因作为牙根发育不良疾病致病候选基因，
通过PCR及单链构象多态性(SSCP)分析、基因测序等方法进行
突变基因的检测。结果发现:
病例1的致病候选基因ALPL塞因Exons出现异常，测序
第四军医大学博士学位论文
发现583位点出现基因突变G一A，并且为杂合子结构，密码子
CGU一CAu可能部分引起第1 36个氨基酸的改变，精氨酸变成组
氨酸。该突变型为583G>A，R136H。因此可以确定致病候选基
因ALPL是本病例的致病相关基因。
病例2的致病候选基因ALPL基因的ExonlZ存在一个C一G
变化，该基因为纯合子结构，该基因1581位点出现基因突变。
密码子CCU一GCU引起第446个氨基酸的改变，脯氨酸( Pro，P)
变成丙氨酸(Gly，G)。该突变型为1581C>G，P446G。因此结合
临床症状可以确定ALPL是病例2的致病基因。
病例3的致病候选基因EDA基因的Ex。ng存在一个T一C
变化，该基因为杂合子结构，来自父系或母系染色体该基因
1231位点可能出现基因突变。密码子CUG一CCU可能部分引起
第330个氨基酸的改变，亮氨酸(Leu，L)变成脯氨酸(Pr。，P)。
该突变型为1231T>C，L33OP。结果说明EDA基因是该患儿的
一个致病相关基因。
经过数据库和文献检索，本实验发现3种突变型均未见相
同报道，初步认为是与牙根发育不良表型相关的新的突变型。
第三部分牙根发育不良致病相关基因的克隆及筛选研究
由于病例1具有较为典型的牙根发育缺陷症状，与以往牙
齿型低碱性磷酸酩酶症的症状不完全相符。牙根发育不良的临
床表型是否为低碱性磷酸醋酶症的新口腔表现形式，还是作为
一种独立疾病与低碱性磷酸酷酶症同时在患者身上发病，仍难
以解释。本研究通过差减克隆策略的改良消减杂交技术筛选该
患儿与健康兄弟对照之间发育表达差异基因，探寻牙根发育的
致病相关基因。
首先构建了牙根发育不良疾病差异表达基因文库，文库总
浓度为4.0x1O2。随机挑取100个白色克隆，ECoR工酶切后，
含有插段的克隆90个，占90%。56obp以上的片段有72个。
Hypoplasia of teeth root (HTR) disease is a disorder with physiological development of teeth root. According to clinical manifestation, it was classified into many subgroup: Dentinogenesis defect of root; cementinogenesis defect of root; shape anomaly; defect of teeth root attachment organ and so on.. It was reported that HTR could occur with an incidence of 1-10% in deciduous and permanent dentition, which resulted in non-root or short root. HTR could affect not only normal dentition function but also appearance of face, which brings severe harms to physiological and psychological activities. So it was a interested topic for stomatological workers. However up to now, the causative genes of HTR weren't reported, and the mechanism of teeth root formation were unknown. It needed further research if it existed specific genes initiating root development. So Aims of my research is to screen and clone causative genes of HTR. Firstly patients with 3 kind of HTR phenotype were collected to screen candidate gene by c
linic, laboratory, pathologic examination, then by fuctional cloning strategy of molecular genetic research. Furtherly candidate genes were inspected to find mutation sites for identifying genetic change of genes. Then from differentially expressed auxo-genes libarary between HTR children and normal control children causative genes were defined by subtractive cloning method, finally which are studied to explore molecular mechanism regulating
teeth root formation by location of timing and tissue expessing, moreover cell
transfection of Eukaryotic expression vector. So it is wished to clarify molecular genetic backgroud of HTR disease, and provide new theory about molecular mechanism of teeth root biological development, and obtain practicable and theoretical results for clinical diagnosis, therapeutics and prevention of HTR disease. The following experiments were carried out.
Section one : The inspection and analysis of HTR patients In this experiment, three children with various HTR phenotype were inspected clinically and by laboratory test, then their shedding teeth were examined by pathologic test. Finally abnormal results were found to diagnose the entity of disease, furthermore to pick up the suspected candidate gene by functional clone strategy. The results of clinic and laboratory test were analyzed to show that 3 kind of HTR diseases are probably the inherited disease with mutated gene or the dysplasia disease. They were separately and preliminarily assessed as a novel hypophosphatasia with HTR phenotype, a typical children hypophosphatasia, a novel syndrome (including Dentinogenesis imperfecta DGI, hypohidrotic ectodermal dysplasia HED, HTR). Pathologic test of patient one showed that teeth crown grew well but without teeth root structure, with sub-mineralized dentin, resorption, and abnormal dentin stacking in apical area, so it was HTR. Pathologic test of patient two showed that pulp chamber and root canal enlarged as "shell or bubble"structure, cementin disappeared with sub-mineralized dentin, incremental line were increasing, Caliber and arrangement of dentinal tubule weren't average. So it was diagnosed as odontophosphatasia. Pathologic test of patient three showed that enamel-dentinal junction teeth arranged as line structure with broad interspace, dentin formed in a great mess, and pulp chamber was filled without root structure. So it was diagnosed as a complex
syndrome.
Section two : Mutation screening of candidate genes for HTR patient
Based on the results of section one, ALPL and EDA gene were picked up as candidate genes for HTR patient by functional clone strategy. Gene mutation were screened by PCR-SSCP technique and gene sequencing. Results of Patient one showed that Exon5 of ALPL gene were abnormal by electrophoresis methods, then sequencing revealed a G--A transition of 583rd site and heterozygote structure. Codon CCU-GCU was predicted to result in a substitution of Arginine by Histidine at 136th ammo acid. Mutation type was 583G>A, R136H. Results of Patient two revealed a C-G
引文
1. Apajalahti S, Arte S, Pirinen S. Short root anomaly in families and its association with other dental anomalies. Eur J Oral Sci 1999;107(2):97-101]
2. Apajalahti S, Holtta P, Turtola L, etal. Prevalence of short-root anomaly in healthy young adults. Acta odontol Scand 2002;60(1):56-59
3. Brown H. Hypoplasia of the dentition. Am. J. Orth. Oral Surg 1944;30:102-103
4. Lind V. Short root anomaly. Scand. J. Dent. Res. 1972; 80:85-93
5. Edwards DM, Roberts GJ. Short root anomaly. Br Dent J 1990;169(9):292-293
6. Witkop CJ. Clinical aspects of dental anomalies. Int Dent J 1976;26:378-390
7. Wesley R, Wysocki G, Mintz S, etal. Dentin dysplasia type I. Oral Surg. 1976; 41:516-523
8. Melnick M, Levin L, Brady J. Dentin dysplasia type I: a scanning electron microscopic analysis of the primary dentition. Oral Surg 1980;50:335-339
9. Witkop C. Hereditary defects of dentin. Dent. Clin. N. Am. 1975;19:25-45
10. Morris M, Augsburger R. Dentine dysplasia with sclerotic bone and skeletal anomalies inherited as an autosomal dominant trait.Oral Surg 1977; 43:267-283
11. Teree T, Klein L. Hypophosphatasia: clinical and metabolic studies. J. Pediat. 1968;72:41-50
12. Wolff C, Zabransky S. Hypophosphatasia congenita letalis. Eur J Pediat.1982;138:197-199
13. Beertsen W, Vandenbos T, Everts V, etal. Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation. J Dent Res 1999:78:1221-1229
14. Olsson A, Matsson L, Blomquist H, etal. Hypophosphatasia affecting the permanent dentition. J. Oral Path. Med. 1996;25:343-347
15. Greenberg CR, Taylor C, Haworth JC, etal. A homoallelic gly317-to-asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Genomics 1993;17:215-217
16. Whyte M, mcAlister W, Patton L, etal. Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J. Pediar. 1984;105:926-933
17. Feigenbaum A, Kumar A, Weksberg R. Singleton-Merten syndrome: autosomal dominant transmission with variable expression. Am J Hum Genet 1988;43:A48
18. Pope FM, Komorowska A, Lee KW, etal. Ehlers-Danlos syndrome type I with novel dental features. J Oral Pathol Med 1992; 21:418-421
19. Michalickova K, Susic M, Willing MC, etal. Mutation of the alpba-2(Ⅴ) chain of type Ⅴ collagen impair matrix assembly and produce Ehlers-Danlos syndrome type Ⅰ. Hum Molec Genet 1998; 7:249-255
20. Lerman RL, Gold R. Idiopathic short root anomaly.J pedod 1977;1:327-333
21. kantaputra PN. A newly recognized syndrome of skeletal
dysplasia with opalescent and rootless teeth. Oral Surg Oral Med Oral pathaol Oral radiaol Endod 2001;92(3):303-307
22. De Man K. Abnormal root development, probably due to erythema multiforme (Stevens-Johnson syndrome), Int J Oral Surg 1979;8:381-385
23. Thornton JB, Worley SL. Short root anomaly in a patient with a history of Stevens-Johnson syndrome: report of case. ASDC J Dent For Children 1991; 58(3): 256-259
24. Brin I, ZilbermanY, Galili D, etal. Eruption of rootless teeth in congenital renal disease. Oral Surg Oral Med Oral pathaol 1985; 60(1): 61-64
25. Takinami S, Kaga M, Yahata H, etal. Radiation-induced hypoplasia of the teeth and mandible: a case report. Oral Surg Oral Med Oral pathaol 1994; 78:382-384
26. Nasman M, Bjork O, Soderhall S, etal. Disturbances in the oral cavity in pediatric long-term survivors after different forms of antineoplastic theraphy. Peditr Dent 1994; 16:217-223
27. Seow WK. Root agenesis associated with chronic infection and traumatic curettage the maxillary sinus. Peditr Dent 1994; 16(3): 227-230
28. Janson GR, Luca Canto G, Martins DR, etal. A radiographic comparison of apical root resorption after orthodontic treatment with 3 different fixed appliance techniques. Am J Orthod Dentofac Orthop 2000; 118:262-273
29．文玲英，杨富生主编．临床儿童口腔科学．西安：世界图书出版社，2001：40-68．
30．徐衍，刘谦。DNA复制调控与人类重大疾病。中国生物工程杂志，2003；23(10)：1—7
2. Apajalahti S, Holtta P, Turtola L, etal. Prevalence of short-root anomaly in healthy young adults. Acta odontol Scand 2002;60(1):56-59
3. Brown H. Hypoplasia of the dentition. Am. J. Orth. Oral Surg 1944;30:102-103
4. Lind V. Short root anomaly. Scand. J. Dent. Res. 1972; 80:85-93
5. Edwards DM, Roberts GJ. Short root anomaly. Br Dent J 1990;169(9):292-293
6. Witkop CJ. Clinical aspects of dental anomalies. Int Dent J 1976;26:378-390
7. Wesley R, Wysocki G, Mintz S, etal. Dentin dysplasia type I. Oral Surg. 1976; 41:516-523
8. Melnick M, Levin L, Brady J. Dentin dysplasia type I: a scanning electron microscopic analysis of the primary dentition. Oral Surg 1980;50:335-339
9. Witkop C. Hereditary defects of dentin. Dent. Clin. N. Am. 1975;19:25-45
10. Morris M, Augsburger R. Dentine dysplasia with sclerotic bone and skeletal anomalies inherited as an autosomal dominant trait.Oral Surg 1977; 43:267-283
11. Teree T, Klein L. Hypophosphatasia: clinical and metabolic studies. J. Pediat. 1968;72:41-50
12. Wolff C, Zabransky S. Hypophosphatasia congenita letalis. Eur J Pediat.1982;138:197-199
13. Beertsen W, Vandenbos T, Everts V, etal. Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation. J Dent Res 1999:78:1221-1229
14. Olsson A, Matsson L, Blomquist H, etal. Hypophosphatasia affecting the permanent dentition. J. Oral Path. Med. 1996;25:343-347
15. Greenberg CR, Taylor C, Haworth JC, etal. A homoallelic gly317-to-asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Genomics 1993;17:215-217
16. Whyte M, mcAlister W, Patton L, etal. Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J. Pediar. 1984;105:926-933
17. Feigenbaum A, Kumar A, Weksberg R. Singleton-Merten syndrome: autosomal dominant transmission with variable expression. Am J Hum Genet 1988;43:A48
18. Pope FM, Komorowska A, Lee KW, etal. Ehlers-Danlos syndrome type I with novel dental features. J Oral Pathol Med 1992; 21:418-421
19. Michalickova K, Susic M, Willing MC, etal. Mutation of the alpba-2(Ⅴ) chain of type Ⅴ collagen impair matrix assembly and produce Ehlers-Danlos syndrome type Ⅰ. Hum Molec Genet 1998; 7:249-255
20. Lerman RL, Gold R. Idiopathic short root anomaly.J pedod 1977;1:327-333
21. kantaputra PN. A newly recognized syndrome of skeletal
dysplasia with opalescent and rootless teeth. Oral Surg Oral Med Oral pathaol Oral radiaol Endod 2001;92(3):303-307
22. De Man K. Abnormal root development, probably due to erythema multiforme (Stevens-Johnson syndrome), Int J Oral Surg 1979;8:381-385
23. Thornton JB, Worley SL. Short root anomaly in a patient with a history of Stevens-Johnson syndrome: report of case. ASDC J Dent For Children 1991; 58(3): 256-259
24. Brin I, ZilbermanY, Galili D, etal. Eruption of rootless teeth in congenital renal disease. Oral Surg Oral Med Oral pathaol 1985; 60(1): 61-64
25. Takinami S, Kaga M, Yahata H, etal. Radiation-induced hypoplasia of the teeth and mandible: a case report. Oral Surg Oral Med Oral pathaol 1994; 78:382-384
26. Nasman M, Bjork O, Soderhall S, etal. Disturbances in the oral cavity in pediatric long-term survivors after different forms of antineoplastic theraphy. Peditr Dent 1994; 16:217-223
27. Seow WK. Root agenesis associated with chronic infection and traumatic curettage the maxillary sinus. Peditr Dent 1994; 16(3): 227-230
28. Janson GR, Luca Canto G, Martins DR, etal. A radiographic comparison of apical root resorption after orthodontic treatment with 3 different fixed appliance techniques. Am J Orthod Dentofac Orthop 2000; 118:262-273
29．文玲英，杨富生主编．临床儿童口腔科学．西安：世界图书出版社，2001：40-68．
30．徐衍，刘谦。DNA复制调控与人类重大疾病。中国生物工程杂志，2003；23(10)：1—7