摘要
本研究采用鹌鹑-鸡胚移植法探索咽囊内胚层对中耳听骨发育(诱导／成形)及中耳听骨与内耳整合的作用。首先在整体标本中观察了正常鸡胚中耳听骨——耳小柱(columella)发育过程中的形态学改变与特征，建立了正常参照标准。然后，采用鹌鹑-鸡胚移植法，研究了菱脑第4节(R4区)咽囊内胚层对耳小柱的诱导和成形作用、R4R5区咽囊内胚层对耳小柱和内耳(半规管)包囊的整合作用、以及R4区咽囊内胚层对R3至中脑(MB)区其它软骨的诱导和成形作用，证实了咽囊内胚层能诱导中耳听骨发育及中耳听骨与内耳的整合作用，并且确切定位了诱导耳小柱各部位发育及其与内耳整合作用的咽囊内胚层组织，从而阐明了中、内耳正常胚胎发育与畸形形成的部分机制。
第一部分：正常鸡胚听骨发育中的解剖形态
目的 掌握正常鸡胚发育过程中耳小柱解剖形态与特征，建立正常参照标准，同时探索在整体标本中清晰显示位置深在的耳小柱的方法。方法 收取胚胎发育第5-19天(E5-E19)鸡胚头部，4％PFA固定，阿辛蓝软骨染色，甲醛水杨酸软组织透明后，解剖显微镜下观察中耳腔内耳小柱及其周围软骨发育情况，包括方形软骨、关节软骨、鳞骨、下颌软骨(Meckel软骨)、内耳(半规管)包囊以及舌骨。结果 耳小柱约在E7出现，E8-E10呈分离的内、外侧2部分，E11已具成熟期形态(内、外侧2部分融为一体，外侧段有3个突起)，E12形态更清晰，E13软组织内血管影出现，E14软骨开始钙化，E15
This research explored the role of pharyngeal pouch endoderm (PPE) to development (induction and patterning ) of chick ossicle and integration of the ossicle and inner ear by quail-chick chimera. First, we observed the normal morphology of chick ossicle, columella, during development in the whole mount, and established a criterion for normal columella. Then, investigated the induction and patterning of R4 PPE to columella, R4R5 to the integration of columella and inner ear by isotopic transplantation, and R4 to cartilages originating from R3 to Midbrain (MB) by ectopic transplantation. And proved PPE can induce development of columella and integration of columella and inner ear, and located the sites of the PPE, which elucidate partly mechanism of normal development and defect formation of the middle and inner ear.
The 1st part: Normal morphology of chick columella during development. Objective master normal morphology and establish a criterion of chick columella during its development, and find a method which can show the configuration of deep located columella in the whole mount. Method Collected E5- E19 chick embryos' heads, fixed specimen with 4%PFA, stained cartilages with Alcian Blue, cleared tissue with methyl salicylate, and observed columella and other cartilages around, such as quadrate, articular, squamosum, Meckel' s cartilage, inner
引文
1．杜佳梅，庄洪兴，蒋海越，等．先天性小耳畸形患者及其家属心理状况调查研究．中华整形外科杂志 2005，21(3)：218
2. Kountakis SE, Helidonis E, Jahrsdoerfer RA. Microtia grade as an indicator of middle ear development in aural atresia. Arch Otolaryngol Head Neck Surg. 1995 Aug; 121(8):885-6.
3. Forrester MB, Merz RD. Descriptive epidemiology of anotia and microtia, Hawaii, 1986-2002. Congenit Anom (Kyoto).2005 Dec;45(4):119-24.
4. Harris J, Kallen B, Robert E. The epidemiology of anotia and microtia. J Med Genet 1996,33:809-813
5．朱军，王艳萍，梁娟，等．1988-1992年全国先天性无耳和小耳畸形发病率的抽样调查．中华耳鼻咽喉科杂志 2000，35：62-65
6. Couly G, Creuzet S, Bennaceur S, Vincent C, Le Douarin NM. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development. 2002 Feb;129(4):1061-73.
7. Ruhin B, Creuzet S, Vincent C, Benouaiche L, Le Douarin NM, Couly G .Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm. Development Dynamics. 2003 Oct; 228(2):239-46.8. Kontges G, Lumsden A. Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996 0ct;122(10):3229-42.
9. Lumsden A, Sprawson N, Graham A. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development. 1991 Dec;113(4):1281-91.
10. Lumsden A, Keynes R. Segmental patterns of neuronal development in the chick hindbrain. Nature. 1989 Feb 2;337(6206):424-8.
11. Teillet MA, Ziller C, Le Douarin NM. Quail-chick chimeras. Methods Mol Biol. 1999;97:305-18.1. Hall JW 3rd. Development of the ear and hearing. J Perinatol. 2000 Dec;20(8 Pt 2):S12-20.
2．孔维佳．耳的生理学．In：黄选兆，汪吉宝．实用耳鼻咽喉科学．北京：人民卫生出版社．1998年 第一版：645-690
3. Lim DJ. Structure and function of the tympanic membrane: a review. Acta Otorhinolaryngol Belg. 1995;49(2):101-15.
4. Funnell WR, Laszlo CA. A critical review of experimental observations on ear-drum structure and function. ORL J Otorhinolaryngol Relat Spec. 1982;44(4):181-205.
5. Bent LR, McFadyen BJ, Inglis JT. Vestibular contributions during human locomotor tasks. Exerc Sport Sci Rev. 2005 Jul;33(3):107-13.
6. Lannigan FJ, O'Higgins P, McPhie P .Remodelling of the normal incus. Clin Otolaryngol Allied Sci. 1993 Jun;18(3):155-60.
7. Graham MD, Perkins R. A scanning electron microscopic study of the normal human stapes. Ann Otol Rhinol Laryngol Suppl. 1979 Nov-Dec;88(6 Pt 5 Suppl 64):2-14.
8. Sanchez-Fernandez JM, Saint-Gerons S, Sanchez del Rey A. A microanalytical study on human auditory ossicles in normal and pathological conditions. Acta Otolaryngol. 1992;112(2):317-21.9．龚树生．耳的临床解剖学．In：黄选兆，汪吉宝．实用耳鼻咽喉科学．北京：人民卫生出版社．1998年 第一版：613-645
10. Hartwein JH, Rauchfuss A. The development of the ossicular ligaments in the human middle ear. Arch Otorhinolaryngol. 1987;244(1):23-5.
11. Huttenbrink KB. The functional significance of the suspending ligaments of the ear ossicle chain. haryngorhinootologie. 1989 Mar;68(3):146-51.
12. Huttenbrink KB. The mechanics and function of the middle ear. Part 1: The ossicular chain and middle ear muscles haryngorhinootologie. 1992 Nov;71(11):545-51.
13. Granstrom G, Jacobsson C. First and second branchial arch syndrome: aspects on the embryogenesis, elucidations, and rehabilitation using the osseointegration concept. Clin Implant Dent Relat Res. 1999;1(2):59-69.
14. William JL. Development of the Ears. In: William JL.. Human Embryology. Churchill Livingstone. 3ird edition Philadelphia, Pennsylvania : 391-417
15. Louryan S. Development of the auditory ossicles in the human embryo: correlations with data obtained in mice Bull Assoc Anat (Nancy). 1993 Mar;77(236):29-32.
16. Bontemps C, Cannistra C, Hannecke V, Michel P, Fonzi L, Barbet JP The first appearance of Meckel's cartilage in the fetus Bull Group Int Rech Sci Stomatol Odontol. 2001 Sep-Dec;43(3):94-9.17. Rodriguez-Vazquez JF, Merida-Velasco JR, Verdugo-Lopez S, Sanchez-Montesinos I, Merida-Velasco JA Morphogenesis of the second pharyngeal arch cartilage (Reichert' s cartilage) in human embryos J Anat. 2006 Feb;208(2) :179-89.
18. Yokoyama T, Iino Y, Kakizaki K, Murakami Y Human temporal bone study on the postnatal ossification process of auditory ossicles Laryngoscope. 1999 Jun;109(6):927-30.
19. Mallo M. Embryological and genetic aspects of middle ear development. Int J Dev Biol. 1998 Jan;42(1):11-22. Review.
20. Rodriguez-Vazquez JF. Development of the stapes and associated structures in human embryos. J Anat. 2005 Aug; 207 (2): 165-73.
21.Masuda Y, Saito R, Endo Y, Kondo Y, Ogura Y. Histological development of stapes footplate in human embryos Acta Med Okayama. 1978 Jun;32(2): 109-17.
22. Kontges G, Lumsden A Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996 Oct;122(10):3229-42.
23. Lumsden A, Sprawson N, Graham A. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development. 1991 Dec; 113(4) : 1281-91.
24. Couly G, Creuzet S, Bennaceur S, Vincent C, Le Douarin NM. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development. 2002Feb;129 (4):1061-73.
25. Ruhin B, Creuzet S, Vincent C, Benouaiche L, Le Douarin NM, Couly G . Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm. Development Dynamics. 2003 Oct;228(2):239-46.
26. Masuda Y, Honjo H, Naito M, Ogura Y. Normal development of the middle ear in the mouse: a light microscopic study of serial sections Acta Med Okayama. 1986 Aug;40(4):201-7.
27. Masuda Y, Honjo H, Naito M, Ogura Y. Bilaminar structure of the developing stapedial footplate in the mouse—a histological study using a light microscope. Auris Nasus Larynx. 1987; 14(1) : 1-7.
28. Whyte JR, Gonzalez L, Cisneros AI, Yus C, Torres A, Sarrat R. Fetal development of the human tympanic ossicular chain articulations. Cells Tissues Organs. 2002;171(4):241-9.
29. Amin S, Tucker AS. Joint formation in the middle ear: Lessons from the mouse and guinea pig. Dev Dyn. 2006 Jan 19; [Epub ahead of print]
30. Whyte J, Cisneros A, Yus C, Blasco J, Torres A, Sarrat R Contribution to the development of the stapedius muscle structure in human fetuses Anat Histol Embryol. 2001 Jun;30(3):175-8.
31. Olszewski J, Antoszewska M. Development of the stapedius muscle in human fetuses. Folia Morphol (Warsz).1987; 46(1-2): 25-9.
32. Palva T, Northrop C, Ramsay H. Supratubal recess in neonates and infants. Int J Pediatr Otorhinolaryngol. 1999 Oct 25; 50(2): 99-107.
33. Marovitz WF, Porubsky ES. The embryological development of the middle ear space—a new concept Ann Otol Rhinol Laryngol. 1971 Jun; 80(3): 384-9.
34. Rood SR, Doyle WJ. Morphology of tensor veli palatini, tensor tympani, and dilatator tubae muscles. Ann Otol Rhinol Laryngol. 1978 Mar-Apr; 87(2 Pt 1): 202-10.
35．武汉医学院第一附属医院耳鼻喉科学教研组．耳鼻咽喉科学．北京：人民卫生出版社．1978年：591
36. Piza J, Northrop C, Eavey RD. Embryonic middle ear mesenchyme disappears by redistribution. Laryngoscope. 1998 Sep; 108(9): 1378-81.
37. Jaskoll TF, Maderson PF.A histological study of the development of the avian middle ear and tympanum. Anat Rec. 1978 Feb; 190(2): 177-99.
38. Kil SH, Collazo A. A review of inner ear fate maps and cell lineage studies. J Neurobiol. 2002 Nov 5; 53(2): 129-42.
39. Torres M, Giraldez F. The development of the vertebrate inner ear. Mech Dev. 1998 Feb;71(1-2):5-21.
40. Bissonnette JP, Fekete DM. Standard atlas of the gross anatomy of the developing inner ear of the chicken. J Comp Neurol. 1996 May 13; 368(4): 620-30.41. Nishimura Y, Kumoi T The embryologic development of the human external auditory meatus. Preliminary report Acta Otolaryngol. 1992; 112(3): 496-503.
42. Wright CG Development of the human external ear J Am Acad Audiol. 1997 Dec; 8(6): 379-82.
43．庄洪兴，王炜．1999．胚胎发育障碍与耳畸形．见：王炜主编．整形外科学．杭州：浙江科学技术出版社．1064
44. Mallo M, Gridley T. Development of the mammalian ear: coordinate regulation of formation of the tympanic ring and the external acoustic meatus. Development. 1996 Jan; 122(1): 173-9.
45. Mallo M, Schrewe H, Martin JF, Olson EN, Ohnemus S. Assembling a functional tympanic membrane: signals from the external acoustic meatus coordinate development of the malleal manubrium. Development. 2000 Oct; 127(19): 4127-36.
46. Tatsuo M, Eiko H. Family lateral semicircular canal malformation with external and middle ear abnormalities. American Journal of Medical Genetics 2003; 116A: 360-367.
47. Kountakis SE, Helidonis E, Jahrsdoerfer RA. Microtia grade as an indicator of middle ear development in aural atresia. Arch Otolaryngol Head Neck Surg. 1995 Aug; 121(8): 885-6.
48. Orliaguet T, Dechelotte P, Scheye T, Vanneuville G. The relationship between Meckel's cartilage and the??development of the human fetal mandible. Surg Radiol Anat. 1993; 15 (2): 113-8.
49. Lorentowicz-Zagalak M, Przystanska A, Wozniak W. The development of Meckel' s cartilage in staged human embryos during the 5th week. 1: Folia Morphol (Warsz). 2005 Feb; 64(1): 23-8
50. http://222. 28. 98. 3/elite/oralpath/content/facedev-bone .htm : 下颌骨的发育
51. Diewert VM. Growth movements during prenatal development of human facial morphology. Prog Clin Biol Res. 1985; 187:57-66.
52. Bennun RD, Mulliken JB, Kaban LB, Murray JE Microtia: a microform of hemifacial microsomia. Plast Reconstr Surg. 1985 Dec;76(6):859-65.
53. Saunders JC. Auditory structure and function in the bird middle ear: an evaluation by SEM and capacitive probe. Hear Res. 1985 Jun; 18(3) : 253-68.
54.Chin K, Kurian R, Saunders JC. Maturation of tympanic membrane layers and collagen in the embryonic and post-hatch chick (Gallus domesticus). J Morphol. 1997 Sep; 233(3): 257-66.
55. von Bartheld CS. Functional morphology of the paratympanic organ in the middle ear of birds. Brain Behav Evol. 1994; 44 (2): 61-73.
56. Roberts DS, Miller SA. Apoptosis in cavitation of middleear space. Anat Rec. 1998 Jul;251(3):286-9.
57. Hogg DA. The development of pneumatisation in the skull of the domestic fowl (Gallus gallus domesticus). J Anat. 1990 Apr;169:139-51.
58. Kil SH, Collazo A. A review of inner ear fate maps and cell lineage studies. J Neurobiol. 2002 Nov 5;53(2):129-42.
59. Peusner KD. Development of the gravity sensing system. J Neurosci Res. 2001 Jan 15:63(2): 103-8.
60. Rebillard G, Rubel EW. Electrophysiological study of the maturation of auditory responses from the inner ear of the chick. Brain Res. 1981 Dec 14;229(1) : 15-23.
61. Cohen YE, Hernandez HN, Saunders JC. Middle-ear development: II. Morphometric changes in the conducting apparatus of the chick. J Morphol. 1992Jun;212(3) :257-67.
62. Kuratani S, Matsuo I, Aizawa S. Developmental patterning and evolution of the mammalian viscerocranium: genetic insights into comparative morphology. Dev Dyn. 1997 Jun; 209 (2): 139-55.
63. Francis-West P, Ladher R, Barlow A, Grave son A. Signalling interactions during facial development. Mech Dev. 1998 Jul; 75(1-2): 3-28.
64. James CS, Duncan RK, Doan DE, Werner YL. Structure of the bird middle ear. In: The middle ear of reptiles and birds: 45-62.
65. Cohen GM, Hersing W. Development of the chick's auditoryossicle, the columella. Physiologist. 1993 Feb;36(1 Suppl):S75-6.
66. Hamburger V, Hamilton HL. A seriea of normal stages in the development of the chick embryo. J Morphol 1951(88) :49-92
67. Noden DM. Interactions and fates of avian craniofacial mesenchyme. Development. 1988;103 Suppl:121-40..
68. Hunt P, Clarke JD, Buxton P, Ferretti P, Thorogood P. Segmentation, crest prespecification and the control of facial form. Eur J Oral Sci. 1998 Jan;106 Suppl 1:12-8.
1. Saunders JC. Auditory structure and function in the bird middle ear: an evaluation by SEM and capacitive probe. Hear Res. 1985 Jun; 18(3) : 253-68.
2. Chin K, Kurian R, Saunders JC. Maturation of tympanic membrane layers and collagen in the embryonic and post-hatch chick (Gallus domesticus). J Morphol. 1997 Sep; 233(3): 257-66.
3. von Bartheld CS. Functional morphology of the paratympanic organ in the middle ear of birds. Brain Behav Evol. 1994; 44 (2): 61-73.
4. Roberts DS, Miller SA. Apoptosis in cavitation of middle ear space. Anat Rec. 1998 Jul;251 (3) : 286-9.
5. Hogg DA. The development of pneumatisation in the skull of the domestic fowl (Gallus gallus domesticus). J Anat. 1990 Apr;169:139-51.
6. Kil SH, Collazo A. A review of inner ear fate maps and cell lineage studies. J Neurobiol. 2002 Nov 5;53(2):129-42.
7. Peusner KD. Development of the gravity sensing system. J Neurosci Res. 2001 Jan 15;63(2):103-8.
8. Rebillard G, Rubel EW. Electrophysiological study of the maturation of auditory responses from the inner ear of the chick. Brain Res. 1981 Dec 14;229(1):15-23.
9. Cohen YE, Hernandez HN, Saunders JC. Middle-ear development: Ⅱ. Morphometric changes in the conducting apparatus of the chick. J Morphol. 1992 Jun; 212(3): 257-67.
10. Cohen GM, Hersing W. Development of the chick's auditory ossicle, the columella. Physiologist. 1993 Feb; 36(1 Suppl): S75-6.
11. James CS, Duncan RK, Doan DE, Werner YL. Structure of the bird middle ear. In: The middle ear of reptiles and birds: 45-62.
12. Jaskoll TF, Maderson PF. A histological study of the development of the avian middle ear and tympanum. Anat Rec. 1978 Feb; 190(2): 177-99.
13．吴康，蔡文琴．原位杂交组织化学常用试剂及处理 http://www.bioon.com/experiment/situ/200404/8653.html
14. Elliott E, Dennison C, Fortgens PH, Travis J. Paraformaldehyde fixation of neutrophils for immunolabeling of granule antigens in cryoultrasections. J Histochem Cytochem. 1995 Oct; 43(10): 1019-25.
15. http://www.bioon.com/experiment/sol/86231.shtml: Ringer's solution
16. Ojeda JL, Barbosa E, Bosque PG. Selective skeletal staining in whole chicken embryos; a rapid Alcian blue technique. Stain Technol. 1970 May; 45(3): 137-8.
17. Dingerkus G, Uhler LD. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of??cartilage. Stain Technol. 1977 Jul;52(4):229-32.
18. French NA. Modeling incubation temperature: the effects of incubator design, embryonic development, and egg size. Poult Sci. 1997 Jan;76(1): 124-33.
19. Peebles ED, Brake J, Gildersleeve RP. Effects of eggshell cuticle removal and incubation humidity on embryonic development and hatchability of broilers. Poult Sci. 1987 May; 66 (5): 834-40.
20. Buhr RJ. Incubation relative humidity effects on allantoic fluid volume and hatchability. Poult Sci. 1995 May; 74 (5): 874-84.
21. Hullinger RL. The avian embryo responding to microgravity of space flight. Physiologist. 1993 Feb;36(1 Suppl) :S42-5.
22. Berman E, Chacon L, House D, Koch BA, Koch WE, Leal J, Lovtrup S, Mantiply E, Martin AH, Martucci GI, et al. Development of chicken embryos in a pulsed magnetic field. Bioelectromagnetics. 1990;11 (2):169-87.
23. Elibol O, Braket J. Effect of frequency of turning from three to eleven days of incubation on hatchability of broiler hatching eggs. Poult Sci. 2003 Mar;82(3):357-9.
24. Fasenko GM, Robinson FE, Whelan AI, Kremeniuk KM, Walker JA. Prestorage incubation of long-term stored broiler breeder eggs: 1. Effects on hatchability. Poult Sci. 2001 Oct; 80 (10):1406-11.
25. Noramly S, Grainger RM. Determination of the embryonicinner ear. J Neurobiol. 2002 Nov 5; 53(2): 100-28.
26. Francis-West PH, Ladher RK, Schoenwolf GC. Development of the sensory organs. Sci Prog. 2002; 85(Pt 2): 151-73.
27. Kuratani S, Matsuo I, Aizawa S. Developmental patterning and evolution of the mammalian viscerocranium: genetic insights into comparative morphology. Dev Dyn. 1997 Jun; 209(2): 139-55.
28. Francis-West P, Ladher R, Barlow A, Graveson A. Signalling interactions during facial development. Mech Dev. 1998 Jul; 75(1-2): 3-28.
29．王光辉；王力红；梁传余；李文．定向诱导骨髓间充质干细胞—藻酸盐复合物构建软骨组织．四川大学学报(医学版)，36(6)：830-833
30. Liu J, Kumar VP, Shen Y, Lau HK, Pereira BP, Pho RW Modified Sihler's technique for studying the distribution of intramuscular nerve branches in mammalian skeletal muscle. Anat Rec. 1997 Jan;247(1):137-44.
31. Gulabivala K, Aung TH, Alavi A, Ng YL. Root and canal morphology of Burmese mandibular molars. Int Endod J. 2001 Jul; 34(5): 359-70.
32. http://www.chemicalland21.com/lifescience/foco/METHYL％20SALICYLATE.htm
33. Kontges G, Lumsden A Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996 Oct; 122(10): 3229-42.34. Lumsden A, Sprawson N, Graham A. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development. 1991 Dec;113(4):1281-91.
35. Lumsden A, Keynes R. Segmental patterns of neuronal development in the chick hindbrain. Nature. 1989 Feb 2;337(6206): 424-8.
36. Lumsden A. Multipotent cells in the avian neural crest. Trends Neurosci. 1989 Mar;12(3):81-3.
37. Couly G, Creuzet S, Bennaceur S, Vincent C, Le Douarin NM. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development. 2002 Feb; 129(4): 1061-73.
38. Ruhin B, Creuzet S, Vincent C, Benouaiche L, Le Douarin NM, Couly G .Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm. Development Dynamics. 2003 Oct;228(2):239-46.
39. Coutifaris C, Omigbodun A, Coukos G. Integrins, endometrial maturation, & human embryo implantation. Semin Reprod Endocrinol. 1998; 16(3):219-29.
40. Tarin D. Embryonic induction. A review of current information on the mode of signal transmission and target response. Med Biol. 1978 Dec; 56 (6):380-5.
41. Saxen L Embryonic induction. Clin Obstet Gynecol. 1975 Dec; 18(4): 149-75.
42. Anniko M, Wikstrom SO, Wroblewski R. Microanalytic and light microscopic studies on the developing otic capsule. Acta Otolaryngol. 1987 Nov-Dec;104(5-6):429-38
43. Le Lievre CS Participation of neural crest-derived cells in the genesis of the skull in birds J Embryol Exp Morphol. 1978 Oct;47:17-37.
44. Couly GF, ColteyPM, Le Douarin NM The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development. 1993 Feb;117(2):409-29
45. Hamburger V, Hamilton HL. A seriea of normal stages in the development of the chick embryo. J Morphol 1951(88) :49-92.
46. OhtaY, KiddMT. Optimum site for in ovo amino acid injection in broiler breeder eggs. Poult Sci. 2001 Oct;80(10) : 1425-9.
47. Hogers B, DeRuiter MC, Baasten AM, Gittenberger-de Groot AC, Poelmann RE. Intracardiac blood flow patterns related to the yolk sac circulation of the chick embryo. Circ Res. 1995 May;76(5):871-7.
48. http://www. invitrogen. com/content/sfs/manuals/3742. pdf
49.Moraes AM, Pessoa CN, Vommaro RC, De Souza W, de Mello FG, Hokoc JN. Cultured embryonic retina systems as a model for the study of underlying mechanisms of Toxoplasma gondii infection. Invest Ophthalmol Vis Sci. 2004 Aug; 45(8):2813-21
50. http://nhjy. hzau. edu. cn/kech/zwsl/syzd/showdoc. asp?Id=1 15: 柠檬酸缓液
51. 丁濂，陈啸梅，许增禄．组织化学．见：方福德，周吕，丁濂，等，主编．现代医学实验技巧全书．上册．第一版．北京：北京医科大学中国协和医科大学联合出版社，1995年．105-176
52. Nagata M, Matsumura T. Action of the bacterial neutral protease, dispase, on cultured cells and its application to fluid suspension culture with a review on biomedical application of this protease. Jpn J Exp Med. 1986 Dec; 56(6): 297-307
53. Bednarczyk M, Lakota P, Siwek M. Improvement of hatchability of chicken eggs injected by blastoderm cells. Poult Sci. 2000 Dec; 79(12):1823-8.
54. Krieg R, Halbhuber KJ Recent advances in catalytic peroxidase histochemistry. Cell Mol Biol (Noisy-le-grand). 2003 Jun; 49(4): 547-63.
55. Teillet MA, Ziller C, Le Douarin NM. Quail-chick chimeras. Methods Mol Biol. 1999; 97: 305-18.
56. Le Douarin N, Dieterlen-Lievre F, Teillet MA. Quail-chick transplantations. Methods Cell Biol. 1996; 51: 23-59.
57. LeClair EE, Tuan RS. Quail-chick transplantation in the embryonic limb bud. Methods Mol Biol. 2000; 135: 387-96.
58. Alvarado-Mallart RM. The chick/quail transplantation model: discovery of the isthmic organizer center. Brain Res Brain Res Rev. 2005 Sep;49(2):109-13. Epub 2005 Apr 15.
59. Yan HF, Shao BN, Trefil P, Guo XX, Wu XL Achievements and applications in making chicken chimeras using BCs. Yi Chuan.??2004 Jul; 26 (4): 537-43.
60.Speksnijder G, Ivarie R. A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs. Poult Sci. 2000 Oct; 79(10): 1430-3.
61.Andacht T, Hu W, Ivarie R. Rapid and improved method for windowing eggs accessing the stage X chicken embryo. Mol Reprod Dev. 2004 Sep;69(1):31-4.
62. Bednarczyk M, Lakota P, Siwek M. Improvement of hatchability of chicken eggs injected by blastoderm cells. Poult Sci. 2000 Dec;79(12):1823-8.
63. Boettger T, Knoetgen H, Wittier L, Kessel M. The avian organizer. Int J Dev Biol. 2001 ;45(1) :281-7.
64. Asashima M, Kinoshita K, Ariizumi T, Malacinski GM. Role of activin and other peptide growth factors in body patterning in the early amphibian embryo. Int Rev Cytol. 1999:191:1-52.
65. Fedak TJ, Hall BK. Perspectives on hyperphalangy: patterns and processes J Anat. 2004 Mar;204(Pt 3):151-63.