压力及BDNF对体外培养的牛眼小梁细胞的影响
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摘要
目的：
1．探索并改进体外牛眼小梁细胞的培养方法。
2．研究压力对体外培养的牛眼小梁细胞的影响。
3．研究BDNF对体外培养的牛眼小梁细胞的影响。
4．研究BDNF对体外培养的加压条件下的牛眼小梁细胞的影响。
方法：
第一部分：牛眼小梁细胞体外培养的建立
取当地屠宰场1岁以内小牛眼，4℃运至实验室。无菌条件下分离小梁组织，剪成小块，接种于25cm~2培养瓶中；或将分离下来的小梁组织用2ml胰蛋白酶及胶原酶等量混合液孵箱内进行消化，收集细胞以10~4 cell／cm~2接种于50 ml培养瓶。培养液为含20％胎牛血清、100 U／ml青霉素、100 U／ml链霉素和2.5 μg／ml二性霉素B的DMEM。待细胞长满后用0.25％胰蛋白酶消化传代。
第二部分：压力对小梁细胞的影响
传三代牛眼小梁细胞以等量10~4 cell／cm~2接种于10ml培养瓶，约一周即至汇合期，换液并用胶塞塞紧培养瓶口，一注射器通过三通管接4号针头注入无菌空气，三通管另接压力计测量压力，设加压1.33、2.67、5.33、10.67KPa(1 KPa=7.5mmHg)四个实验组，未加压仅紧塞瓶口的细胞组设为对照组，此组压力为0，每组重复6瓶。
第 四 军 医 大 学 硕 士 学位 论 文
第三部分：BDNF对小梁细胞的影响
对传三代小梁细胞的培养液中加有 50 ng／ml 的 BDNF
作为实验组，不含BDNF 的空白培养液进行培养的为对照
组，测生长曲线；实验组与对照组分别加压 5．33 KPa 与
10．67 KPa，作用 24 h。
结果：
1．成功培养出性状良好的牛眼小梁细胞并传代，培养出的牛小
梁细胞生长良好，传代后稳定。
2．当作用于细胞的压力为 1．33、2．67 KPa，作用时间为 48 h
时，与对照组比较，小梁细胞形态结构无明显变化，台盼兰计数无
显著性差异（P＞0．05h而当压力为5．33＊pa，作用时间为24 h时，
细胞形态结构有轻度的损伤；压力为5．33 KPa作用48 h或10石7
KPa作用 24 h，细胞的损伤程度也进一步加重，甚至出现细胞脱壁
死亡。
3．50 ng＊l的BDNF的培养液明显促进传三代的小梁细胞达
汇合期。
4．加有 50 ng／ml BDNF培养液后，对于不同压力作用下的体外
培养的小梁细胞数量及活性均有保护作用，经统计学检验，有无生
长因于组间及不同压力组间均有统计学意义（P＜O．OS）。
结论：
1．消化法与组织块法均成功培养出性状良好的牛眼小梁细胞。
较之传统的组织块法，消化法简便易行，可以迅速获得大量供实验
用细胞，节约实验时间。
4
第 四 军 医 大 学 硕 土 学 位 论 文
2．小梁细胞承受压力的能力是有限的，压力过高或受压时间过
长都会对细胞造成损伤。
3．BDNF有促进正常小梁细胞生长的作用。
4．BDNF能够保护高压情况下小梁细胞的数量及活性，从而能
够维持小梁细胞正常生理功能而减轻高服压对眼组织的损害。
PUPROSE:
1. To improve the cell culture method of BTM cells in vitro.
2. To study the effects of pressures on cultured BTM cells in vitro.
3. To study the effects of BDNF on cultured BTM cells in vitro.
4. To study the effects of BDNF on cultured BTM cells under different pressures in vitro.
METHODS:
Part 1: Establishment of the cultured BTM cells in vitro
Bovine eyes were enucleated and carried to the lab under 4 ℃ . We separated TM tissues from bovine eyes under sterile conditions, inoculated to 25 cm2 culture bottles, or digested the TM tissues with 2 ml mixture which was composed of 0.25% Trypsin and 0.3 % Collagenase equally. Collected the cells and seeded into 50 ml culture bottle with 104 cell/cm2. The culture medium was DMEM containing 20% fetus serum, lOOU/ml penicillin, 100 U/ml streptomycin and 2.5 ng/ml amphotericin B. The cells were passaged when
they came to be confluent.
Part 2: The effects of pressures on cultured BTM cells in vitro
The third passaged BTM cells were seeded into 10 ml culture bottles with 104 cell/cm2, and about 1 week later they became to be confluent. We changed the culture medium, blocked the culture bottles and injected sterilized airs into the bottles through a T-shaped tube whose another entrance connected with a baresthesiometer. Four experimental groups were blocked and raised air pressures to 1.33, 2.67, 5.33 and 10.67Kpa (1 Kpa=7.5 mmHg) respectively. In control group, cells were only blocked in bottles. 6 bottles were repeated in every group.
Part 3: The effects of BDNF on cultured BTM cells in vitro The cells of experimental groups were cultured in the culture medium with 50 ng/ml BDNF, on the contrary the cells of the control group were cultured without BDNF. The cells' growth curve was drawn. Next, all groups were observed under the pressures of 5.33 KPa and 10.67 KPa for 24 h.
RESULTS:
1. BTM cells grew well and passaged normally by our culture method.
2. Compared with the control group, the cells under the pressure of 1.33 KPa or 2.67 KPa lasted 48 h had no
remarkable difference. There were slight changes in the group under 5.33 KPa pressure lasted 24 h. The cellular damages appeared more severe or even the cells appeared to death if the pressure was 5.33 KPa lasted 48 h or elevated to 10.67 KPa lasted 24 h.
3. The culture medium containing 50 ng/ml BDNF could obviously improve the third passaged BTM cells to be confluent.
4. Compared with the control group, BDNF could protect the cells' quantities and activities under different pressures. There was significant difference between the control group and the experimental groups (P<0.05).
CONCLUSIONS:
1. Not only digestion culture method but also tissue culture method could obtain well BTM cells. Compared with tissue culture method, digestion culture method was easier and more convenient to harvest enough cells for studies.
2. BTM cells kept alive only under a certain pressure level, and the cells would be damaged if the pressure was too high or the exposure time was too long.
3. BDNF could greatly improve BTM cells growth.
4. BDNF could protect the quantities and activities of BTM cells under a certain high pressure, thus could protect their normal physical functions and protect the ocular
tissues from being damaged by too high IOP.
1．探索并改进体外牛眼小梁细胞的培养方法。
2．研究压力对体外培养的牛眼小梁细胞的影响。
3．研究BDNF对体外培养的牛眼小梁细胞的影响。
4．研究BDNF对体外培养的加压条件下的牛眼小梁细胞的影响。
方法：
第一部分：牛眼小梁细胞体外培养的建立
取当地屠宰场1岁以内小牛眼，4℃运至实验室。无菌条件下分离小梁组织，剪成小块，接种于25cm~2培养瓶中；或将分离下来的小梁组织用2ml胰蛋白酶及胶原酶等量混合液孵箱内进行消化，收集细胞以10~4 cell／cm~2接种于50 ml培养瓶。培养液为含20％胎牛血清、100 U／ml青霉素、100 U／ml链霉素和2.5 μg／ml二性霉素B的DMEM。待细胞长满后用0.25％胰蛋白酶消化传代。
第二部分：压力对小梁细胞的影响
传三代牛眼小梁细胞以等量10~4 cell／cm~2接种于10ml培养瓶，约一周即至汇合期，换液并用胶塞塞紧培养瓶口，一注射器通过三通管接4号针头注入无菌空气，三通管另接压力计测量压力，设加压1.33、2.67、5.33、10.67KPa(1 KPa=7.5mmHg)四个实验组，未加压仅紧塞瓶口的细胞组设为对照组，此组压力为0，每组重复6瓶。
第 四 军 医 大 学 硕 士 学位 论 文
第三部分：BDNF对小梁细胞的影响
对传三代小梁细胞的培养液中加有 50 ng／ml 的 BDNF
作为实验组，不含BDNF 的空白培养液进行培养的为对照
组，测生长曲线；实验组与对照组分别加压 5．33 KPa 与
10．67 KPa，作用 24 h。
结果：
1．成功培养出性状良好的牛眼小梁细胞并传代，培养出的牛小
梁细胞生长良好，传代后稳定。
2．当作用于细胞的压力为 1．33、2．67 KPa，作用时间为 48 h
时，与对照组比较，小梁细胞形态结构无明显变化，台盼兰计数无
显著性差异（P＞0．05h而当压力为5．33＊pa，作用时间为24 h时，
细胞形态结构有轻度的损伤；压力为5．33 KPa作用48 h或10石7
KPa作用 24 h，细胞的损伤程度也进一步加重，甚至出现细胞脱壁
死亡。
3．50 ng＊l的BDNF的培养液明显促进传三代的小梁细胞达
汇合期。
4．加有 50 ng／ml BDNF培养液后，对于不同压力作用下的体外
培养的小梁细胞数量及活性均有保护作用，经统计学检验，有无生
长因于组间及不同压力组间均有统计学意义（P＜O．OS）。
结论：
1．消化法与组织块法均成功培养出性状良好的牛眼小梁细胞。
较之传统的组织块法，消化法简便易行，可以迅速获得大量供实验
用细胞，节约实验时间。
4
第 四 军 医 大 学 硕 土 学 位 论 文
2．小梁细胞承受压力的能力是有限的，压力过高或受压时间过
长都会对细胞造成损伤。
3．BDNF有促进正常小梁细胞生长的作用。
4．BDNF能够保护高压情况下小梁细胞的数量及活性，从而能
够维持小梁细胞正常生理功能而减轻高服压对眼组织的损害。
PUPROSE:
1. To improve the cell culture method of BTM cells in vitro.
2. To study the effects of pressures on cultured BTM cells in vitro.
3. To study the effects of BDNF on cultured BTM cells in vitro.
4. To study the effects of BDNF on cultured BTM cells under different pressures in vitro.
METHODS:
Part 1: Establishment of the cultured BTM cells in vitro
Bovine eyes were enucleated and carried to the lab under 4 ℃ . We separated TM tissues from bovine eyes under sterile conditions, inoculated to 25 cm2 culture bottles, or digested the TM tissues with 2 ml mixture which was composed of 0.25% Trypsin and 0.3 % Collagenase equally. Collected the cells and seeded into 50 ml culture bottle with 104 cell/cm2. The culture medium was DMEM containing 20% fetus serum, lOOU/ml penicillin, 100 U/ml streptomycin and 2.5 ng/ml amphotericin B. The cells were passaged when
they came to be confluent.
Part 2: The effects of pressures on cultured BTM cells in vitro
The third passaged BTM cells were seeded into 10 ml culture bottles with 104 cell/cm2, and about 1 week later they became to be confluent. We changed the culture medium, blocked the culture bottles and injected sterilized airs into the bottles through a T-shaped tube whose another entrance connected with a baresthesiometer. Four experimental groups were blocked and raised air pressures to 1.33, 2.67, 5.33 and 10.67Kpa (1 Kpa=7.5 mmHg) respectively. In control group, cells were only blocked in bottles. 6 bottles were repeated in every group.
Part 3: The effects of BDNF on cultured BTM cells in vitro The cells of experimental groups were cultured in the culture medium with 50 ng/ml BDNF, on the contrary the cells of the control group were cultured without BDNF. The cells' growth curve was drawn. Next, all groups were observed under the pressures of 5.33 KPa and 10.67 KPa for 24 h.
RESULTS:
1. BTM cells grew well and passaged normally by our culture method.
2. Compared with the control group, the cells under the pressure of 1.33 KPa or 2.67 KPa lasted 48 h had no
remarkable difference. There were slight changes in the group under 5.33 KPa pressure lasted 24 h. The cellular damages appeared more severe or even the cells appeared to death if the pressure was 5.33 KPa lasted 48 h or elevated to 10.67 KPa lasted 24 h.
3. The culture medium containing 50 ng/ml BDNF could obviously improve the third passaged BTM cells to be confluent.
4. Compared with the control group, BDNF could protect the cells' quantities and activities under different pressures. There was significant difference between the control group and the experimental groups (P<0.05).
CONCLUSIONS:
1. Not only digestion culture method but also tissue culture method could obtain well BTM cells. Compared with tissue culture method, digestion culture method was easier and more convenient to harvest enough cells for studies.
2. BTM cells kept alive only under a certain pressure level, and the cells would be damaged if the pressure was too high or the exposure time was too long.
3. BDNF could greatly improve BTM cells growth.
4. BDNF could protect the quantities and activities of BTM cells under a certain high pressure, thus could protect their normal physical functions and protect the ocular
tissues from being damaged by too high IOP.
引文
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57. Wordinger RJ, Clark AF, Agarwal R, Lambert W, McNatt L, Wilson SE, Qu Z, Fung BK. Cultured human trabecular meshwork cells express functional growth factor receptors. Invest Ophthalmol Vis Sci. 1998; 39(9): 1575-1589.
58. Wordinger RJ, Clark AF, Agarwal R, Lambert W, Wilson SE. Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork. Invest Ophthalmol Vis Sci. 1999; 40(1): 242-247.
59. Reim H, Rohen JW, Dittrich JK. Clinical picture, histological and electron microscopy ophthalmic findings in an infant with Pfaundler-Hurler mucopolysaccharidosis. Klin Monatsbl Augenheilkd. 1971; 159(4): 444-456.
60. Shay-Whey M, Tse-Hsiang Y, Steven M, Margaret L, Eve J, Douglas e, Beatrice Y. Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth, Invest Ophthalmol Vis Sci. 1997; 38(13): 2781-2791.
61. Tripathi BJ, Tripathi RC, Livingston AM, Borisuth NS. The role of growth factors in the embryogenesis and differentiation of the eye. Am J Anat 1991; 192(4): 442-471.
62. Tripathi RC, Tripathi BJ, Jakobier FA. Ocular Anatomy Emrbyology Terarology. Harper and Row, 1982; 5(2): 197-284.
63. Barde YA, Edgar D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1982;
1(5): 549-553.
64. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A. Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron. 1991; 7(5): 857-866.
65. Ip NY, Ibanez CF, Nye SH, McClain J, Jones PF, Gies DR, Belluscio L, Le Beau MM, Espinosa R 3rd, Squinto SP, et al. Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci. 1992; 89(15): 3060-3064.
66. Wordinger RJ, Lambert W, Agarwal R, Talati M, Clark AF. Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk). Invest Ophthalmol Vis Sci. 2000; 41(12): 3833-3341.
67. Pereira SP, Araujo EG. Veratridine increases the survival of retinal ganglion cells in vitro. Braz J Med Biol Res. 1997; 30(12): 1467-1470.
68. Theoret H, Herbin M, Boire D, Ptito M. Transneuronal retrograde degeneration of retinal ganglion cells following cerebral hemispherectomy in cats. Brain Res. 1997; 14 (1-2): 203-208.
69. Wong RO, Yamawaki RM, Shatz CJ. Synaptic Contacts and the Transient Dendritic Spines of Developing Retinal Ganglion Cells. Eur J Neurosci. 1992; 4(12): 1387-1397.
70. Yu W, Miller RF. Hyperosmotic activation of transmitter
release from presynaptic terminals onto retinal ganglion cells. J Neurosci Methods. 1995; 62(1-2): 159-168.
71. Mey J, Thanos S. Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo. Brain Res. 1993; 602(5): 304-317.
72. McConnell P, Skaper S, Williams LR, Varon S. Purification of adult rat sciatic nerve ciliary neuronotrophic factor. Brain Res. 1986; 3(2): 362-365.
73. Peinado-Ramon P, Salvador M, Villegas-Perez MP, et al. Effects of axotomy and intraocular administration of NT-4, NT-3 and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. Invest Ophthalmol Vis Sci, 1996, 37(6): 489-500.
74. Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron. 1995; 15(4): 805-819.
75. Rothe T, Grantyn R. Retinal ganglion neurons express a toxin-resistant developmentally regulated novel type of high-voltage-activated calcium channel. J Neurophysiol. 1994; 72(5): 2542-2546.
76. Ary-Pires R, Nakatani M, Rehen SK, Linden R. Developmentally regulated release of intraretinal neurotrophic factors in vitro. Int J Dev Neurosci. 1997; 15(2): 239-255.
77．胡诞宁，Robert Ritch，神经营养因子与生长因子在体外对人视网膜神经节细胞之作用．中华眼底病杂志，1999；15(3)：149-152．
78. Takahashi N, Cummins D, Caprioli J. Rat retinal ganglion cells in culture. Exp Eye Res, 1991; 5(3): 565-572.
79. Cohen A, Bray GM, Aguayo AJ. Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro. J Neurobiol. 1994; 25(8): 953-959.
80. Mansour-Robaey S, Clarke DB, Wang YC, Bray GM, Aguayo AJ. Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci. 1994; 91(12): 1632-1636.
81. Stefan Isenmann, Nikolaj Klcker, Claude Gravel and Mathias Bihr. Short communication protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo. European Journal of Neuroscience. 1998; 10(9): 2751-2756
82．李凤鸣主编，眼科全书，人民卫生出版社(北京)，1996：35-46．
83. Cui Q, Harvey AR. At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats. J Neurosci. 1995; 15(12): 8143-8155.
84. Mei-Lan Ko, Dan-Ning Hu, Robert Ritch, Sansar C, Sharma. The combined effect of Brain-Derived Neurotrophic Factor and a free radical scavenger in experimental glaucoma.
Invest Ophthalmol Vis Sci. 2000; 41(10): 2967-2971.
85. Ebendal T, Hedlund KO, Norrgren G. Nerve growth factors in chick tissues. JNeurosci Res. 1982; 8(2-3): 153-164.
86. Iok-Hou Pang, PhD, Mitchell D. McCartney, PhD, H. Thomas Steely, PhD, and Abbot F. Clark, PhD. Human ocular perfusion organ culture: a versatile ex vivo model for glaucoma research. Journal of Glaucoma. 2000; 9(4): 468-479.
1．杨新光，李美玉．牛眼小梁网细胞体外培养及吞噬功能的研究．中华眼科杂志，1996；32(2)：136-139．
2．葛坚，林明楷，卓业鸿．糖皮质激素性青光眼患者小梁细胞体外培养和超微结构研究．中华眼科杂志，2000；36(3)：238-240．
3．钟丽春，李美玉．小梁细胞的培养与原发性开角型青光眼的病因研究．国外医学眼科学分册，1995；19(1)：46-51．
4. Coroneo MT, Korbmacher C, Flugel C, Stiemer B, Lutjen-Drecoll E, Wiederholt M. Electrical and morphological evidence for heterogeneous populations of cultured bovine trabecular meshwork cells. Exp Eye Res. 1991; 52(3): 375-388.
5. Anderson PJ, Wang J, Epstein DL. Metabolism of calf trabecular (reticular) meshwork. Invest Ophthalmol Vis Sci.
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11．杨新光，李美玉，张东杲．牛眼前房小梁细胞体外培养的建立．第四军医大学学报，1995；16(3)：236-237．
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22．葛坚，卓业鸿．人眼小梁细胞体外培养及生物学特性研究．眼科学报，1996；12(1)：64-69．
23．王林，魏厚仁，曾水清．人眼小梁细胞组织培养及超微结构观察．中华眼科杂志，1989；2(1)：97-99．
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50. Liu X, Cai S, Glasser A, Volberg T, Polansky JR, Fauss DJ, Brandt CR, Geiger B, Kaufman PL. Effect of H-7 on cultured
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51. Tumminia SJ, Mitton KP, Arora J, Zelenka P, Epstein DL, Russell P. Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 1998; 39(8): 1361-1371.
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55. Cai S, Liu X, Glasser A, Volberg T, Filla M, Geiger B, Polansky JR, Kaufman PL. Effect of latrunculin-A on morphology and actin-associated adhesions of cultured human trabecular meshwork cells. Mol Vis. 2000; 28(16): 6132-6143.
56. Gills JP, Roberts BC, Epstein DL. Microtubule disruption leads to cellular contraction in human trabecular meshwork
cells. Invest Ophthalmol Vis Sci. 1998; 39(3): 653-658.
57. Wordinger RJ, Clark AF, Agarwal R, Lambert W, McNatt L, Wilson SE, Qu Z, Fung BK. Cultured human trabecular meshwork cells express functional growth factor receptors. Invest Ophthalmol Vis Sci. 1998; 39(9): 1575-1589.
58. Wordinger RJ, Clark AF, Agarwal R, Lambert W, Wilson SE. Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork. Invest Ophthalmol Vis Sci. 1999; 40(1): 242-247.
59. Reim H, Rohen JW, Dittrich JK. Clinical picture, histological and electron microscopy ophthalmic findings in an infant with Pfaundler-Hurler mucopolysaccharidosis. Klin Monatsbl Augenheilkd. 1971; 159(4): 444-456.
60. Shay-Whey M, Tse-Hsiang Y, Steven M, Margaret L, Eve J, Douglas e, Beatrice Y. Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth, Invest Ophthalmol Vis Sci. 1997; 38(13): 2781-2791.
61. Tripathi BJ, Tripathi RC, Livingston AM, Borisuth NS. The role of growth factors in the embryogenesis and differentiation of the eye. Am J Anat 1991; 192(4): 442-471.
62. Tripathi RC, Tripathi BJ, Jakobier FA. Ocular Anatomy Emrbyology Terarology. Harper and Row, 1982; 5(2): 197-284.
63. Barde YA, Edgar D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1982;
1(5): 549-553.
64. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A. Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron. 1991; 7(5): 857-866.
65. Ip NY, Ibanez CF, Nye SH, McClain J, Jones PF, Gies DR, Belluscio L, Le Beau MM, Espinosa R 3rd, Squinto SP, et al. Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci. 1992; 89(15): 3060-3064.
66. Wordinger RJ, Lambert W, Agarwal R, Talati M, Clark AF. Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk). Invest Ophthalmol Vis Sci. 2000; 41(12): 3833-3341.
67. Pereira SP, Araujo EG. Veratridine increases the survival of retinal ganglion cells in vitro. Braz J Med Biol Res. 1997; 30(12): 1467-1470.
68. Theoret H, Herbin M, Boire D, Ptito M. Transneuronal retrograde degeneration of retinal ganglion cells following cerebral hemispherectomy in cats. Brain Res. 1997; 14 (1-2): 203-208.
69. Wong RO, Yamawaki RM, Shatz CJ. Synaptic Contacts and the Transient Dendritic Spines of Developing Retinal Ganglion Cells. Eur J Neurosci. 1992; 4(12): 1387-1397.
70. Yu W, Miller RF. Hyperosmotic activation of transmitter
release from presynaptic terminals onto retinal ganglion cells. J Neurosci Methods. 1995; 62(1-2): 159-168.
71. Mey J, Thanos S. Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo. Brain Res. 1993; 602(5): 304-317.
72. McConnell P, Skaper S, Williams LR, Varon S. Purification of adult rat sciatic nerve ciliary neuronotrophic factor. Brain Res. 1986; 3(2): 362-365.
73. Peinado-Ramon P, Salvador M, Villegas-Perez MP, et al. Effects of axotomy and intraocular administration of NT-4, NT-3 and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. Invest Ophthalmol Vis Sci, 1996, 37(6): 489-500.
74. Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron. 1995; 15(4): 805-819.
75. Rothe T, Grantyn R. Retinal ganglion neurons express a toxin-resistant developmentally regulated novel type of high-voltage-activated calcium channel. J Neurophysiol. 1994; 72(5): 2542-2546.
76. Ary-Pires R, Nakatani M, Rehen SK, Linden R. Developmentally regulated release of intraretinal neurotrophic factors in vitro. Int J Dev Neurosci. 1997; 15(2): 239-255.
77．胡诞宁，Robert Ritch，神经营养因子与生长因子在体外对人视网膜神经节细胞之作用．中华眼底病杂志，1999；15(3)：149-152．
78. Takahashi N, Cummins D, Caprioli J. Rat retinal ganglion cells in culture. Exp Eye Res, 1991; 5(3): 565-572.
79. Cohen A, Bray GM, Aguayo AJ. Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro. J Neurobiol. 1994; 25(8): 953-959.
80. Mansour-Robaey S, Clarke DB, Wang YC, Bray GM, Aguayo AJ. Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci. 1994; 91(12): 1632-1636.
81. Stefan Isenmann, Nikolaj Klcker, Claude Gravel and Mathias Bihr. Short communication protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo. European Journal of Neuroscience. 1998; 10(9): 2751-2756
82．李凤鸣主编，眼科全书，人民卫生出版社(北京)，1996：35-46．
83. Cui Q, Harvey AR. At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats. J Neurosci. 1995; 15(12): 8143-8155.
84. Mei-Lan Ko, Dan-Ning Hu, Robert Ritch, Sansar C, Sharma. The combined effect of Brain-Derived Neurotrophic Factor and a free radical scavenger in experimental glaucoma.
Invest Ophthalmol Vis Sci. 2000; 41(10): 2967-2971.
85. Ebendal T, Hedlund KO, Norrgren G. Nerve growth factors in chick tissues. JNeurosci Res. 1982; 8(2-3): 153-164.
86. Iok-Hou Pang, PhD, Mitchell D. McCartney, PhD, H. Thomas Steely, PhD, and Abbot F. Clark, PhD. Human ocular perfusion organ culture: a versatile ex vivo model for glaucoma research. Journal of Glaucoma. 2000; 9(4): 468-479.
1．杨新光，李美玉．牛眼小梁网细胞体外培养及吞噬功能的研究．中华眼科杂志，1996；32(2)：136-139．
2．葛坚，林明楷，卓业鸿．糖皮质激素性青光眼患者小梁细胞体外培养和超微结构研究．中华眼科杂志，2000；36(3)：238-240．
3．钟丽春，李美玉．小梁细胞的培养与原发性开角型青光眼的病因研究．国外医学眼科学分册，1995；19(1)：46-51．
4. Coroneo MT, Korbmacher C, Flugel C, Stiemer B, Lutjen-Drecoll E, Wiederholt M. Electrical and morphological evidence for heterogeneous populations of cultured bovine trabecular meshwork cells. Exp Eye Res. 1991; 52(3): 375-388.
5. Anderson PJ, Wang J, Epstein DL. Metabolism of calf trabecular (reticular) meshwork. Invest Ophthalmol Vis Sci.
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