维拉帕米诱导人视网膜色素上皮细胞凋亡的实验研究
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摘要
目的
增殖性玻璃体视网膜病变(简称PVR)是孔源性视网膜脱离(简称RRD)的常见并发症和手术失败的主要原因，尽管玻璃体视网膜手术技术不断提高，但复发率并未改善，同时玻切手术本身就是诱导PVR形成的一个危险因素，因此，如何在PVR形成的早期就应用药物抑制其发生和发展是眼科医生急需解决的问题。视网膜色素上皮细胞(简称RPE)是PVR形成时最重要的细胞。抑制PVR的关键就是抑制RPE细胞的增殖。近期PVR研究的热点是借助凋亡途径控制细胞的增殖，主要是施加凋亡诱导剂，使迁移增殖的RPE细胞发生凋亡，从根本上阻止PVR膜的形成和进一步发展。现今用于诱导RPE细胞凋亡的药物主要是抗代谢药，这些药副作用大，患者不易接受。维拉帕米是一种可靠的钙通道阻滞剂，副作用小、方便、价廉、易于得到。研究证明，维拉帕米对其他组织细胞的凋亡具有双重调节作用，同时钙离子在凋亡中的作用也不十分清楚，且有关维拉帕米对RPE凋亡的诱导作用及机制国内外均未见报道。因此本实验进行了人视网膜色素上皮细胞原代和传代培养，施加维拉帕米诱导其凋亡，来确定诱导RPE凋亡的有效作用浓度和时间，从而为今后探讨维拉帕米在增殖性疾病中的临床应用奠定理论基础。
方法
一、组织来源及准备
在无菌条件下，取我校一院眼科角膜移植后的供体眼球。
二、人RPE细胞的培养
无菌状态下，采用酶消化法，分离和种植人RPE细胞于24孔
板内，待细胞融合后，胰酶传代。
三机’I’f比色法测定药物对RPE增生的影响
将第 3代 RPE细胞以 2 X 10’个／00gi的细胞密度接种于 96
IL塑料培养板中，加人40m牙L，80m珍L，160m才L，320m牙L的维
拉帕米，培养24小时后，应用M’I’f比色法，在全自动酶标仪上测
其在490urn处的吸光值A。计算细胞增生抑制率，并对数据进行
统计学分析。
四、凋亡的检测
将第3代的RPE细胞分三组进行凋亡检测。第一和第二组
分别加人维拉帕米80mg／L和 160 mg／L，第三组作为对照组未加
药。采用HE染色、AO荧光染色以及透射电镜进行观察。以
80mg／L，160 mg／L浓度的维拉帕米，作用 12－72小日后，力人 PI
染液后，经流式细胞仪检测。通过计算凋亡区（亚二倍体峰）细胞
所占百分比得出凋亡率。
结 果
在测定药物对RPE增生影响的实验中，细胞抑制率分别为
13．55％、16．71％、42．34％和51．of％，随着浓度增大，细胞抑
制率不断上升，结合方差分析和t检验证明维拉帕米对人RPE细
胞增殖有明显的抑制作用，且呈明显的剂量依赖关系。结合光镜。
电镜、荧光显微镜和流式细胞仪检测凋亡所得结果为80 mgll和
160mg／l维拉帕米均可诱导RPE凋亡。在同一浓度时，随着时间
延长，凋亡率不断增加，在同一时间内，药物浓度增加，凋亡率也不
断增大，经统计学分析证明其差异具有显著性。结果显示 160mg／
L维拉帕米作用 72小时，凋亡率可达 49．3％。
结 论
1．维拉帕米能有效抑制体外培养的人RPE细胞增生，且呈明
·2·
显的剂量依赖关系。
2．维拉帕米能够诱导体外培养的人RPE细胞凋亡，本实验条
件下，160mg／L是诱导凋亡的最适浓度，72 ／J’时是凋亡的最佳作
用时间。
Purpose
Proliferative vitreoretinopathy(PVR) is the common complication and the main cause of failure of rhegmatogenous retinal detachment (RRD) operation. Although the technique of vitreoretinal operation promoted gradually, the recurrence rate is not improved. And Vitrecto-my itself is a main risk factor in inducing the formation of PVR. Therefore, how to use medicines to inhibit the PVR& formation and development in the earlier period is an important problem need to solve. Retinal pigment epithelium ( RPE) is the most important cell in developing PVR. The key point of blocking PVR is inhibiting the proliferation of RPE. Recently,the hot research spot in PVR is controlling the proliferation of RPE by apoptosis . That is Adding the induced ?apoptosis agents to make the RPEs with migration and proliferation apoptosis. so that the formation and further development of PVR membrane are blocked fundamentally. At present, the drug of inducing RPE apoptosis is mainly Antimetabolite, which has great side effect and is not
easy to accepted by patients. Verapamil is a kind of reliable calcium channel blocking agent, which has small side effect. And it is cheap, convenient and easy to be obtained. It is known that verapamil has double adjustment effect to the apoptosis of other tissue cells. Meanwhile, the function of the Ca + in apoptosis is not very clear. As we know, till now, there is no report about the induced function of verapamil to the
RPE apoptosis and how it works in domestic and oversea areas. Therefore , we successfully complete primitive culture and subculture for human retinal pigment epithelium, and add Verapamil to induce the apoptosis in order to explore the effective concentration and time in inducing apoptosis. Thus it builds the theoretical foundation for the future research of the clinic application of verapamil to the proliferative disease.
Material and Method
1. Tissue source and preparation
Under sterile condition, we obtain the donator after the cornea transplantation from department of ophthalmology in the first affiliated hospital in China Medical University.
2. The culture of Human RPE
Under sterile condition, using enzyme digestive method to isolate and complete plantation of humans RPE by 0. 25% trypsin to 24 - pore culture plate. The cells is transfered by 0. 25% trypsin after they become a confluent monolayer.
3. Determination of the drug influnce to the proliferation of RPE through MTT colorimetric metrod
The cultured RPEs at density of 2 × 104/100μJ are plantated in 96 - pore culture plate. Adding verapamil at the concentration of 40mg/L,80mg/L,160mg/L,320mg/L to the cell in 96 -pore culture plate, after 24 hours, we measure the absorbency in 490nm wavelength on auto - enzyme - labeled meter using MTT colorimetric method . Statistics analysis is carried out to the data obtained.
4. Verification of apoptosis
We choose three groups of the third generation RPE to compare
the apoptosis result. The first and the second group are added verapamil at the concentration of 80mg/L and 160mg/L respectively, and the third group is as a standard without verapamil. Three methods are used to observe the process. They are HE stain, Acridine Orange (AO) fluorescent stain and transmission electron microscopy. Adding PI stain to the RPEs which use verapamil within 12-72 hours, the specimens are measured by the flow cytometer. The apoptosis rate is obtained through calculating the quantity of cells in the apoptosis area
Result
The inhibiting rate is 13. 55% , 16. 71% , 42. 34% and 51. 01% respectively in the experiment of determination of the drug influence to the proliferation of RPE. . With the increase of the concentration of verapamil, the inhibiting rates rise. Based on the analysis of variance and t - test, it is verified that verapamil has strong inhibition on Human RPE ( HRPE) and an obvious dosage - dependent relationship. According to the results from the light microscopy, electron microscopy,fluorescence microscope and flow cytometer, the verapamil at the con
增殖性玻璃体视网膜病变(简称PVR)是孔源性视网膜脱离(简称RRD)的常见并发症和手术失败的主要原因，尽管玻璃体视网膜手术技术不断提高，但复发率并未改善，同时玻切手术本身就是诱导PVR形成的一个危险因素，因此，如何在PVR形成的早期就应用药物抑制其发生和发展是眼科医生急需解决的问题。视网膜色素上皮细胞(简称RPE)是PVR形成时最重要的细胞。抑制PVR的关键就是抑制RPE细胞的增殖。近期PVR研究的热点是借助凋亡途径控制细胞的增殖，主要是施加凋亡诱导剂，使迁移增殖的RPE细胞发生凋亡，从根本上阻止PVR膜的形成和进一步发展。现今用于诱导RPE细胞凋亡的药物主要是抗代谢药，这些药副作用大，患者不易接受。维拉帕米是一种可靠的钙通道阻滞剂，副作用小、方便、价廉、易于得到。研究证明，维拉帕米对其他组织细胞的凋亡具有双重调节作用，同时钙离子在凋亡中的作用也不十分清楚，且有关维拉帕米对RPE凋亡的诱导作用及机制国内外均未见报道。因此本实验进行了人视网膜色素上皮细胞原代和传代培养，施加维拉帕米诱导其凋亡，来确定诱导RPE凋亡的有效作用浓度和时间，从而为今后探讨维拉帕米在增殖性疾病中的临床应用奠定理论基础。
方法
一、组织来源及准备
在无菌条件下，取我校一院眼科角膜移植后的供体眼球。
二、人RPE细胞的培养
无菌状态下，采用酶消化法，分离和种植人RPE细胞于24孔
板内，待细胞融合后，胰酶传代。
三机’I’f比色法测定药物对RPE增生的影响
将第 3代 RPE细胞以 2 X 10’个／00gi的细胞密度接种于 96
IL塑料培养板中，加人40m牙L，80m珍L，160m才L，320m牙L的维
拉帕米，培养24小时后，应用M’I’f比色法，在全自动酶标仪上测
其在490urn处的吸光值A。计算细胞增生抑制率，并对数据进行
统计学分析。
四、凋亡的检测
将第3代的RPE细胞分三组进行凋亡检测。第一和第二组
分别加人维拉帕米80mg／L和 160 mg／L，第三组作为对照组未加
药。采用HE染色、AO荧光染色以及透射电镜进行观察。以
80mg／L，160 mg／L浓度的维拉帕米，作用 12－72小日后，力人 PI
染液后，经流式细胞仪检测。通过计算凋亡区（亚二倍体峰）细胞
所占百分比得出凋亡率。
结 果
在测定药物对RPE增生影响的实验中，细胞抑制率分别为
13．55％、16．71％、42．34％和51．of％，随着浓度增大，细胞抑
制率不断上升，结合方差分析和t检验证明维拉帕米对人RPE细
胞增殖有明显的抑制作用，且呈明显的剂量依赖关系。结合光镜。
电镜、荧光显微镜和流式细胞仪检测凋亡所得结果为80 mgll和
160mg／l维拉帕米均可诱导RPE凋亡。在同一浓度时，随着时间
延长，凋亡率不断增加，在同一时间内，药物浓度增加，凋亡率也不
断增大，经统计学分析证明其差异具有显著性。结果显示 160mg／
L维拉帕米作用 72小时，凋亡率可达 49．3％。
结 论
1．维拉帕米能有效抑制体外培养的人RPE细胞增生，且呈明
·2·
显的剂量依赖关系。
2．维拉帕米能够诱导体外培养的人RPE细胞凋亡，本实验条
件下，160mg／L是诱导凋亡的最适浓度，72 ／J’时是凋亡的最佳作
用时间。
Purpose
Proliferative vitreoretinopathy(PVR) is the common complication and the main cause of failure of rhegmatogenous retinal detachment (RRD) operation. Although the technique of vitreoretinal operation promoted gradually, the recurrence rate is not improved. And Vitrecto-my itself is a main risk factor in inducing the formation of PVR. Therefore, how to use medicines to inhibit the PVR& formation and development in the earlier period is an important problem need to solve. Retinal pigment epithelium ( RPE) is the most important cell in developing PVR. The key point of blocking PVR is inhibiting the proliferation of RPE. Recently,the hot research spot in PVR is controlling the proliferation of RPE by apoptosis . That is Adding the induced ?apoptosis agents to make the RPEs with migration and proliferation apoptosis. so that the formation and further development of PVR membrane are blocked fundamentally. At present, the drug of inducing RPE apoptosis is mainly Antimetabolite, which has great side effect and is not
easy to accepted by patients. Verapamil is a kind of reliable calcium channel blocking agent, which has small side effect. And it is cheap, convenient and easy to be obtained. It is known that verapamil has double adjustment effect to the apoptosis of other tissue cells. Meanwhile, the function of the Ca + in apoptosis is not very clear. As we know, till now, there is no report about the induced function of verapamil to the
RPE apoptosis and how it works in domestic and oversea areas. Therefore , we successfully complete primitive culture and subculture for human retinal pigment epithelium, and add Verapamil to induce the apoptosis in order to explore the effective concentration and time in inducing apoptosis. Thus it builds the theoretical foundation for the future research of the clinic application of verapamil to the proliferative disease.
Material and Method
1. Tissue source and preparation
Under sterile condition, we obtain the donator after the cornea transplantation from department of ophthalmology in the first affiliated hospital in China Medical University.
2. The culture of Human RPE
Under sterile condition, using enzyme digestive method to isolate and complete plantation of humans RPE by 0. 25% trypsin to 24 - pore culture plate. The cells is transfered by 0. 25% trypsin after they become a confluent monolayer.
3. Determination of the drug influnce to the proliferation of RPE through MTT colorimetric metrod
The cultured RPEs at density of 2 × 104/100μJ are plantated in 96 - pore culture plate. Adding verapamil at the concentration of 40mg/L,80mg/L,160mg/L,320mg/L to the cell in 96 -pore culture plate, after 24 hours, we measure the absorbency in 490nm wavelength on auto - enzyme - labeled meter using MTT colorimetric method . Statistics analysis is carried out to the data obtained.
4. Verification of apoptosis
We choose three groups of the third generation RPE to compare
the apoptosis result. The first and the second group are added verapamil at the concentration of 80mg/L and 160mg/L respectively, and the third group is as a standard without verapamil. Three methods are used to observe the process. They are HE stain, Acridine Orange (AO) fluorescent stain and transmission electron microscopy. Adding PI stain to the RPEs which use verapamil within 12-72 hours, the specimens are measured by the flow cytometer. The apoptosis rate is obtained through calculating the quantity of cells in the apoptosis area
Result
The inhibiting rate is 13. 55% , 16. 71% , 42. 34% and 51. 01% respectively in the experiment of determination of the drug influence to the proliferation of RPE. . With the increase of the concentration of verapamil, the inhibiting rates rise. Based on the analysis of variance and t - test, it is verified that verapamil has strong inhibition on Human RPE ( HRPE) and an obvious dosage - dependent relationship. According to the results from the light microscopy, electron microscopy,fluorescence microscope and flow cytometer, the verapamil at the con
引文
1. The Retina Society Terminology Committee. The Classification of Retinal Detachment with Proliferative Vitreoretinopathy. Ophthalmology 1983;90:121-125.
2. Minihan M, Tanner V, Williamson TH. Primary rhegmatogenous retinal detachment: 20 years of change. Br J Ophthalmol 2001 May;85(5):546-8.
3. Campochiaro PA Pathogenic mechanisms in proliferative vitreoretinopathy. Arch Ophthalmol, 1997, 115:237-241
4. Mandelcorn MS, Machemer R, Fineberg E, Hersch SB. Proliferation and metaplasia of intravitreal retinal pigment epithelium cell autotransplants. Am J Ophthalmol 1975;80:227-237.
5. Preston MR. Effects of Calcium Channel Blockers on Cellar Apoptosis. CANCER 1999 May;85(10):2093-2102.
6．鄂征 组织培养和分子细胞学技术1994．12：142-143
7. Glaser BM, Vidaurri-Leal J, Michels RG. Cryotherapy during surgery for giant retinal tears and intravitreal dispersion of viable retinal pigment epithelial cells. Ophthalmology 1993;100:466-470.
8. H K Muller. Genesis of apoptosis. BMJ 1994;308:1441
9．李秋明，宋绣雯，刘新华等。维拉帕米抗人巩膜成纤维细胞增殖的实验研究。中华眼底病杂志，1996，12(1)：98
10．姜彩辉，张卯年，张鲲等。维拉帕米对兔视网膜色素上皮细胞增殖的抑制作用。军医进修学报1999：3(20)
11．彭黎明，王曾礼。细胞凋亡的基础与临床。2000，39-40。
12．喻晓兵，吴景天。维甲酸诱导的视网膜色素上皮细胞凋亡。中华眼底病杂志，1998，14：153-155
13．曹泽毅。激素受体及临床应用。北京医科大学、中国协和医
科大学联合出版社，1993：67-70。
14．洪晶，吴景天。低温冷冻对培养的人RPE细胞内CAMP及Ca~(2+)浓度的影响。中国眼科杂志，1998，34：62-64。
15．洪晶，吴景天。地塞米松·对低温冷冻后人视网膜色素上皮细胞游离Ca~(2+)浓度的影响。中华眼底病杂志，1997，13：86-88
16．陈君，洪晶，张忠志等。三氟啦嗪对视网膜色素上皮细胞游离钙和三磷酸肌醇的影响。中华眼底病杂志，2001，17：63。
17. Hoffman S, Gopalakrishna R, Gundimeda U et al. Verapamil inhibits proliferation, migration and protein kinase C activity in human retinal pigment epithelial cells. Exp Eye Res 1998 Jul; 67(1):45-52.
18. Hackett S, friedman Z, Peter A. Cyclic3',5'-adenosine monophosphate modulates retinal pignment epithelial cell migration in vitro. Arch Ophthalmol 1986; 104(8): 1688
2. Minihan M, Tanner V, Williamson TH. Primary rhegmatogenous retinal detachment: 20 years of change. Br J Ophthalmol 2001 May;85(5):546-8.
3. Campochiaro PA Pathogenic mechanisms in proliferative vitreoretinopathy. Arch Ophthalmol, 1997, 115:237-241
4. Mandelcorn MS, Machemer R, Fineberg E, Hersch SB. Proliferation and metaplasia of intravitreal retinal pigment epithelium cell autotransplants. Am J Ophthalmol 1975;80:227-237.
5. Preston MR. Effects of Calcium Channel Blockers on Cellar Apoptosis. CANCER 1999 May;85(10):2093-2102.
6．鄂征 组织培养和分子细胞学技术1994．12：142-143
7. Glaser BM, Vidaurri-Leal J, Michels RG. Cryotherapy during surgery for giant retinal tears and intravitreal dispersion of viable retinal pigment epithelial cells. Ophthalmology 1993;100:466-470.
8. H K Muller. Genesis of apoptosis. BMJ 1994;308:1441
9．李秋明，宋绣雯，刘新华等。维拉帕米抗人巩膜成纤维细胞增殖的实验研究。中华眼底病杂志，1996，12(1)：98
10．姜彩辉，张卯年，张鲲等。维拉帕米对兔视网膜色素上皮细胞增殖的抑制作用。军医进修学报1999：3(20)
11．彭黎明，王曾礼。细胞凋亡的基础与临床。2000，39-40。
12．喻晓兵，吴景天。维甲酸诱导的视网膜色素上皮细胞凋亡。中华眼底病杂志，1998，14：153-155
13．曹泽毅。激素受体及临床应用。北京医科大学、中国协和医
科大学联合出版社，1993：67-70。
14．洪晶，吴景天。低温冷冻对培养的人RPE细胞内CAMP及Ca~(2+)浓度的影响。中国眼科杂志，1998，34：62-64。
15．洪晶，吴景天。地塞米松·对低温冷冻后人视网膜色素上皮细胞游离Ca~(2+)浓度的影响。中华眼底病杂志，1997，13：86-88
16．陈君，洪晶，张忠志等。三氟啦嗪对视网膜色素上皮细胞游离钙和三磷酸肌醇的影响。中华眼底病杂志，2001，17：63。
17. Hoffman S, Gopalakrishna R, Gundimeda U et al. Verapamil inhibits proliferation, migration and protein kinase C activity in human retinal pigment epithelial cells. Exp Eye Res 1998 Jul; 67(1):45-52.
18. Hackett S, friedman Z, Peter A. Cyclic3',5'-adenosine monophosphate modulates retinal pignment epithelial cell migration in vitro. Arch Ophthalmol 1986; 104(8): 1688