视网膜色素上皮细胞体外凋亡过程中钙离子平衡与caspase-3基因表达
Int J Ophthalmol,Vo1.11,No.1,Jan.2011 www.UO.cn
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· Original article。
[Ca2+]i homeostasis and caspase-3 gene expression in
verapamil..induced retinalaucea _
apoptosis ln vitro 一 ‘ ● --
Dong.Bo Pang1
.
Jing Hong2
p...
Int J Ophthalmol,Vo1.11,No.1,Jan.2011 www.UO.cn
Tel:029-82245172 83085628 Email:Uo.2O !c0
· Original article。
[Ca2+]i homeostasis and caspase-3 gene expression in
verapamil..induced retinalaucea _
apoptosis ln vitro 一 ‘ ● --
Dong.Bo Pang1
.
Jing Hong2
pigment epithelium cells
Foundation item : National Natural Science Foundation of
China(No.30471865)
Department of Ophthalmology.the First Hospital Affiliated to
Liaoning Medical University, Jinzhou 121001, Liaoning
Province,China
Department of Ophthalmology,the Third Hospital Affiliated
to Beijing Medical University,Beijing 100191,China
Correspondence to: Dong—Bo Pang. Department of
Ophthalmology, the First Hospital Affiliated to Liaoning
Medical University, Jinzhou 121001, Liaoning Province,
China.pang2000@ 163.com
Received:2010—10—08 Accepted:2010—12—10
Abstract
·AIM:T0 study caspase-3 gene expression and[Ca“]i
homeostasis in verapamiI(Ver).induced human retinaI
pigment epithelium (RPE)cells apoptosis.
·METHODS:Ver 80mg/L was applied in cultured human
RPE cells for 1 2.24 and 48 hours to induce RPE cells
apoptosis. The expression of apoptotic effector gene
caspase一3 was assessed by reverse transcription
polymerase chain reaction (RT—PCR).Single cell was
measu red using fIuorescence indicator Fura一3/AM with
MetaFluo4.5/coolsnapfx/IX70 intracellular Ca“ fluorescence
imaging system .
·RESULTS:High Ievels of expression of caspase一3 mRNA
were observed in normaI RPE cells and it significantly
increased after co.cultured with Ver.The flu0rescence ln
resting RPE cells was strong and distributed throughout
the cells.The nucleus appeared more fluorescent than the
cytoplasm.Calcium fluorescence of RPE cells attenuated
after co—cultured with Ver.
· CONCLUS10N: Up—regulation of caspase一3 gene
expression and disturbance of『Ca“ ]i homeostasis might
play pivotal roles in Ver—induced RPE cells apoptosis.
·KEY、/\,ORDS:[Ca“ ]i homeostasis;caspase一3 gene;
Ver.induced RPE cells apoptosis
D0I:10.3969/i.issn.1672-5l23.201 1.01.001
Pang DB,Hong J.[ca ]i homeostasis and caspase-3 gene
expression in verapamil—induced retinal pigment epithelium cells
apoptosis in vitro.Guoji Yanke Zazbi rInt J Ophthalmo] 2011;11
(1):1—3
INTRODUCTIoN
he retinal pigment epithelium(RPE)cell is a mosaic of
上 polygonal cells interposed between the choroid and the
neural retina and serves as the outer blood—retinal barrier
regulating retinal homeostasis and visual function ’ .
Normally RPE cells form acquiescent monolayer,but they
retain the ability to divide and do so when placed in culture or
when participating in wound repair .After severe injury that
may be associated with ocular trauma or retinal detachment,
RPE cells can be detached and consequently found in the
vitreous.Once in this new environment,RPE ceils have been
shown to dedifferentiate and exhibit a pseudometaplastic
transformation into fibroblast-like, spindle—shaped cells,
which become actively dividing and migratory. These
processes are considered to be key events in the onset of
proliferative vitreoretinopathy(PVR) .PVR is an excess
proliferation which ocular tissue is in response to repair in
trauma,immigration and proliferation of RPE can promote
PVR and RPE are the most important cells in form ing
proliferation membrane of PVR .It is necessary to apply
drags or viteoretinal operation combined with drugs to inhibit
the formation and development of PVR in the earlier period.
W e previously applied calcium channel blocker-verapamil to
induce cultured human RPE vitro apoptosis successfully .
but the mechanism of apoptosis and which apoptotic signals
have involved in is unknown.Although it is widely accepted
that intracellular calcium signaling and DNA damage might be
the common triggers implicated in denomination of apoptosis,
gene caspase一3 may play an important role in the executive
phase of apoptosis. In this study, we further characterize
human RPE cells apoptosis induced by Ver and explore
whether caspase一3 gene expression and the calcium messenger
system are involved in the regulation of verapamil(Ver)一
induced cells apoptosis.
M ATERIA[ S AND M ETHoDS
Materials Trizol agent,Super Script TM One—Step RT·PeR
System was purchased from Takara.Caspase·3 primers (5 一
ITGTGAAGTGCAAATGTFeTAAAGG.3 .5 .CAAGAAATCT
CCCGTGAAATGTC.3 ).and actin primers (5 .AAATCGTG
CGTGACATFAA一3 . 5 一CTCGTCATACTCCTGCTFG。3 ) were
obtained from Baoshengwu Co. Fura一3/AM was purchased
from Eugene Oregon.Tissue source was obtained from the
donator after the cornea transplantation from the department of
ophthalmology in the first affiliated hospital of China Medical
University.Their ages were between 24 and 38 years.Under
sterile condition,using enzyme digestive method to isolate and
complete plantation of human RPE cells by 2.5g/L trypsin to
1
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0.actin
381bp }-一-·.---;I 500bp caspase一3 301bp 100bp
Figure 1 Caspase-3 mRNA level in RPE cells treated with Ver.
Table 1 The level of caspase-3 mRNA and[ca ]i of RPE
cells in each group (nmol/L, ± )
Figure 2 Intracellular calcium fluorescence in control cultured RPE cells(Fluo-3/AM ×400) A:Control;B:12 houm;C:24
hours:D:48 hours.
24一pore culture plate. RPE cells were cultured in DEME
containing 200mL/L fetal calf serum. Cultures were
maintained at 37 oC in a humidified incubator under the
atmosphere of 900mL/L air and 100mL/L C0 .
M ethotis
Caspase-3 gene expression Total RNA was extracted by
TriZOl agents.The following conditions were used for 35 cycles
of PCR amplification:caspase.3:30 seconds denaturation at
94℃ ,30 seconds annealing at 56oC.and 2 minutes extension
at 72℃ .rI’he amplified product was 301 bp;actin:30 seconds
denaturation at 94℃ 。30 seconds annealing at 63℃ .and 2
minutes extension at 72℃ .The amplified product Was 38 l bD.
The amplified products were resolved by gel electrophoresis on
20 L agarose.
[Ca ]i in RPE cells Planting the third period human RPE
cells on 3.5 cm culture dish,applying 80mg/L Ver on
cultured RPE cells f0r 1 2,24 and 48 hours,control group
Was established simultaneously.After an overnight period of
attachment,the medium was removed and the cells were
Ioaded for 30 minutes at 37oC with Fura-3/AM in PBS.The
cells were then washed to remove extracellular Fura一3/AM and
placed on MetaFluo4.5/coolsnapfx/IX70 intraeellular Ca“
fluorescence imaging system.Automatic running gel imaging
system was used to scan the straps of PCR,optical density
(OD)of each strap was read by FluorChen V.2.0 system.
Calcium concentration of individual RPE cells was calculated
by[ca“]i=Kd(F 一F)/(F—F ),Kd=400,F is
fluorescence value.
Statistical Analysis Data were expressed as mean±SD.
Analysis of variance Was adopted to conduct statistics test.P<
0.05 Was considered significant.Statistics Was conducted by
SPSS 11.5 software.
2
RESULTS
Caspase-3 Expression A low
nlRNA was detected in norma】
caspase一3
increased
H .兰 k m d n C ∞ E 叫
lnt J Ophthalmol,Vo1.11,No.1,Jan.2011 www.HO.cn
Tel:029-82245172 83085628 Email:UO.2000@ 163.corn
precipitating the dramatic morphological changes of apoptosis.
Our results showed significant increase in the expression of
caspase一3 gene following exposure to Vet,suggesting Ver—
induced apoptosis in the RPE cells via caspase 3 dependent
pathway.This is coherent with our previous results that Ver
could down—regulate the expression of bcl-2 protein in the
RPE cells . Gene bc/-2. as the negative regu lator of
caspase一3, exerts anti·apoptosis action at or before the
processing of certain caspases to their catalytically active
forms .Changes in[ca ]i provide a chemical signal for
early cell death pathway.If f Ca“1 i can be elevated for a
sustained period,cells are induced to undergo apoptosis
But when『Ca ]i decreased,cells are also induced to
undergo apoptosis.Homeostasis disorder of calcium signaling
system could be a mechanism of apoptosis ⋯.Our experiment
demonstrated that Ver elicited a sustained decrease of『Ca 1 i
which was a very early effect compared to morphological
changes(cell rounding and shrinkage).Thus,we concluded
that『Ca“ ]i might be another early initiator in connection
with apoptosis.Although the precise mechanism needs further
study,our findings provide the evidence for an important role
of caspase一3 and homeostasis of calcium signaling system in
the regulation of Ver·induced apoptosis.
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视网膜色素上皮细胞体外凋亡过程中钙离子平
衡与 caspase-3基因表达
庞 东渤 .洪 晶
基金项目:中国国家自然科学基金资助项目(No.30471865)
(作者单位: 121001中国辽宁省锦州市,辽宁医学院附属第一医
院眼科; 100191中国北京市,北京医科大学附属第 医院眼科)
作者简介:庞东渤,男,博士,副教授,硕士研究生导师,研究方
向:眼底病研究。
通讯作者:庞东渤.pang2000@163.tom
摘要
目的:研究钙通道拮抗剂一维拉帕米(verapamil,Ver)诱导
视网膜色素上皮(retinal pigment epithelium,RPE)细胞凋
亡过程中钙离子及凋亡基因caspase一3变化。
方法:应用 80mg/L的Ver分别作用健康人眼 RPE细胞
12,24及48h诱导凋亡,设立对照组。逆转录聚合酶链反
应(RT—PCR)检测凋亡基因caspase一3的表达,采用Fluo.3/AM
负载技术,MetaFluo4.5/coolsnapfx/IX70细胞内钙离子荧
光成像系统测定每组 20个 RPE细胞钙荧光值,并计算
RPE细胞内钙浓度([ca“]i)。
结果:对照组 RPE细胞 Ca“荧光分布胞核最强,胞质次
之。Ver作用 12,24及48h后,细胞 内[Ca ]i明显降低
(P<0.01)。对照组 RPE细胞可见 caspase.3的mRNA有
少量的表达。Ver作用 12h后,可见caspase-3的mRNA有
较高的表达,与对照组比较,具有显著性差异(P<0.01)。
随着 Ver作用时间的延长,caspase.3的mRNA表达逐渐增
强,在48h时有所下降。
结论:Caspase一3基因表达上调及 RPE细胞内钙离子稳态
失调可能在 Ver诱导 RPE细胞凋亡中起关键作用。
关键词:钙离子平衡;caspase一3基因;Ver诱导视网膜色素
上皮细胞凋亡
3
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