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 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 国际眼科杂志 2011年 1月 第 11卷 第1期 WWW．UO．crl 电话：029—82245172 83085628 电子信箱：UO．2000@163．com 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． REFERENCES 1 Grierson I，Hiscott P，Hogg P，Robey H， Mazure A，Larkin G． Development，repair and regeneration of the retinal pigment epithelium． e 1994；8(Pt 2)：255-262 2 Zhao S，Rizzoln LJ，Barnstable CJ．Differentiation and transdifferen— tiation of the retinal pigment epithelium．／nt Rev Cytol 1997；171：225-266 3 Campochiam PA，Hackett SF．Corneal endothelial cell matrix promotes expression of differentiated features of retinal pigmented epithelial cells： implication of laminin and basic fibroblast growth factor as active components．Exp e Res 1993；57(5)：539—547 4 Hiseott P，Sheridan C，Magee RM，Griemon I．Matrix and the retinal pigm ent epithelium in proliferative retinal disease．Prog Retinal Eye Res 1999；18(2)：167—190 5 Chaaeris DG． Proliferative vitreoretinopathy：pathobiology，surgical management，and adjunctive treatment．Br J Ophthalmol 1995；79(1O)： 953-960 6 Pang DB．Hong J．Expression of Bcl-2 and bFGF on apoptosis of cultured human retinal pigment epithelial cells induced by verapamil． Yanke X Jinzhan 2005；25(5)：400-403 7 Hyoh Y，Ishizaka S，Horii T，Fujiwara A，Tegoshi T，Yamada M， Afizono N．Activation of caspases in intestinal villus epithelial cells of norma1 and nematode infeeted rats．Gut 2002；50(1)：7l-77 8 Nakayama M，Ishidoh K，Kayagaki N，Kojima Y，Yamaguchi N， Nakano H，Kominami E，Okumura K，Yagita H．Multiple pathways of TWEAK．induced cell death．J Immunol 2002：l68(2)：734-743 9 Droin N，Dubrez L，Eymin B，Renvoiz C，Br ard J，Dimanche—Boitrel MT．Solary E．Upregulation of CASP genes in human tumor cells undergoing etoposide—induced apoptosis．Oncogene 1998：16(22)：2885-2894 lO KIuck RM ．Bossy WE．Green DR，Newmeyer DD．The release of cytochrome c from mitochondria：a primary site for Bcl-2 regulation of apoptosis．Science l997；275(5303)：l132一l136 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