CAJ | 학술논문

背景玻璃体视网膜手术后视网膜色素上皮(RPE)细胞的增生和移行是增生性玻璃体视网膜病变(PVR)发生的主要病理机制,干预RPE细胞的生物学行为对于PVR的靶向治疗有重要意义. 目的观察整合素连接激酶(ILK)对RPE细胞生长、凋亡及分泌血管内皮生长因子(VEGF)的影响. 方法用低糖DMEM培养基常规培养和传代人RPE D407细胞系,选择50代以内的细胞进行实验.应用特异性敲减ILK的小干扰RNA(siRNA) (ILK-siRNA)与100μl无血清培养基和RPE细胞共培养的方式转染RPE细胞作为实验组,应用非特异性siRNA转染RPE细胞作为对照组,分别将24孔板置于37℃、体积分数5％ CO2孵箱中孵育.转染48 h后,应用MTT比色法检测各组RPE细胞活力；应用流式细胞仪检测RPE细胞的生长周期及细胞凋亡情况；采用Western blot法检测并比较各组RPE细胞中cyclin D1和caspase-3表达水平的变化；应用ELISA法检测并比较各组RPE细胞分泌VEGF水平的变化. 结果转染siRNA 48 h后,MTT法检测见实验组和对照组RPE细胞活力分别为(43.69±0.89)％和(73.95±1.20)％,实验组比对照组降低了40.92％,差异有统计学意义(t=49.524,P=0.000).流式细胞术检测发现,实验组处于G1期的细胞数为(83.30±1.26)％,对照组为(47.10±0.93)％；实验组S期细胞数量显著减少,为(12.63±0.92)％,对照组为(36.25±1.21)％；实验组G2期细胞数量显著减少,为(4.07±1.40)％,对照组为(16.65±1.53)％,两组间G1、S、G2期细胞比例的差异均有统计学意义(t=56.624、-38.130、-14.860,均P=0.000).实验组的细胞凋亡率为(15.18±1.22)％,对照组为(2.20±0.15)％,二者间差异有统计学意义(t=25.742,P=0.000).Western blot 检测结果示,实验组cyclin D1的表达量明显降低,而caspase-3的表达量则明显增高.ELISA法检测结果示,实验组RPE细胞的VEGF分泌量为(1314.49±147.23) ng/L,比对照组的(3251.27± 113.87) ng/L降低了59.6％,差异有统计学意义(t=-31.217,P=0.000).结论体外培养的RPE细胞敲减ILK后导致细胞增生活力下降,凋亡增加,且RPE细胞分泌VEGF的功能受到抑制,有望为PVR的靶向防治提供理论基础. 揹景玻璃體視網膜手術後視網膜色素上皮(RPE)細胞的增生和移行是增生性玻璃體視網膜病變(PVR)髮生的主要病理機製,榦預RPE細胞的生物學行為對于PVR的靶嚮治療有重要意義. 目的觀察整閤素連接激酶(ILK)對RPE細胞生長、凋亡及分泌血管內皮生長因子(VEGF)的影響. 方法用低糖DMEM培養基常規培養和傳代人RPE D407細胞繫,選擇50代以內的細胞進行實驗.應用特異性敲減ILK的小榦擾RNA(siRNA) (ILK-siRNA)與100μl無血清培養基和RPE細胞共培養的方式轉染RPE細胞作為實驗組,應用非特異性siRNA轉染RPE細胞作為對照組,分彆將24孔闆置于37℃、體積分數5％ CO2孵箱中孵育.轉染48 h後,應用MTT比色法檢測各組RPE細胞活力；應用流式細胞儀檢測RPE細胞的生長週期及細胞凋亡情況；採用Western blot法檢測併比較各組RPE細胞中cyclin D1和caspase-3錶達水平的變化；應用ELISA法檢測併比較各組RPE細胞分泌VEGF水平的變化. 結果轉染siRNA 48 h後,MTT法檢測見實驗組和對照組RPE細胞活力分彆為(43.69±0.89)％和(73.95±1.20)％,實驗組比對照組降低瞭40.92％,差異有統計學意義(t=49.524,P=0.000).流式細胞術檢測髮現,實驗組處于G1期的細胞數為(83.30±1.26)％,對照組為(47.10±0.93)％；實驗組S期細胞數量顯著減少,為(12.63±0.92)％,對照組為(36.25±1.21)％；實驗組G2期細胞數量顯著減少,為(4.07±1.40)％,對照組為(16.65±1.53)％,兩組間G1、S、G2期細胞比例的差異均有統計學意義(t=56.624、-38.130、-14.860,均P=0.000).實驗組的細胞凋亡率為(15.18±1.22)％,對照組為(2.20±0.15)％,二者間差異有統計學意義(t=25.742,P=0.000).Western blot 檢測結果示,實驗組cyclin D1的錶達量明顯降低,而caspase-3的錶達量則明顯增高.ELISA法檢測結果示,實驗組RPE細胞的VEGF分泌量為(1314.49±147.23) ng/L,比對照組的(3251.27± 113.87) ng/L降低瞭59.6％,差異有統計學意義(t=-31.217,P=0.000).結論體外培養的RPE細胞敲減ILK後導緻細胞增生活力下降,凋亡增加,且RPE細胞分泌VEGF的功能受到抑製,有望為PVR的靶嚮防治提供理論基礎.
배경 파리체시망막수술후시망막색소상피(RPE)세포적증생화이행시증생성파리체시망막병변(PVR)발생적주요병리궤제,간예RPE세포적생물학행위대우PVR적파향치료유중요의의. 목적 관찰정합소련접격매(ILK)대RPE세포생장、조망급분비혈관내피생장인자(VEGF)적영향. 방법 용저당DMEM배양기상규배양화전대인RPE D407세포계,선택50대이내적세포진행실험.응용특이성고감ILK적소간우RNA(siRNA) (ILK-siRNA)여100μl무혈청배양기화RPE세포공배양적방식전염RPE세포작위실험조,응용비특이성siRNA전염RPE세포작위대조조,분별장24공판치우37℃、체적분수5％ CO2부상중부육.전염48 h후,응용MTT비색법검측각조RPE세포활력；응용류식세포의검측RPE세포적생장주기급세포조망정황；채용Western blot법검측병비교각조RPE세포중cyclin D1화caspase-3표체수평적변화；응용ELISA법검측병비교각조RPE세포분비VEGF수평적변화. 결과 전염siRNA 48 h후,MTT법검측견실험조화대조조RPE세포활력분별위(43.69±0.89)％화(73.95±1.20)％,실험조비대조조강저료40.92％,차이유통계학의의(t=49.524,P=0.000).류식세포술검측발현,실험조처우G1기적세포수위(83.30±1.26)％,대조조위(47.10±0.93)％；실험조S기세포수량현저감소,위(12.63±0.92)％,대조조위(36.25±1.21)％；실험조G2기세포수량현저감소,위(4.07±1.40)％,대조조위(16.65±1.53)％,량조간G1、S、G2기세포비례적차이균유통계학의의(t=56.624、-38.130、-14.860,균P=0.000).실험조적세포조망솔위(15.18±1.22)％,대조조위(2.20±0.15)％,이자간차이유통계학의의(t=25.742,P=0.000).Western blot 검측결과시,실험조cyclin D1적표체량명현강저,이caspase-3적표체량칙명현증고.ELISA법검측결과시,실험조RPE세포적VEGF분비량위(1314.49±147.23) ng/L,비대조조적(3251.27± 113.87) ng/L강저료59.6％,차이유통계학의의(t=-31.217,P=0.000).결론 체외배양적RPE세포고감ILK후도치세포증생활력하강,조망증가,차RPE세포분비VEGF적공능수도억제,유망위PVR적파향방치제공이론기출.
Background The induction of proliferative and migratory potential in retinal pigmenepithelial (RPE) cellfollowing vitreouand retinal surgery ithe majocause of proliferative vitreoretinopathy (PVR).Managing the biological behaviouof RPE celliimportanfothe targeting treatmenof PVR.Objective Thistudy wato investigate the effecof integrin-linked kinase(ILK) on the proliferation and apoptosiof human RPE celland theisecretion of vasculaendothelial growth factor(VEGF)in vitro.MethodThe RPE D407 line wacultured and passaged in loweglucose DMEM,and the cellwere used up to thei50th generation in the study.ILK-small interfering RNA(siRNA) transfected into RPE cellin DMEM waused athe experimental group,while nonspecifisiRNtransfected into RPE cellwaused athe control group.The viability of the cellwaassayed by MT48 houraftetransfection.The cell cycle progression and apoptosiof the cellwere examined using flow cytometry.The expression of cyclin D1 and caspase-3 proteinin RPE cellwaevaluated by Western blot,and VEGF level in the cell supernatanwadetermined by ELISA.The resultwere compared between the experimental group and the control group using the independensample test.Resu1tFourty-eighhouraftetransfection of ILK-siRNA,the viability of the RPE cellwa(43.69 ±0.89)％ in the experimental group and(73.95 ± 1.20)％ in the control group,showing decline of 40.92％ in the experimental group compared to the control group(t=49.524,P=0.000).In the experimental group,the proportionof cellin G1,and G2 phase were (83.30 ± 1.26) ％,(12.63 ± 0.92) ％ and (4.07±1.40) ％,respectively,and those in the control group were(47.10±0.93) ％,(36.25±1.21) ％ and(16.65± 1.53)％,with significandifferencebetween the two groups(=56.624,-38.130,-14.860,all aP =0.000).The apoptotirate was(15.18±1.22) ％ in the experimental group,which wasignificantly lowethan thain the control group((2.20±0.15)％)(t=25.742,P =0.000).In addition,the expression of the cyclin D1 protein reduced and thaof caspase-3 protein elevated in the experimental group compared with the control group.VEGF contenin the cell supernatanwa(1314.49± 147.23)ng/L in the experimental group and thain the control group wa(3251.27 ± 113.87)ng/L,showing reduction of 59.6％ in the experimental group(t=-31.217,P=0.000).ConclusionKnockdown of ILK in RPE cellinhibitthe proliferation and promoteapoptosiin human RPE cellin vitro.decreased level of ILK also reducethe secretion of VEGF in human RPE cells.These resultsuggesthathe knockdown of ILK in RPE cellmay be novel approach to the prevention and treatmenof PVR.