背景 研究表明盘状结构域受体2(DDR2)和基质金属蛋白酶-13(MMP-13)在肿瘤新生血管发生中具有重要作用,是相关疾病治疗的关键靶点,但DDR2和MMP-13在脉络膜新生血管(CNV)发生中的作用尚不清楚.目的 探讨DDR2和MMP-13在大鼠CNV中的表达特点以及MEK/ERK和PI3 K/Akt通路对DDR2及MMP-13表达的调控作用.方法 采用532 nm倍频激光视网膜光凝法制备棕色挪威(BN)大鼠CNV模型,将78只BN模型鼠按照随机数字表法分为正常对照组(n=7)、模型对照组(n=39)、PD98059(MEK1抑制剂)组(n=16)和LY294002(PI3K抑制剂)组(n=16),PD98059组和LY294002组大鼠分别于光凝后1d、7d玻璃体内注射5 mmol/L PD98059 3μl或5 mmol/L LY294002 3μl.分别于光凝前和光凝后1、3、7、14 d用过量麻醉法处死动物并制备样本和切片,采用Western blot法检测各组大鼠眼杯中ERK、p-ERK、Akt和p-Akt蛋白的表达变化,采用逆转录PCR(RT-PCR)法检测各组大鼠眼杯中DDR2 mRNA和MMP-13 mRNA的表达变化,采用组织病理学方法测定各组大鼠CNV厚度,采用免疫组织化学和免疫荧光染色法分别定位和测定大鼠CNV区域DDR2和MMP-13的表达.结果 光凝后3d,光凝局部细胞增生,光凝后7 d CNV形成,至光凝后14 d CNV趋于稳定.免疫组织化学染色结果显示,DDR2在正常大鼠视网膜血管内皮细胞层、视网膜神经节细胞(RGCs)层和内核层细胞中呈弱表达,但在CNV中呈强阳性表达;MMP-13在正常对照组大鼠视网膜内界膜层、光感受器层和巩膜层均呈强阳性表达,在模型对照组中呈强阳性表达.免疫荧光结果显示,CNV组织中DDR2和MMP-13共表达.RT-PCR结果显示,模型对照组光凝后1、3、7、14 d DDR2 mRNA相对表达水平分别为55.22±4.03、47.74±2.23、14.82±4.56和5.59±2.47,MMP-13 mRNA相对表达水平分别为25.54±3.83、43.51±4.36、10.90±4.00和5.23±3.23,与正常对照组相比,差异均有统计学意义(均P＜0.05).免疫荧光结果显示,模型对照组光凝后3、7、14 d大鼠CNV区DDR2相对荧光密度单位(RFU)值分别为2.73±0.53、5.21±0.31和3.22±0.33,MMP-13 RFU值分别为1.66±0.17、3.57±0.44和2.67±0.21,组间总体比较差异均有统计学意义(F=81.01,P＜0.05;F=40.31,P＜0.01);PD98059组和LY294002组光凝后14 d CNV区DDR2 RFU值分别为1.14±0.19和1.03±0.14,与模型对照组比较,差异均有统计学意义(均P＜0.05),MMP-13RFU值分别为1.37±0.25和1.24±0.20,与模型对照组比较,差异均有统计学意义(均P＜0.05).Western blot结果显示,模型对照组光凝后7d眼杯中p-ERK和p-Akt水平升高,与正常对照组大鼠相比差异均有统计学意义(均P＜0.05);至光凝后14 d恢复至近正常水平,与正常对照组比较差异均无统计学意义(均P＞0.05).PD98058组大鼠眼杯p-ERK水平明显下降,注射后7d、14 d PD98058组大鼠眼杯中p-ERK表达的抑制率分别为71.58％和65.21％,PD98058组7d与模型对照组7d比较、PD98058组14 d与模型对照组14 d比较,差异均有统计学意义(均P＜0.05).LY294002组大鼠眼杯中p-Akt表达水平明显下降,LY294002注射后7d、14d大鼠眼杯中p-Akt表达的抑制率分别为65.62％和67.04％,LY294002组7d与模型对照组7d比较、LY294002组14d与模型对照组14 d比较,差异均有统计学意义(均P＜0.05).各组眼杯中ERK和Akt的表达水平总体比较差异均无统计学意义(F=0.62,P＞0.05;F=0.81,P＞0.05).模型对照组、PD98059组和LY294002组光凝后14 d大鼠CNV厚度分别为(119.19±18.03)、(51.00±11.29)和(59.18±9.00) μm,总体比较差异有统计学意义(F=29.376,P＜0.05),与模型对照组比较,PD98059和LY294002注射后CNV厚度分别减少57.21％和50.34％,差异均有统计学意义(均P＜0.05).结论 DDR2和MMP-13在大鼠CNV中表达上调,参与CNV的形成;MEK/ERK和PI3K/Akt通路通过参与调控DDR2和MMP-13在CNV局部的表达而抑制CNV的发生和进展.
揹景 研究錶明盤狀結構域受體2(DDR2)和基質金屬蛋白酶-13(MMP-13)在腫瘤新生血管髮生中具有重要作用,是相關疾病治療的關鍵靶點,但DDR2和MMP-13在脈絡膜新生血管(CNV)髮生中的作用尚不清楚.目的 探討DDR2和MMP-13在大鼠CNV中的錶達特點以及MEK/ERK和PI3 K/Akt通路對DDR2及MMP-13錶達的調控作用.方法 採用532 nm倍頻激光視網膜光凝法製備棕色挪威(BN)大鼠CNV模型,將78隻BN模型鼠按照隨機數字錶法分為正常對照組(n=7)、模型對照組(n=39)、PD98059(MEK1抑製劑)組(n=16)和LY294002(PI3K抑製劑)組(n=16),PD98059組和LY294002組大鼠分彆于光凝後1d、7d玻璃體內註射5 mmol/L PD98059 3μl或5 mmol/L LY294002 3μl.分彆于光凝前和光凝後1、3、7、14 d用過量痳醉法處死動物併製備樣本和切片,採用Western blot法檢測各組大鼠眼杯中ERK、p-ERK、Akt和p-Akt蛋白的錶達變化,採用逆轉錄PCR(RT-PCR)法檢測各組大鼠眼杯中DDR2 mRNA和MMP-13 mRNA的錶達變化,採用組織病理學方法測定各組大鼠CNV厚度,採用免疫組織化學和免疫熒光染色法分彆定位和測定大鼠CNV區域DDR2和MMP-13的錶達.結果 光凝後3d,光凝跼部細胞增生,光凝後7 d CNV形成,至光凝後14 d CNV趨于穩定.免疫組織化學染色結果顯示,DDR2在正常大鼠視網膜血管內皮細胞層、視網膜神經節細胞(RGCs)層和內覈層細胞中呈弱錶達,但在CNV中呈彊暘性錶達;MMP-13在正常對照組大鼠視網膜內界膜層、光感受器層和鞏膜層均呈彊暘性錶達,在模型對照組中呈彊暘性錶達.免疫熒光結果顯示,CNV組織中DDR2和MMP-13共錶達.RT-PCR結果顯示,模型對照組光凝後1、3、7、14 d DDR2 mRNA相對錶達水平分彆為55.22±4.03、47.74±2.23、14.82±4.56和5.59±2.47,MMP-13 mRNA相對錶達水平分彆為25.54±3.83、43.51±4.36、10.90±4.00和5.23±3.23,與正常對照組相比,差異均有統計學意義(均P＜0.05).免疫熒光結果顯示,模型對照組光凝後3、7、14 d大鼠CNV區DDR2相對熒光密度單位(RFU)值分彆為2.73±0.53、5.21±0.31和3.22±0.33,MMP-13 RFU值分彆為1.66±0.17、3.57±0.44和2.67±0.21,組間總體比較差異均有統計學意義(F=81.01,P＜0.05;F=40.31,P＜0.01);PD98059組和LY294002組光凝後14 d CNV區DDR2 RFU值分彆為1.14±0.19和1.03±0.14,與模型對照組比較,差異均有統計學意義(均P＜0.05),MMP-13RFU值分彆為1.37±0.25和1.24±0.20,與模型對照組比較,差異均有統計學意義(均P＜0.05).Western blot結果顯示,模型對照組光凝後7d眼杯中p-ERK和p-Akt水平升高,與正常對照組大鼠相比差異均有統計學意義(均P＜0.05);至光凝後14 d恢複至近正常水平,與正常對照組比較差異均無統計學意義(均P＞0.05).PD98058組大鼠眼杯p-ERK水平明顯下降,註射後7d、14 d PD98058組大鼠眼杯中p-ERK錶達的抑製率分彆為71.58％和65.21％,PD98058組7d與模型對照組7d比較、PD98058組14 d與模型對照組14 d比較,差異均有統計學意義(均P＜0.05).LY294002組大鼠眼杯中p-Akt錶達水平明顯下降,LY294002註射後7d、14d大鼠眼杯中p-Akt錶達的抑製率分彆為65.62％和67.04％,LY294002組7d與模型對照組7d比較、LY294002組14d與模型對照組14 d比較,差異均有統計學意義(均P＜0.05).各組眼杯中ERK和Akt的錶達水平總體比較差異均無統計學意義(F=0.62,P＞0.05;F=0.81,P＞0.05).模型對照組、PD98059組和LY294002組光凝後14 d大鼠CNV厚度分彆為(119.19±18.03)、(51.00±11.29)和(59.18±9.00) μm,總體比較差異有統計學意義(F=29.376,P＜0.05),與模型對照組比較,PD98059和LY294002註射後CNV厚度分彆減少57.21％和50.34％,差異均有統計學意義(均P＜0.05).結論 DDR2和MMP-13在大鼠CNV中錶達上調,參與CNV的形成;MEK/ERK和PI3K/Akt通路通過參與調控DDR2和MMP-13在CNV跼部的錶達而抑製CNV的髮生和進展.
배경 연구표명반상결구역수체2(DDR2)화기질금속단백매-13(MMP-13)재종류신생혈관발생중구유중요작용,시상관질병치료적관건파점,단DDR2화MMP-13재맥락막신생혈관(CNV)발생중적작용상불청초.목적 탐토DDR2화MMP-13재대서CNV중적표체특점이급MEK/ERK화PI3 K/Akt통로대DDR2급MMP-13표체적조공작용.방법 채용532 nm배빈격광시망막광응법제비종색나위(BN)대서CNV모형,장78지BN모형서안조수궤수자표법분위정상대조조(n=7)、모형대조조(n=39)、PD98059(MEK1억제제)조(n=16)화LY294002(PI3K억제제)조(n=16),PD98059조화LY294002조대서분별우광응후1d、7d파리체내주사5 mmol/L PD98059 3μl혹5 mmol/L LY294002 3μl.분별우광응전화광응후1、3、7、14 d용과량마취법처사동물병제비양본화절편,채용Western blot법검측각조대서안배중ERK、p-ERK、Akt화p-Akt단백적표체변화,채용역전록PCR(RT-PCR)법검측각조대서안배중DDR2 mRNA화MMP-13 mRNA적표체변화,채용조직병이학방법측정각조대서CNV후도,채용면역조직화학화면역형광염색법분별정위화측정대서CNV구역DDR2화MMP-13적표체.결과 광응후3d,광응국부세포증생,광응후7 d CNV형성,지광응후14 d CNV추우은정.면역조직화학염색결과현시,DDR2재정상대서시망막혈관내피세포층、시망막신경절세포(RGCs)층화내핵층세포중정약표체,단재CNV중정강양성표체;MMP-13재정상대조조대서시망막내계막층、광감수기층화공막층균정강양성표체,재모형대조조중정강양성표체.면역형광결과현시,CNV조직중DDR2화MMP-13공표체.RT-PCR결과현시,모형대조조광응후1、3、7、14 d DDR2 mRNA상대표체수평분별위55.22±4.03、47.74±2.23、14.82±4.56화5.59±2.47,MMP-13 mRNA상대표체수평분별위25.54±3.83、43.51±4.36、10.90±4.00화5.23±3.23,여정상대조조상비,차이균유통계학의의(균P＜0.05).면역형광결과현시,모형대조조광응후3、7、14 d대서CNV구DDR2상대형광밀도단위(RFU)치분별위2.73±0.53、5.21±0.31화3.22±0.33,MMP-13 RFU치분별위1.66±0.17、3.57±0.44화2.67±0.21,조간총체비교차이균유통계학의의(F=81.01,P＜0.05;F=40.31,P＜0.01);PD98059조화LY294002조광응후14 d CNV구DDR2 RFU치분별위1.14±0.19화1.03±0.14,여모형대조조비교,차이균유통계학의의(균P＜0.05),MMP-13RFU치분별위1.37±0.25화1.24±0.20,여모형대조조비교,차이균유통계학의의(균P＜0.05).Western blot결과현시,모형대조조광응후7d안배중p-ERK화p-Akt수평승고,여정상대조조대서상비차이균유통계학의의(균P＜0.05);지광응후14 d회복지근정상수평,여정상대조조비교차이균무통계학의의(균P＞0.05).PD98058조대서안배p-ERK수평명현하강,주사후7d、14 d PD98058조대서안배중p-ERK표체적억제솔분별위71.58％화65.21％,PD98058조7d여모형대조조7d비교、PD98058조14 d여모형대조조14 d비교,차이균유통계학의의(균P＜0.05).LY294002조대서안배중p-Akt표체수평명현하강,LY294002주사후7d、14d대서안배중p-Akt표체적억제솔분별위65.62％화67.04％,LY294002조7d여모형대조조7d비교、LY294002조14d여모형대조조14 d비교,차이균유통계학의의(균P＜0.05).각조안배중ERK화Akt적표체수평총체비교차이균무통계학의의(F=0.62,P＞0.05;F=0.81,P＞0.05).모형대조조、PD98059조화LY294002조광응후14 d대서CNV후도분별위(119.19±18.03)、(51.00±11.29)화(59.18±9.00) μm,총체비교차이유통계학의의(F=29.376,P＜0.05),여모형대조조비교,PD98059화LY294002주사후CNV후도분별감소57.21％화50.34％,차이균유통계학의의(균P＜0.05).결론 DDR2화MMP-13재대서CNV중표체상조,삼여CNV적형성;MEK/ERK화PI3K/Akt통로통과삼여조공DDR2화MMP-13재CNV국부적표체이억제CNV적발생화진전.
Background It is estimated that discoidin domain receptor 2 (DDR2) and matrix metalloproteinase-13 (MMP-13) play an important role in the development of tumor angiogenesis.However,their effects on choroidal neovascularizaiton (CNV) have not been clarified yet.Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV.Methods CNV models were established in 78 Brown Norway (BN) rats by retinal photocoagulation with 532 nm laser.Then the animals were randomly divided into the normal control group (n =7),the model control group (n =39),PD98059 (MEK1 inhibitor) group (n =16) and LY294002 (PI3K inhibitor) group (n =16),and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group.The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR (RT-PCR),and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay.CNV thickness was measured by pathological examination 14 days after photocoagulation,and the changes of CNV thickness,the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group,PD98059 group and LY294002 group.Results Three days after photocoagulation,the cells within the lasered lesions proliferated,then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation.Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer,inner nuclear layer and vascular endothelial cells;while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer,photoreceptor layer and sclera layer.Both DDR2 and MMP-13 were strongly expressed in CNV area.Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV area.RT-PCR revealed that the relative DDR2 mRNA levels at 1 day,3 days,7 days and 14 days after photocoagulation were 55.22±4.03,47.74±2.23,14.82±4.56 and 5.59±2.47 respectively,while the relative MMP-13 mRNA levels were 25.54±3.83,43.51±4.36,10.90±4.00 and 5.23±3.23 respectively.Compared with the normal control group,the expression of DDR2 and MMP-13 were significantly increased (all at P＜0.05).Immunofluorescence staining showed that the relative fluorescence unit (RFU) values at 1 day,3 days,7 days and 14 days after photocoagulation were 2.73±0.53,5.21±0.31 and 3.22±0.33 for DDR2 and 1.66±0.17,3.57±0.44,2.67±0.21 for MMP-13,respectively.The RFU values in the PD98059 group and LY294002 group 14 days after photocoagulation were 1.14±0.19,1.03±0.14 for DDR2 and 1.37±0.25,1.24±0.20 for MMP-13,respectively.Compared with the model control group,the differences were statistically significant (both at P＜0.05).Western blot results showed that,compared to the normal control group,the expression levels of p-ERK and p-Akt pretein increased at day 7 after photocoagulation (both at P＜0.05),and returned back to the baseline at day 14 after photocoagulation (both at P＞0.05).Both PD98059 and LY294002 treatment were able to attenuate the thickness of CNV to 57.21％ and 50.34％ at day 14 after photocoagulation and further decrease the expression levels of DDR2 and MMP-13 in CNV (all at P＜0.05).Conclusions The expressions of DDR2 and MMP-13 up-regulate in laser-induced CNV.MEK/ERK and PI3K/Akt pathways suppress the development of CNV by regulating the expression of DDR2 and MMP-13.
