中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
1期
10-15
,共6页
脉络膜新生血管%基质金属蛋白酶%骨髓细胞%微小RNA
脈絡膜新生血管%基質金屬蛋白酶%骨髓細胞%微小RNA
맥락막신생혈관%기질금속단백매%골수세포%미소RNA
Choroidal neovascularization%Matrix metalloproteinase%Bone marrow cell%MicroRNA
背景 基质金属蛋白酶-2(MMP-2)/MMP-13在脉络膜新生血管(CNV)生成过程中发挥重要作用,但CNV原位细胞中MMP-2/MMP-13的表达有限.骨髓来源细胞(BMCs)参与CNV生成,可能是CNV中MMP-2/MMP-13的重要来源,而微小RNA188-5p(miR188-5p)可能是调控BMCs表达MMP-2/MMP-13的关键节点. 目的 探讨参与CNV生成的BMCs表达MMP-2/MMP-13的情况及BMCs表达的MMP-2/MMP-13是否受miR188-5p调控.方法 将表达绿色荧光蛋白(GFP)的转基因雌性小鼠的骨髓细胞移植到野生型雌性C57BL/6J小鼠以建立C57BL/6J.GFP嵌合体小鼠模型,流式细胞术检测嵌合度大于85％的42只小鼠纳入实验作为实验组,未进行骨髓细胞移植的野生型雌性C57BL/6J小鼠作为对照组.两组小鼠均以视网膜激光光凝法诱导CNV,两个组分别于光凝前及光凝后第1、3、5、7、10、14、28天各取3只小鼠分离视网膜脉络膜组织,采用ELISA法检测小鼠视网膜脉络膜组织匀浆中MMP-2/MMP-13质量浓度的变化;采用实时荧光定量PCR(RT-qPCR)法检测上述时间点小鼠视网膜脉络膜组织中miR188-5p mRNA的相对表达量变化;采用免疫荧光染色和原位杂交技术观察上述时间点小鼠CNV区GFP阳性细胞中MMP-2/MMP-13和miR188-5p的表达情况,并进行定量分析.结果 实验组C57BL/6J.GFP嵌合体模型小鼠外周血单核细胞中表达GFP细胞所占比例平均为(90.67±3.02)％,符合实验要求.ELISA检测显示,对照组与实验组小鼠视网膜脉络膜匀浆中MMP-2/MMP-13的表达趋势一致,总体比较差异均无统计学意义(MMP-2:F=0.060,P=0.810;MMP-13:F=0.012,P=0.915).ELISA和免疫荧光均显示在诱导CNV后1d,实验组和对照组MMP-2表达均快速增加,3d时表达量最高,实验组占总表达量的64.21％,随后两组MMP-2表达均下降.两组MMP-13表达量在诱导CNV后1d缓慢增加,7d时表达量最高,实验组占总表达量的79.61％,随后MMP-13总体表达下降.靶基因预测结果可显示MMP-2和MMP-13的3'非编码区有miR188-5p的互补结合位点.RT-qPCR和原位杂交检测结果均显示在诱导CNV后1d,视网膜脉络膜中miR188-5p表达量快速下降,7d时表达量最低.小鼠CNV区BMCs中miR188-5p的动态表达与BMCs中MMP-13的动态表达呈负相关(r=-0.868,P＜0.05);CNV诱导后5d内BMCs中miR188-5p的动态表达与BMCs中MMP-2的动态表达呈负相关(r=-0.997,P＜0.05). 结论 在激光诱导的小鼠CNV模型中,MMP-2/MMP-13的表达随时间发生动态变化,BMCs对其表达的迅速上调起主要作用.CNV区BMCs中miR188-5p表达随时间变化的趋势与MMP-13相反,与在CNV形成早期MMP-2表达趋势相反.miR188-5p的调控靶基因是MMP-2/MMP-13,提示BMCs的MMP-2/MMP-13表达可能受miR188-5p调控.
揹景 基質金屬蛋白酶-2(MMP-2)/MMP-13在脈絡膜新生血管(CNV)生成過程中髮揮重要作用,但CNV原位細胞中MMP-2/MMP-13的錶達有限.骨髓來源細胞(BMCs)參與CNV生成,可能是CNV中MMP-2/MMP-13的重要來源,而微小RNA188-5p(miR188-5p)可能是調控BMCs錶達MMP-2/MMP-13的關鍵節點. 目的 探討參與CNV生成的BMCs錶達MMP-2/MMP-13的情況及BMCs錶達的MMP-2/MMP-13是否受miR188-5p調控.方法 將錶達綠色熒光蛋白(GFP)的轉基因雌性小鼠的骨髓細胞移植到野生型雌性C57BL/6J小鼠以建立C57BL/6J.GFP嵌閤體小鼠模型,流式細胞術檢測嵌閤度大于85％的42隻小鼠納入實驗作為實驗組,未進行骨髓細胞移植的野生型雌性C57BL/6J小鼠作為對照組.兩組小鼠均以視網膜激光光凝法誘導CNV,兩箇組分彆于光凝前及光凝後第1、3、5、7、10、14、28天各取3隻小鼠分離視網膜脈絡膜組織,採用ELISA法檢測小鼠視網膜脈絡膜組織勻漿中MMP-2/MMP-13質量濃度的變化;採用實時熒光定量PCR(RT-qPCR)法檢測上述時間點小鼠視網膜脈絡膜組織中miR188-5p mRNA的相對錶達量變化;採用免疫熒光染色和原位雜交技術觀察上述時間點小鼠CNV區GFP暘性細胞中MMP-2/MMP-13和miR188-5p的錶達情況,併進行定量分析.結果 實驗組C57BL/6J.GFP嵌閤體模型小鼠外週血單覈細胞中錶達GFP細胞所佔比例平均為(90.67±3.02)％,符閤實驗要求.ELISA檢測顯示,對照組與實驗組小鼠視網膜脈絡膜勻漿中MMP-2/MMP-13的錶達趨勢一緻,總體比較差異均無統計學意義(MMP-2:F=0.060,P=0.810;MMP-13:F=0.012,P=0.915).ELISA和免疫熒光均顯示在誘導CNV後1d,實驗組和對照組MMP-2錶達均快速增加,3d時錶達量最高,實驗組佔總錶達量的64.21％,隨後兩組MMP-2錶達均下降.兩組MMP-13錶達量在誘導CNV後1d緩慢增加,7d時錶達量最高,實驗組佔總錶達量的79.61％,隨後MMP-13總體錶達下降.靶基因預測結果可顯示MMP-2和MMP-13的3'非編碼區有miR188-5p的互補結閤位點.RT-qPCR和原位雜交檢測結果均顯示在誘導CNV後1d,視網膜脈絡膜中miR188-5p錶達量快速下降,7d時錶達量最低.小鼠CNV區BMCs中miR188-5p的動態錶達與BMCs中MMP-13的動態錶達呈負相關(r=-0.868,P＜0.05);CNV誘導後5d內BMCs中miR188-5p的動態錶達與BMCs中MMP-2的動態錶達呈負相關(r=-0.997,P＜0.05). 結論 在激光誘導的小鼠CNV模型中,MMP-2/MMP-13的錶達隨時間髮生動態變化,BMCs對其錶達的迅速上調起主要作用.CNV區BMCs中miR188-5p錶達隨時間變化的趨勢與MMP-13相反,與在CNV形成早期MMP-2錶達趨勢相反.miR188-5p的調控靶基因是MMP-2/MMP-13,提示BMCs的MMP-2/MMP-13錶達可能受miR188-5p調控.
배경 기질금속단백매-2(MMP-2)/MMP-13재맥락막신생혈관(CNV)생성과정중발휘중요작용,단CNV원위세포중MMP-2/MMP-13적표체유한.골수래원세포(BMCs)삼여CNV생성,가능시CNV중MMP-2/MMP-13적중요래원,이미소RNA188-5p(miR188-5p)가능시조공BMCs표체MMP-2/MMP-13적관건절점. 목적 탐토삼여CNV생성적BMCs표체MMP-2/MMP-13적정황급BMCs표체적MMP-2/MMP-13시부수miR188-5p조공.방법 장표체록색형광단백(GFP)적전기인자성소서적골수세포이식도야생형자성C57BL/6J소서이건립C57BL/6J.GFP감합체소서모형,류식세포술검측감합도대우85％적42지소서납입실험작위실험조,미진행골수세포이식적야생형자성C57BL/6J소서작위대조조.량조소서균이시망막격광광응법유도CNV,량개조분별우광응전급광응후제1、3、5、7、10、14、28천각취3지소서분리시망막맥락막조직,채용ELISA법검측소서시망막맥락막조직균장중MMP-2/MMP-13질량농도적변화;채용실시형광정량PCR(RT-qPCR)법검측상술시간점소서시망막맥락막조직중miR188-5p mRNA적상대표체량변화;채용면역형광염색화원위잡교기술관찰상술시간점소서CNV구GFP양성세포중MMP-2/MMP-13화miR188-5p적표체정황,병진행정량분석.결과 실험조C57BL/6J.GFP감합체모형소서외주혈단핵세포중표체GFP세포소점비례평균위(90.67±3.02)％,부합실험요구.ELISA검측현시,대조조여실험조소서시망막맥락막균장중MMP-2/MMP-13적표체추세일치,총체비교차이균무통계학의의(MMP-2:F=0.060,P=0.810;MMP-13:F=0.012,P=0.915).ELISA화면역형광균현시재유도CNV후1d,실험조화대조조MMP-2표체균쾌속증가,3d시표체량최고,실험조점총표체량적64.21％,수후량조MMP-2표체균하강.량조MMP-13표체량재유도CNV후1d완만증가,7d시표체량최고,실험조점총표체량적79.61％,수후MMP-13총체표체하강.파기인예측결과가현시MMP-2화MMP-13적3'비편마구유miR188-5p적호보결합위점.RT-qPCR화원위잡교검측결과균현시재유도CNV후1d,시망막맥락막중miR188-5p표체량쾌속하강,7d시표체량최저.소서CNV구BMCs중miR188-5p적동태표체여BMCs중MMP-13적동태표체정부상관(r=-0.868,P＜0.05);CNV유도후5d내BMCs중miR188-5p적동태표체여BMCs중MMP-2적동태표체정부상관(r=-0.997,P＜0.05). 결론 재격광유도적소서CNV모형중,MMP-2/MMP-13적표체수시간발생동태변화,BMCs대기표체적신속상조기주요작용.CNV구BMCs중miR188-5p표체수시간변화적추세여MMP-13상반,여재CNV형성조기MMP-2표체추세상반.miR188-5p적조공파기인시MMP-2/MMP-13,제시BMCs적MMP-2/MMP-13표체가능수miR188-5p조공.
Background Matrix metalloproteinases (MMPs) play important roles in the formation of choroidal neovascularization (CNV),but its mail origin is not ocular cells in situ.Bone marrow-derived cells (BMCs) participate in the formation of CNV and is probably a primary source of expressing MMPs in CNV.MMP-2/MMP-13 is speculated to be the regulating target genes of miR188-5p.Objective This study was to verify whether BMCs are the main source of MMPs,and whether the MMP-2/MMP-13 expression is potentially regulated by miR188-5p.Methods BMCs expressed green fluorescent protein (GFP) from transgenic female C57BL/6J mice were transplanted to female wild-type C57BL/6J mice to establish C57BL/6J.GFP chimeras models,and 42 mice with chimerisms more than 85％ by flow cytometry were included as the experimental group.Other 42 wild-type C57BL/6J mice without the BMCs transplantation were enrolled as the control group.CNV was induced by laser coagulation of retinas on the mice of both groups.MMP-2/MMP-13 levels in the retinochoroid tissue were quantified by ELISA at day 1,3,5,7,10,14,and 28 after photocoagulation.The expression of miR188-5p mR NA in the retinochoroid tissue was assayed by real-time quantitative PCR (RT-qPCR).Immunofluorescence stain and fluorescent in situ hybridization were used to identify the MMP-2/MMP-13 and miR188-5p expressed by GFP-positive BMCs in CNV,and the expression level was quantified by images analysis.Results The proportion of GFP+ mouse mononuclear cells was (90.67±3.02) ％ in the C57BL/6J.GFP chimeras.The concentration changes of MMP-2/MMP-13 in retinochoroid homogenate showed a same tendency with the lapse of time between the experimental group and the control group (MMP-2:F=0.060,P =0.810 ; MMP-13:F =0.012,P =0.915).The expression level was zoomed in retinochoroid tissue after induce of CNV with the maximal value on the third day in both groups,and the proportion in the experimental group was 64.21％ ;while the expression level of MMP-13 was slowly raised after induce of CNV with the peak at the seventh day,and the proportion in the experimental group was 79.61％.A complementary association point of miR188-5p was exhibited in the 3 '-untranslated regions of MMP-2 or MMP-13 by target gene prediction.The expression level of miR188-5p mRNA in the BMCs of CNV area was sharply declined after induce of CNV with the lowest value on the seventh day.A negative correlation was found between the expressing level of miR188-5p and MMP-13 protein (r=-0.868,P＜0.05) as well as early stage of expression level of MMP-2 protein (r=-0.997,P＜ 0.05).Conclusions The elevation of MMP-2/MMP-13 expression levels is associated with the formation of CNV,and the regulation of miR188-5p expression in the BMCs of CNV area is responsible for increase of MMP-2/MMP-13 expression.The tendency of miR188-5p expression is inversed with MMP-2/MMP-13.
