中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
3期
206-210
,共5页
角膜新生血管/化学诱导%趋化因子%受体%巨噬细胞/代谢%碱%烧伤,化学/病理
角膜新生血管/化學誘導%趨化因子%受體%巨噬細胞/代謝%堿%燒傷,化學/病理
각막신생혈관/화학유도%추화인자%수체%거서세포/대사%감%소상,화학/병리
Corneal neovascularization/chemically induced%Chemokine%Receptor%Macrophage/metabolism%Alkali%Burn,chemical/pathology
背景 研究发现巨噬细胞及其分泌的趋化因子与角膜新生血管(CNV)的发生相关.巨噬细胞具有异质性,在不同的微环境以及不同的刺激条件下发挥不同的作用. 目的 检测C-X3-C趋化因子配体1(CX3CL1)和C-C趋化因子配体2(CCL2)对巨噬细胞增生的作用,并探讨其意义. 方法 7～8周龄雄性BALB/c小鼠20只,用角膜碱烧伤的方法构建左眼CNV模型,术后4d在裂隙灯显微镜下检查小鼠CNV情况,颈椎脱臼法处死小鼠并制备角膜切片.用荧光免疫组织化学法检测小鼠角膜组织中巨噬细胞表面两种趋化因子受体C-C趋化因子受体2(CCR2)和CX3CR1的表达.用密度梯度离心法分离人外周血单核细胞,在含质量分数10％胎牛血清(FBS)的RPMI-1640培养液中加入30 μg/L粒细胞巨噬细胞刺激因子(GM-CSF),诱导和培养人外周血单核细胞来源的巨噬细胞.将培养的细胞分为CD68+CCR2组和CD68+CX3CR1组,分别加入CD68+CCR2抗体和CD68+CX3CR1抗体,每组2管,其中一管作为IgG同型对照.采用流式细胞仪分别检测小鼠碱烧伤角膜组织中巨噬细胞和人外周血巨噬细胞中CX3CR1以及CCR2的表达.分别用人重组CX3CL1和CCL2蛋白刺激巨噬细胞,实时定量PCR(real-time PCR)法检测干预后巨噬细胞中血管内皮生长因子(VEGF)、血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶1(ADAMTS-1)、凝血酶敏感蛋白1(TSP-1)等血管生成相关因子的表达.结果 角膜碱烧伤后4d,裂隙灯显微镜下可见角膜局部有新生血管发生,CNV沿角膜缘向角膜中心区域延伸.荧光免疫组织化学法检测发现,角膜组织内有F4/80阳性的巨噬细胞浸润,其表面可见CCR2和CX3CR1分子的表达,呈绿色荧光.未受GM-CSF诱导培养的巨噬细胞能维持生长约2周,但死亡细胞较多,而经GM-CSF诱导培养的细胞生长稳定,细胞数量增加,无明显悬浮死亡现象.培养的巨噬细胞中CX3CR1表达率平均约为75％,CCR2表达率为45％.Real-time PCR法检测发现,CCL2干预后巨噬细胞中VEGF mRNA的相对表达量明显增加,其中150 mg/L CCL2组VEGF mRNA的相对表达量明显高于对照组,差异有统计学意义(t=-5.09,P=0.03),而ADAMTS-1 mRNA、TSP-1 mRNA表达下降,其中150 mg/L CCL2组ADAMTS-1 mRNA、TSP-1 mRNA表达明显低于对照组,差异有统计学意义(t=3.01,P=0.04;t=4.27,P=0.02).CX3CL1干预后巨噬细胞中VEGF mRNA表达量明显下降,而ADAMTS-1mRNA、TSP-1 mRNA的表达水平升高,与对照组相比,150 mg/L CX3CL1组VEGF mRNA表达量下降,ADAMTS-1 mRNA、TSP-1mRNA表达量下降,差异均有统计学意义(t=6.35,P=0.02；t=-2.92,P=0.04；t=-3.81,P=0.03). 结论 CCL2、CX3CL1能通过其对应受体影响巨噬细胞VEGF、ADAMTS-1、TSP-1等血管新生相关因子的表达,提示通过CCL2、CX3CL1等干预靶点治疗新生血管性眼病的可能.
揹景 研究髮現巨噬細胞及其分泌的趨化因子與角膜新生血管(CNV)的髮生相關.巨噬細胞具有異質性,在不同的微環境以及不同的刺激條件下髮揮不同的作用. 目的 檢測C-X3-C趨化因子配體1(CX3CL1)和C-C趨化因子配體2(CCL2)對巨噬細胞增生的作用,併探討其意義. 方法 7～8週齡雄性BALB/c小鼠20隻,用角膜堿燒傷的方法構建左眼CNV模型,術後4d在裂隙燈顯微鏡下檢查小鼠CNV情況,頸椎脫臼法處死小鼠併製備角膜切片.用熒光免疫組織化學法檢測小鼠角膜組織中巨噬細胞錶麵兩種趨化因子受體C-C趨化因子受體2(CCR2)和CX3CR1的錶達.用密度梯度離心法分離人外週血單覈細胞,在含質量分數10％胎牛血清(FBS)的RPMI-1640培養液中加入30 μg/L粒細胞巨噬細胞刺激因子(GM-CSF),誘導和培養人外週血單覈細胞來源的巨噬細胞.將培養的細胞分為CD68+CCR2組和CD68+CX3CR1組,分彆加入CD68+CCR2抗體和CD68+CX3CR1抗體,每組2管,其中一管作為IgG同型對照.採用流式細胞儀分彆檢測小鼠堿燒傷角膜組織中巨噬細胞和人外週血巨噬細胞中CX3CR1以及CCR2的錶達.分彆用人重組CX3CL1和CCL2蛋白刺激巨噬細胞,實時定量PCR(real-time PCR)法檢測榦預後巨噬細胞中血管內皮生長因子(VEGF)、血小闆凝血酶敏感蛋白樣模體的解整鏈蛋白金屬蛋白酶1(ADAMTS-1)、凝血酶敏感蛋白1(TSP-1)等血管生成相關因子的錶達.結果 角膜堿燒傷後4d,裂隙燈顯微鏡下可見角膜跼部有新生血管髮生,CNV沿角膜緣嚮角膜中心區域延伸.熒光免疫組織化學法檢測髮現,角膜組織內有F4/80暘性的巨噬細胞浸潤,其錶麵可見CCR2和CX3CR1分子的錶達,呈綠色熒光.未受GM-CSF誘導培養的巨噬細胞能維持生長約2週,但死亡細胞較多,而經GM-CSF誘導培養的細胞生長穩定,細胞數量增加,無明顯懸浮死亡現象.培養的巨噬細胞中CX3CR1錶達率平均約為75％,CCR2錶達率為45％.Real-time PCR法檢測髮現,CCL2榦預後巨噬細胞中VEGF mRNA的相對錶達量明顯增加,其中150 mg/L CCL2組VEGF mRNA的相對錶達量明顯高于對照組,差異有統計學意義(t=-5.09,P=0.03),而ADAMTS-1 mRNA、TSP-1 mRNA錶達下降,其中150 mg/L CCL2組ADAMTS-1 mRNA、TSP-1 mRNA錶達明顯低于對照組,差異有統計學意義(t=3.01,P=0.04;t=4.27,P=0.02).CX3CL1榦預後巨噬細胞中VEGF mRNA錶達量明顯下降,而ADAMTS-1mRNA、TSP-1 mRNA的錶達水平升高,與對照組相比,150 mg/L CX3CL1組VEGF mRNA錶達量下降,ADAMTS-1 mRNA、TSP-1mRNA錶達量下降,差異均有統計學意義(t=6.35,P=0.02；t=-2.92,P=0.04；t=-3.81,P=0.03). 結論 CCL2、CX3CL1能通過其對應受體影響巨噬細胞VEGF、ADAMTS-1、TSP-1等血管新生相關因子的錶達,提示通過CCL2、CX3CL1等榦預靶點治療新生血管性眼病的可能.
배경 연구발현거서세포급기분비적추화인자여각막신생혈관(CNV)적발생상관.거서세포구유이질성,재불동적미배경이급불동적자격조건하발휘불동적작용. 목적 검측C-X3-C추화인자배체1(CX3CL1)화C-C추화인자배체2(CCL2)대거서세포증생적작용,병탐토기의의. 방법 7～8주령웅성BALB/c소서20지,용각막감소상적방법구건좌안CNV모형,술후4d재렬극등현미경하검사소서CNV정황,경추탈구법처사소서병제비각막절편.용형광면역조직화학법검측소서각막조직중거서세포표면량충추화인자수체C-C추화인자수체2(CCR2)화CX3CR1적표체.용밀도제도리심법분리인외주혈단핵세포,재함질량분수10％태우혈청(FBS)적RPMI-1640배양액중가입30 μg/L립세포거서세포자격인자(GM-CSF),유도화배양인외주혈단핵세포래원적거서세포.장배양적세포분위CD68+CCR2조화CD68+CX3CR1조,분별가입CD68+CCR2항체화CD68+CX3CR1항체,매조2관,기중일관작위IgG동형대조.채용류식세포의분별검측소서감소상각막조직중거서세포화인외주혈거서세포중CX3CR1이급CCR2적표체.분별용인중조CX3CL1화CCL2단백자격거서세포,실시정량PCR(real-time PCR)법검측간예후거서세포중혈관내피생장인자(VEGF)、혈소판응혈매민감단백양모체적해정련단백금속단백매1(ADAMTS-1)、응혈매민감단백1(TSP-1)등혈관생성상관인자적표체.결과 각막감소상후4d,렬극등현미경하가견각막국부유신생혈관발생,CNV연각막연향각막중심구역연신.형광면역조직화학법검측발현,각막조직내유F4/80양성적거서세포침윤,기표면가견CCR2화CX3CR1분자적표체,정록색형광.미수GM-CSF유도배양적거서세포능유지생장약2주,단사망세포교다,이경GM-CSF유도배양적세포생장은정,세포수량증가,무명현현부사망현상.배양적거서세포중CX3CR1표체솔평균약위75％,CCR2표체솔위45％.Real-time PCR법검측발현,CCL2간예후거서세포중VEGF mRNA적상대표체량명현증가,기중150 mg/L CCL2조VEGF mRNA적상대표체량명현고우대조조,차이유통계학의의(t=-5.09,P=0.03),이ADAMTS-1 mRNA、TSP-1 mRNA표체하강,기중150 mg/L CCL2조ADAMTS-1 mRNA、TSP-1 mRNA표체명현저우대조조,차이유통계학의의(t=3.01,P=0.04;t=4.27,P=0.02).CX3CL1간예후거서세포중VEGF mRNA표체량명현하강,이ADAMTS-1mRNA、TSP-1 mRNA적표체수평승고,여대조조상비,150 mg/L CX3CL1조VEGF mRNA표체량하강,ADAMTS-1 mRNA、TSP-1mRNA표체량하강,차이균유통계학의의(t=6.35,P=0.02；t=-2.92,P=0.04；t=-3.81,P=0.03). 결론 CCL2、CX3CL1능통과기대응수체영향거서세포VEGF、ADAMTS-1、TSP-1등혈관신생상관인자적표체,제시통과CCL2、CX3CL1등간예파점치료신생혈관성안병적가능.
Background Intracorneal macrophages play a critical role in corneal neovascularization (CNV)by secreting relative chemokines.But macrophages are characteristic by heterogeneity which has different biologic functions under different induction or stimulation from microenvironment.Objective This study was to detect the effects of chemokine (C-X3-C motif) ligand 1 (CX3CL1) and chemokine (C-C motif) ligand 2 (CCL2) on macrophages in vitro.Methods CNV was induced by corneal alkali burn in the left eyes of 20 male BALB/c mice aged 7-8 weeks.The CNV was evaluated under the slit lamp microscope 4 days after alkali burn,and then the corneal sections were prepared after mice were sacrificed.The expressions of CCR2 and CX3CR1 in the corneal specimens were detected by histo-fluorescence staining.Human peripheral blood mononuclear cells were separated using density gradient centrifugation and incubated in RPMI-1640 medium containing 10％ fetal bovine seruml(FBS) with 30 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF).The cells were divided into CD68 +CCR2 group and CD68+CX3CR1 group,and the percentage of the CX3CR1 and CCR2 expressions in the infiltrated macrophages of corneal specimens and human monocyte-derived macrophages was assayed by flow cytometry.The cultured cells were stimulated using human recombinant CX3CL1 and CCL2 proteins,and real-time PCR was used to detect the relative expressions of angiogenesis-related factors in macrophages.Results CNV was found in corneas 4 days after alkali burn and the CNV onsets from corneal limbus to central zone observed by a slit lamp.CCR2 and CX3CR1 were expressed in the F4/80-positive macrophages in alikali burned corneas.The macrophages grew for two weeks and appeared more dead cells in without GM-CSF group,but in GM-CSF induced group,the number of macrophages was increased.The percentage of CX3CR1-positive cells was 75％ and that of CCR2-positive cells was 45％.Real-time PCR showed that expression level of vascular endothelial growth factor (VEGF) mRNA increased and that ADAMTS-1 mRNA or TSP-1 mRNA decreased on macrophages after CCL2 stimulation,with significant differences in the 150 mg/L CCL2 group compared with the control group (t =-5.09,P =0.03 ; t =3.01,P =0.04 ; t =4.27,P =0.02).However,the VEGF mRNA expression decreased and ADAMTS-1 mRNA and TSP-1 mRNA increased after CX3CL1 stimulation,showing significant differences between the 150 mg/L CX3CL1 group and the control group (t=6.35,P=0.O2;t=-2.92,P=0.04; t=-3.81,P=0.03).Conclusions These results suggest that the macrophages have high heterogeneity.CCL2-and CX3CL1-expressing macrophages can regulate the expressions of angiogenesis-related factors.Macrophage chemokine signal may be a good target for treatment of neovascular ocular disease.
