楊寶娣 宋艷萍 陳中山 丁琴

微脈沖半導體激光對兔視網(wǎng)膜色素上皮細胞閾值下光凝的光生物調制效應△

楊寶娣 宋艷萍 陳中山 丁琴

微脈沖激光；視網(wǎng)膜色素上皮細胞；血管內皮生長因子；色素上皮源性因子；堿性成纖維細胞生長因子

目的探討微脈沖激光閾值下光凝對色素兔視網(wǎng)膜形態(tài)結構以及視網(wǎng)膜色素上皮細胞(retinal pigment epithelial cells，RPE)分泌細胞因子的影響。方法微脈沖810 nm激光閾值下光凝色素兔視網(wǎng)膜，光凝后1 d、3 d、7 d、14 d分別行眼底熒光血管造影(fluorescein angiography，F(xiàn)FA)和組織切片技術觀察視網(wǎng)膜結構變化；免疫熒光技術觀察視網(wǎng)膜血管內皮生長因子(vascular endothelial growth factor，VEGF)、色素上皮源性因子(pigment epithelium-derived factor，PEDF)、堿性成纖維細胞生長因子(basic fibroblast growth factor，b-FGF)的表達變化；RT-PCR檢測視網(wǎng)膜組織勻漿中VEGF mRNA、PEDF mRNA、b-FGF mRNA的表達變化。結果光凝后FFA檢查未見熒光素滲漏；組織切片各層結構基本完整。免疫熒光表明，光凝后VEGF、PEDF在視細胞層及RPE層表達明顯增強；b-FGF在神經(jīng)纖維層、節(jié)細胞層也有表達。RT-PCR表明，激光后3種細胞因子mRNA含量均增多，光凝后1 d PEDF mRNA(3.748±0.890)表達最高，3 d次之，與其他時間點比較差異均有統(tǒng)計學意義(均為P<0.05)；b-FGF mRNA光凝后1 d(1.578±0.299)增高最明顯，但與其他時間點比較差異均無統(tǒng)計學意義(均為P>0.05)；光凝后3 d VEGF mRNA(2.301±0.378)表達最高，與其他時間點比較差異均有統(tǒng)計學意義(均為P<0.05)。光凝后14 d，3種細胞因子的升高幅度相近(1.283±0.310、1.662±0.409、1.310±0.184)。結論微脈沖810 nm激光閾值下光凝可刺激正常視網(wǎng)膜RPE細胞協(xié)調表達分泌3種細胞因子，且對視網(wǎng)膜組織結構無損傷。

[眼科新進展，2014，34(1)：5-9]

自從眼底激光器問世，激光治療已成為眾多眼底病的主要治療方法，包括視網(wǎng)膜血管性疾病、視網(wǎng)膜裂孔、眼內腫瘤以及青光眼等，尤其對于視網(wǎng)膜血管性疾病，眼底激光治療是目前最主要的方法之一。傳統(tǒng)激光主要通過激光的熱損傷作用達到治療的目的，包括破壞視網(wǎng)膜組織、改善殘余視網(wǎng)膜的供養(yǎng)狀態(tài)、改變視網(wǎng)膜內血管活性因子的產生等[1-2]。但是這種治療為強激光治療，會引起醫(yī)源性視網(wǎng)膜損傷，限制了激光在黃斑區(qū)疾病的應用[3]。近年來，很多學者提出閾值下光凝，即光凝所采用的激光能量處于損傷閾值(細胞失活)水平以下，在達到治療效果的同時將視網(wǎng)膜損傷降到最小。810 nm激光穿透性較強，主要作用于視網(wǎng)膜色素上皮(retinal pigment epithelium，RPE)層，采用微脈沖模式，調整合適的參數(shù)可進行閾值下光凝。目前研究表明，閾值下微脈沖810 nm 半導體激光可成功治療糖尿病黃斑水腫[4-5]、中心性漿液性脈絡膜視網(wǎng)膜病變[6-7]等，但治療機制仍未完全明確。目前推測，閾值下微脈沖810 nm激光可選擇性作用于視網(wǎng)膜RPE層，通過光生物調制效應刺激RPE 再生，增強其吞噬功能，并促進分泌各種血管活性因子[8-9]，如血管內皮生長因子(vascular endothelial growth factor，VEGF)、堿性成纖維細胞生長因子(basic fibroblast growth factor，b-FGF)、色素上皮源性因子(pigment epithelium-derived factor，PEDF)等，使促血管生長因子與抗血管生長因子達到新的平衡，抑制新生血管的形成，改善視網(wǎng)膜屏障功能，達到治療眼底血管性疾病的目的[10]。本研究我們觀察微脈沖810 nm激光閾值下光凝對色素兔視網(wǎng)膜形態(tài)結構的影響以及激光后RPE細胞表達分泌VEGF、PEGF、b-FGF 的變化，進一步明確閾值下激光治療的作用機制。

1 材料與方法

1.1材料

1.1.1實驗動物健康青紫藍兔12只24眼，體質量2.5～3.0 kg，雌雄不限，由廣州軍區(qū)武漢總醫(yī)院動物實驗中心提供，實驗前雙眼前節(jié)及眼底檢查均正常。

1.1.2主要儀器與試劑810 nm半導體激光器(IRIDEX公司)、眼底熒光造影儀(Heidelberg公司)；小鼠抗兔VEGF單克隆抗體(Abcam公司)、小鼠抗人PEDF單克隆抗體(Abcam公司)、小鼠抗人b-FGF單克隆抗體(Abcam公司)、熒光(Cy3)標記羊抗小鼠IgG(武漢博士德生物工程有限公司)、抗熒光淬滅封片劑(Southernbiotech公司)、Trizol液(Invitrogen公司)、cDNA反轉錄合成試劑盒(Fermentas公司)、SYBR Green熒光定量PCR試劑盒(Fermentas公司)。

1.2實驗方法

1.2.1動物分組、激光造模、眼底熒光血管造影檢查12只(24眼)青紫藍兔隨機分為兩組：實驗組8只(16眼)，對照組4只(8眼)。100 g·L-1水合氯醛3.5 mL·kg-1腹腔注射麻醉，復方托吡卡胺眼液滴眼散瞳，鹽酸奧布卡因眼液滴眼行表面麻醉。實驗組16眼在眼前放置全視網(wǎng)膜鏡，經(jīng)裂隙燈用810 nm半導體激光，參照Sanislo等[11]微脈沖激光對兔眼視網(wǎng)膜形態(tài)學閾能量、組織學閾能量及閾下能量的比值關系和閾值下微脈沖半導體激光治療糖尿病黃斑水腫的相關方法[12-13]確定激光參數(shù)：功率400 mV，光斑200 μm，時間(每次發(fā)射激光的總時間)200 ms，負載系數(shù)10%，避開視盤及有髓神經(jīng)纖維，距離視盤垂直距離1 PD處上下方行視網(wǎng)膜光凝，每眼500點。對照組8眼不做激光光凝。激光光凝后1 d、3 d、7 d、14 d麻醉后行眼底熒光血管造影(fluorescein angiography，F(xiàn)FA)檢查。隨后深麻醉處死，摘取眼球，進行下一步實驗操作。對照組每個時間點分別選取一只兔子進行實驗組同樣的操作。

1.2.2視網(wǎng)膜組織切片HE染色每個時間點各摘取兔右眼球，40 g·L-1多聚甲醛固定，酒精梯度脫水，二甲苯透明，石蠟包埋，切片厚4 μm，脫蠟，脫水，蘇木素-伊紅(HE)染色，中性樹膠封片，顯微鏡下觀察視網(wǎng)膜組織結構改變，采集分析圖像。

1.2.3視網(wǎng)膜組織切片免疫熒光檢測VEGF、PEDF和b-FGF表達視網(wǎng)膜組織切片脫蠟，抗原修復，山羊血清封閉抗原，分別滴加一抗過夜，即小鼠抗兔VEGF單克隆抗體(150)、小鼠抗人PEDF單克隆抗體(1100)、小鼠抗人b-FGF單克隆抗體(1300)，在暗處滴加熒光(Cy3)標記羊抗小鼠IgG，核復染，封片，熒光顯微鏡觀察采集圖像。

1.2.4熒光定量RT-PCR檢測視網(wǎng)膜VEGF、PEDF和b-FGF的mRNA表達采用Trizol一步法提取總RNA，紫外分光光度計檢測RNA的純度與濃度。按照M-MLV逆轉錄酶試劑盒說明書進行逆轉錄反應合成第一鏈cDNA。按照SYBR Green熒光染料試劑盒說明書建立20 μL反應體系，用ABI7900型熒光定量PCR儀進行擴增反應。PCR熱循環(huán)數(shù)：50 ℃ 2 min,95 ℃ 10 min循環(huán)1次，接著變性95 ℃ 30 s，退火和延伸60 ℃ 30 s，循環(huán)40次。所用引物序列：VEGF上游引物5’-CTACCTCCACCATGCCAAGT-3’，下游引物5’-GCACTCCAGGCTTTCATCAT-3’；PEDF上游引物5’-ATCACAGGCAAGCCCATCAA-3’，下游引物5’-GCTGGTTCAGGTGGTAGTCC-3’；b-FGF上游引物5’-GTGCAAACCGTTACCTTGCT-3’，下游引物5’-ACTGCCCAGTTCGTTTCAGT-3’；內參β-actin上游引物5’-AGTGCGACGTGGACATCCG-3’，下游引物5’-TGGCTCTAACAGTCCGCCTAG-3’。每個樣本分別重復3次，以提高實驗的重復性和可信度。目標mRNA的相對含量用相對Ct值法(△△Ct)。

2 結果

2.1光凝前后FFA檢查結果光凝前后，F(xiàn)FA檢查無明顯的熒光素滲漏，光凝后即刻可見不明顯的激光斑樣透見熒光，光凝后14 d透見熒光減弱(圖1)。

2.2光凝前后視網(wǎng)膜組織結構變化光鏡下，正常色素兔視網(wǎng)膜層次結構清晰完整，RPE層連續(xù)規(guī)則。光凝后實驗組兔視網(wǎng)膜各層結構完整，但光凝后1 d，視網(wǎng)膜RPE層局灶性小隆起、欠規(guī)整；光凝后3 d RPE層仍欠規(guī)整，局灶性小隆起較前好轉；光凝后14 d視網(wǎng)膜各層結構恢復正常(圖2)。

Figure 1 FFA results of rabbit’s retina before and after photocoagulation.A:FFA before photocoagulation;B:FFA after photocoagulation instantly;C:FFA at 14 days after photocoagulation 光凝前后兔眼視網(wǎng)膜FFA檢查結果。A：光凝前FFA；B：光凝后即刻FFA；C：光凝后14 d FFA

Figure 2 Changes of retinal tissue of rabbits (HE,×400).A:One day after photocoagulation;B:Three days after photocoagulation;C:Seven days after photocoagulation;D:Fourteen days after photocoagulation 實驗組兔視網(wǎng)膜組織學改變(HE，×400)。A：光凝后1 d；B：光凝后3 d；C：光凝后7 d；D：光凝后14 d

2.3光凝前后視網(wǎng)膜內VEGF、PEDF、b-FGF的表達對照組VEGF在神經(jīng)纖維層(nerve fiber layer,NFL)、節(jié)細胞層(ganglion cell layer,GC)、內叢狀層(inner plexiform layer,IPL)、內核層(inner nuclear layer,INL)、外叢狀層(outer plexiform layer，OPL)、視錐視桿細胞層(rods or cones cell layer,RC)、RPE層表達；實驗組光凝后VEGF在RC及RPE層表達明顯增強，且光凝后3 d表達最強，以后逐漸減弱，光凝后14 d基本恢復正常(圖3)。對照組PEDF在NFL、GC、IPL、INL、RPE層表達；實驗組光凝后PEDF在RC及RPE層表達明顯增強，光凝后1 d表達最強，3 d次之，以后逐漸減弱，但光凝后14 d仍較激光光凝前表達增強(圖4)。對照組b-FGF在RC、RPE層表達；實驗組光凝后b-FGF在NFL、GC也有表達，且光凝后1 d表達更強，此后逐漸減弱，光凝后14 d基本恢復正常(圖5)。

Figure 3 Expression of VEGF in each retinal layer before and after photocoagulation.A：Expression of VEGF in NFL,GC,IPL,INL,OPL,RC and RPE layers in control group;B:Expression of VEGF in RC and RPE layers enhanced obviously at 3 days after photocoagulation in experimental group 光凝前后VEGF在視網(wǎng)膜各層的表達。A：對照組VEGF在NFL、GC、IPL、INL、OPL、RC、RPE層表達；B：實驗組光凝3 d后VEGF在RC及RPE層表達明顯增強

Figure 4 Expression of PEDF in retina before and after photocoagulation.A：Expression of PEDF in control group;B:Expression of PEDF at 1 day after photocoagulation in experimental group;C:Expression of PEDF at 3 days after photocoagulation in experimental group 光凝前后PEDF在視網(wǎng)膜的表達。A：對照組PEDF表達；B：實驗組光凝后1 d PEDF表達；C：實驗組光凝后3 d PEDF表達(×200)

Figure 5 Expression of b-FGF in each retinal layer before and after photocoagulation.A：Expression of b-FGF in RC and RPE layers in control group;B:Expression of b-FGF in NFL and GC layers at 1 day after photocoagulation in experimental group;C：Expression of b-FGF at 7 days after photocoagulation in experimental group 光凝前后b-FGF在視網(wǎng)膜各層的表達。A：對照組b-FGF在RC、RPE層表達；B：實驗組光凝后1 d b-FGF在NFL、GC也有表達；C：實驗組光凝后7 d b-FGF表達

2.4光凝后VEGF、PEDF和b-FGF的mRNA表達微脈沖激光光凝后視網(wǎng)膜組織中VEGF、PEDF和b-FGF mRNA表達均增多，其中VEGF mRNA激光后3 d表達最強，與其他時間點比較差異均有統(tǒng)計學意義(均為P<0.05)，激光后7 d及14 d表達逐漸減弱；PEDF mRNA光凝后1 d表達最強，3 d次之，與其他時間點比較差異均有統(tǒng)計學意義(均為P<0.05)，此后表達逐漸減弱；b-FGF mRNA光凝后表達增強，但各個時間點統(tǒng)計學差異不顯著(均為P>0.05)。光凝后14 d 3種細胞因子的升高幅度相近(表1)。

表1 光凝后VEGF mRNA、PEDF mRNA 和b-FGF mRNA 不同時間點的表達變化Table 1 Expression of VEGF，PEDF，b-FGF mRNA at different time points after laser photocoagulation

3 討論

新生血管的形成是很多眼底病共同的病理改變，如濕性年齡相關性黃斑變性、病理性近視、增殖期糖尿病視網(wǎng)膜病變等，它是機體對缺血、缺氧及炎癥等刺激的一種適應性反應，但極易造成出血、滲出及過度增生等病理改變，損傷眼部結構，引起視功能的嚴重障礙甚至致盲。大量研究表明新生血管形成的直接原因是促血管生長因子與抗血管生長因子之間的平衡失調[10,14-15]。其中VEGF和b-FGF在促血管生長因子中作用顯著，而PEDF在抗血管生長因子中作用顯著。在體內RPE有多種生理功能，包括遮光、構成視網(wǎng)膜-脈絡膜屏障、物質轉運、視黃醛的轉運存儲、吞噬降解脫落的感光細胞外節(jié)、清除氧自由基以及合成多種細胞因子等[16]。正常生理狀態(tài)下,體內的RPE相對靜止，無增殖現(xiàn)象，但是受到炎癥、理化因素及光化學因素的刺激將發(fā)生增殖，并分泌多種細胞因子，如VEGF、PEDF和b-FGF等[10,17]。通過調整合適的激光參數(shù)，可使閾值下微脈沖810 nm激光主要作用于RPE層，對內層神經(jīng)視網(wǎng)膜和外層脈絡膜的影響很小。本研究主要通過觀察閾值下微脈沖激光光凝前后VEGF、PEDF、b-FGF的變化，探討閾值下激光對視網(wǎng)膜RPE細胞的光生物學效應，進一步為視網(wǎng)膜新生血管疾病的治療提供新的思路。

VEGF可誘導內皮細胞的增殖、促進細胞分裂，抑制細胞凋亡，增加血管通透性[18]，具有很強的促血管生長作用。b-FGF屬于肝素結合生長因子家族成員，具有比VEGF更強的促內皮細胞分裂的作用[19-20]，然而，Ozaki等[21]研究表明b-FGF是發(fā)生視網(wǎng)膜新生血管的非必需非充分因素。Wong等[22]實驗結果表明，兔眼玻璃體內同時注射VEGF和b-FGF可使視網(wǎng)膜新生血管長得更快，滲漏更明顯，而VEGF與b-FGF單獨作用均不會出現(xiàn)這種效果。此外，b-FGF可誘導Müller膠質細胞分泌VEGF，并促進細胞增殖[23]。以上研究說明，b-FGF對VEGF有協(xié)同刺激作用。本研究顯示光凝后1 d b-FGF升高最明顯，VEGF在光凝后3 d升高最明顯，這與Hu等[14]的研究結果一致，推測在視網(wǎng)膜新生血管的形成過程中，b-FGF可能對VEGF功能的發(fā)揮起著啟動和允許作用。PEDF具有誘導培養(yǎng)的Y79視網(wǎng)膜母細胞瘤細胞神經(jīng)分化作用。后來的研究表明，PEDF具有神經(jīng)保護[24]和很強的抑制新生血管生長的作用[25-26]。

本研究RT-PCR結果顯示，光凝后1 d、3 d、7 d、14 d，三種細胞因子mRNA表達均升高，其中PEDF、b-FGF光凝后1 d升高最顯著，VEGF光凝后3 d升高最顯著，這與Hu等[14]及Ogata等[8]的實驗結果一致。光凝后14 d三種因子的升高幅度相似，說明閾值下激光可刺激視網(wǎng)膜RPE細胞分泌細胞因子，并保持促血管生長因子與抗血管生長因子的內在平衡，可能起到預防或治療視網(wǎng)膜新生血管的作用。本研究FFA檢查未見血管熒光素滲漏，僅見不明顯的激光斑樣透見熒光，視網(wǎng)膜激光部位病理切片HE染色視網(wǎng)膜各層結構未見明顯損傷，說明采用本實驗激光參數(shù)進行閾值下光凝安全可行，并可以通過及時熒光造影觀察定位激光作用部位。

本研究初步證實了閾值下微脈沖810 nm激光對視網(wǎng)膜RPE層的生物調制效應，促進其分泌細胞因子，并調節(jié)各種因子的內在平衡，發(fā)揮預防或治療視網(wǎng)膜新生血管作用的同時避免造成醫(yī)源性視網(wǎng)膜損傷，逆轉了對傳統(tǒng)激光破壞性治療視網(wǎng)膜血管性疾病的觀點，為黃斑部疾病尤其是血管性疾病的治療提供了新的思路。

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date：Aug 10,2013

National Natural Science Foundation of China(No:61378084)；Scientific Research Foundation of Health Department of Hubei Province(No:JX6C-22)From theOphthalmologyCenterofEntireArmyofClinicalCollegeofWuhanofSouthernMedicalUniversity，WuhanGeneralHospitalofGuangzhouMilitaryRegion，Wuhan430070,HubeiPro-vince，China

Photobiological effects of micropulsed subthreshold 810 nm diode laser photocoagulation on retinal pigment epithelial cells of rabbits

YANG Bao-Di,SONG Yan-Ping,CHEN Zhong-Shan,DING-Qin

micropulsed diode laser;retinal pigment epithelial cell;vascular endothelial growth factor;pigment epithelium-derived factor;basic fibroblast growth factor

Objective To investigate the photobiological effects of micropulsed subthreshold 810 nm diode laser photocoagulation on retinal structure and cell factor secretion under retinal pigment epithelial cells of chinchilla rabbits.Methods Micropulsed subthreshold 810 nm diode laser photocoagulated retina of chinchilla rabbits.By fluorescein angiography (FFA),HE staining of retina,the changes of morphology and vascular of retina were observed at 1 day,3 days,7 days,14 days after photocoagulation.The expression changes of vascular endothelial growth factor (VEGF),pigment epithelium-derived factor (PEDF),basic fibroblast growth factor (b-FGF) in retina were observed by immunofluorescence.The mRNA expression changes of VEGF,PEDF,b-FGF were analyzed in retinal tissue homogenates by RT-PCR.Results After photocoagulation,FFA examination showed no vascular fluorescence leakage;Retinal tissue sections stained with HE showed retina structure was in order generally;Immunofluorescence showed that after laser photocoagulation,the expression of VEGF and PEDF were enhanced significantly in rods or cones cell layer and retinal pigment epithelial layer;b-FGF was also expressed in nerve fiber layer and ganglion cell layer.RT-PCR results showed that after laser photocoagulation the mRNA content of three cytokines increased.PEDF mRNA expression increased to the maximum at 1 day after photocoagulation (3.748±0.890),followed by that at 3 days,which both had significantly difference compared with the other time points(allP<0.05).b-FGF mRNA expression increased to the maximum at 1 day after photocoagulation (1.578±0.299),but for each time point,the difference was not statistically significant(allP>0.05).VEGF mRNA expression increased to the maximum at 3 days after photocoagulation (2.301±0.378),which had significantly difference compared with other time points(allP<0.05).At 14 days after photocoagulation,the increased amplitude of three cytokines was near to each other (1.283±0.310,1.662±0.409,1.310±0.184).Conclusion Micropulsed subthreshold 810 nm diode laser can stimulate normal RPE cellsinvivoto secrete VEGF,PEDF,b-FGF in perfect union with no damage on retinal structure.

楊寶娣，女，1988年11月出生，河南開封人，在讀碩士研究生。聯(lián)系電話：13986005204；E-mail：yangbaodi_8651@163.com

AboutYANGBao-Di:Female,born in November,1988.Postgraduate student.Tel:13986005204;E-mail:yangbaodi_8651@163.com

2013-08-10

國家自然科學基金資助(編號：61378084)；湖北省衛(wèi)生廳科學研究基金資助(編號：JX6C-22)

430070 湖北省武漢市，南方醫(yī)科大學附屬武漢臨床學院，廣州軍區(qū)武漢總醫(yī)院，全軍眼科中心

宋艷萍，E-mail:songyanping@medmail.com.cn

楊寶娣,宋艷萍,陳中山,丁琴.微脈沖半導體激光對兔視網(wǎng)膜色素上皮細胞閾值下光凝的光生物調制效應[J].眼科新進展,2014,34(1):5-9.

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10.13389/j.cnki.rao.2014.0002

修回日期：2013-09-19

本文編輯：方紅玲

Accepteddate:Sep 19,2013

Responsibleauthor:SONG Yan-Ping,E-mail:songyanping@medmail.com.cn

[RecAdvOphthalmol，2014，34(1):5-9]