CAJ | 학술논문

背景各种原因导致滤过手术失败的共同点是滤过通道的成纤维细胞过度增生、分化,导致过度纤维化、瘢痕形成.研究发现,p38丝裂原活化蛋白激酶(MAPK)信号通道在成纤维细胞表型转化过程中发挥重要作用. 目的探讨p38 MAPK抑制剂SB203580对滤过手术后结膜下纤维化反应的抑制效果及其作用机制.方法 12只清洁级新西兰白兔行常规青光眼滤过手术制作双眼滤过手术模型,模型兔按随机数字表法随机分为单纯滤过手术组、SB203580治疗组和丝裂霉素C(MMC)对照组.SB203580治疗组兔眼滤过术毕立即结膜下注射0.2 g/L SB203580溶液1 ml,MMC对照组兔眼术中用浸泡0.2 g/L MMC的棉片置于术区结膜下及巩膜瓣下3 min.各组兔术后进行裂隙灯显微镜观察,术后1、3、7、10、14d使用Icare回弹式眼压计测量眼压,术后14 d抽取房水0.2ml,并获取手术滤过区结膜下组织标本,用ELISA法检测房水及滤过区结膜组织中的α-平滑肌肌动蛋白( α-SMA)、纤维连接蛋白的表达,用荧光实时定量PGR检测各组兔术眼滤过区结膜组织中ACTA2、结缔组织生长因子(CTGF)和Ⅰ型胶原蛋白α2链(COL1 A2) mRNA的表达. 结果术后14d,单纯滤过手术组滤过泡血管化、瘢痕形成,SB203580治疗组滤过泡扁平、弥散,MMC对照组滤过泡呈苍白缺血状、扁平弥散.单纯滤过手术组、SB203580治疗组和MMC对照组兔间术前眼压值差异无统计学意义( F=0.065,P=0.937),术后眼压值随着时间的延长逐渐升高,各时间点的总体比较差异有统计学意义(F=32.873,P=0.030).ELISA法检测SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的α-SMA质量浓度均明显低于单纯滤过手术组,而MMC组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的纤维连接蛋白质量浓度均明显低于单纯滤过手术组,而MMC对照组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).荧光实时定量PCR检测表明,各组兔术眼滤过区结膜下组织ACTA2、CTGF、COL1A2mRNA表达差异均有统计学意义(P＜0.01),以单纯滤过手术组表达量最高,SB203580治疗组次之,MMC对照组最低,差异均有统计学意义(P＜0.05).结论 p38 MAPK抑制剂SB203580能减少肌成纤维细胞分化及细胞外基质合成,减轻兔眼滤过手术后的组织纤维化反应. 揹景各種原因導緻濾過手術失敗的共同點是濾過通道的成纖維細胞過度增生、分化,導緻過度纖維化、瘢痕形成.研究髮現,p38絲裂原活化蛋白激酶(MAPK)信號通道在成纖維細胞錶型轉化過程中髮揮重要作用. 目的探討p38 MAPK抑製劑SB203580對濾過手術後結膜下纖維化反應的抑製效果及其作用機製.方法 12隻清潔級新西蘭白兔行常規青光眼濾過手術製作雙眼濾過手術模型,模型兔按隨機數字錶法隨機分為單純濾過手術組、SB203580治療組和絲裂黴素C(MMC)對照組.SB203580治療組兔眼濾過術畢立即結膜下註射0.2 g/L SB203580溶液1 ml,MMC對照組兔眼術中用浸泡0.2 g/L MMC的棉片置于術區結膜下及鞏膜瓣下3 min.各組兔術後進行裂隙燈顯微鏡觀察,術後1、3、7、10、14d使用Icare迴彈式眼壓計測量眼壓,術後14 d抽取房水0.2ml,併穫取手術濾過區結膜下組織標本,用ELISA法檢測房水及濾過區結膜組織中的α-平滑肌肌動蛋白( α-SMA)、纖維連接蛋白的錶達,用熒光實時定量PGR檢測各組兔術眼濾過區結膜組織中ACTA2、結締組織生長因子(CTGF)和Ⅰ型膠原蛋白α2鏈(COL1 A2) mRNA的錶達. 結果術後14d,單純濾過手術組濾過泡血管化、瘢痕形成,SB203580治療組濾過泡扁平、瀰散,MMC對照組濾過泡呈蒼白缺血狀、扁平瀰散.單純濾過手術組、SB203580治療組和MMC對照組兔間術前眼壓值差異無統計學意義( F=0.065,P=0.937),術後眼壓值隨著時間的延長逐漸升高,各時間點的總體比較差異有統計學意義(F=32.873,P=0.030).ELISA法檢測SB203580治療組和MMC對照組兔房水及濾過區結膜下組織中的α-SMA質量濃度均明顯低于單純濾過手術組,而MMC組α-SMA質量濃度明顯低于SB203580治療組,差異均有統計學意義(P＜0.05).SB203580治療組和MMC對照組兔房水及濾過區結膜下組織中的纖維連接蛋白質量濃度均明顯低于單純濾過手術組,而MMC對照組α-SMA質量濃度明顯低于SB203580治療組,差異均有統計學意義(P＜0.05).熒光實時定量PCR檢測錶明,各組兔術眼濾過區結膜下組織ACTA2、CTGF、COL1A2mRNA錶達差異均有統計學意義(P＜0.01),以單純濾過手術組錶達量最高,SB203580治療組次之,MMC對照組最低,差異均有統計學意義(P＜0.05).結論 p38 MAPK抑製劑SB203580能減少肌成纖維細胞分化及細胞外基質閤成,減輕兔眼濾過手術後的組織纖維化反應.
배경 각충원인도치려과수술실패적공동점시려과통도적성섬유세포과도증생、분화,도치과도섬유화、반흔형성.연구발현,p38사렬원활화단백격매(MAPK)신호통도재성섬유세포표형전화과정중발휘중요작용. 목적 탐토p38 MAPK억제제SB203580대려과수술후결막하섬유화반응적억제효과급기작용궤제.방법 12지청길급신서란백토행상규청광안려과수술제작쌍안려과수술모형,모형토안수궤수자표법수궤분위단순려과수술조、SB203580치료조화사렬매소C(MMC)대조조.SB203580치료조토안려과술필립즉결막하주사0.2 g/L SB203580용액1 ml,MMC대조조토안술중용침포0.2 g/L MMC적면편치우술구결막하급공막판하3 min.각조토술후진행렬극등현미경관찰,술후1、3、7、10、14d사용Icare회탄식안압계측량안압,술후14 d추취방수0.2ml,병획취수술려과구결막하조직표본,용ELISA법검측방수급려과구결막조직중적α-평활기기동단백( α-SMA)、섬유련접단백적표체,용형광실시정량PGR검측각조토술안려과구결막조직중ACTA2、결체조직생장인자(CTGF)화Ⅰ형효원단백α2련(COL1 A2) mRNA적표체. 결과 술후14d,단순려과수술조려과포혈관화、반흔형성,SB203580치료조려과포편평、미산,MMC대조조려과포정창백결혈상、편평미산.단순려과수술조、SB203580치료조화MMC대조조토간술전안압치차이무통계학의의( F=0.065,P=0.937),술후안압치수착시간적연장축점승고,각시간점적총체비교차이유통계학의의(F=32.873,P=0.030).ELISA법검측SB203580치료조화MMC대조조토방수급려과구결막하조직중적α-SMA질량농도균명현저우단순려과수술조,이MMC조α-SMA질량농도명현저우SB203580치료조,차이균유통계학의의(P＜0.05).SB203580치료조화MMC대조조토방수급려과구결막하조직중적섬유련접단백질량농도균명현저우단순려과수술조,이MMC대조조α-SMA질량농도명현저우SB203580치료조,차이균유통계학의의(P＜0.05).형광실시정량PCR검측표명,각조토술안려과구결막하조직ACTA2、CTGF、COL1A2mRNA표체차이균유통계학의의(P＜0.01),이단순려과수술조표체량최고,SB203580치료조차지,MMC대조조최저,차이균유통계학의의(P＜0.05).결론 p38 MAPK억제제SB203580능감소기성섬유세포분화급세포외기질합성,감경토안려과수술후적조직섬유화반응.
Background The main cause of filtering surgery failure is over proliferation of fibroblasts in filtering channels,leading to excessive fibrosis and scar formation.Researches determined that p38 mitogen-activated protein kinase(MAPK) signaling pathway plays an important role in fibroblast phenotype transition. Objective The present study was to investigate the inhibitory effect of p38 MAPK inhibitor on myofibroblasts transdifferentiation and the extracellular matrix synthesis after filtration surgery in rabbit eyes. Methods Trabeculectomy was performed on 24 eyes of 12 clean New Zealand white rabbits to establish the filtering operative models.The models were randomized into model group,SB203580 group and mitomycin C ( MMC ) group.1 ml SB203580 ( 0.2 g/L) was conjunctively injected at the end of operation in the rabbits of the SB203580 group,and the cotton piece with 0.2 g/L MMC solution was placed on the operative area for 3 minutes intraoperatively in the rabbits of the MMC group.The bleb appearances were examined under the slit lamp microscope,and intraocular pressure(IOP) was measured with Icare tonometer I,3,7,10,14 days after operation.0.2 ml aqueous humor was extracted and the conjunctive tissue at the filtering area was obtained 14 days after operation for the detection of α-smooth muscle actin (α-SMA) and fibronectin protein by ELISA.Expression of ACTA2 mRNA,connective tissue growth factor(CTGF) mRNA and alpha2 chain of type Ⅰ collagen( COL1A2 )mRNA in conjunctive tissue was assayed with fluorescence real-time PCR. Results Vascularization of fibrosis of filtering bleb were obvious in the eyes of the model group,and the bleb was flat and diffuse in the eyes of the SB203580 group and MMC group on 14 days following operation.No significant difference was seen in IOP before trabeculectomy among these three groups( F=0.065,P=0.937 ).IOP was gradually elevated with the increase of time after operation ( F =32.873,P =0.030 ).ELISA assay showed that α-SMA level in conjunctiva was lower in the SB203580 group and MMC group compared with the model group,and that of MMC group was significant lower than the SB203580 group( P＜0.05 ).Fibronectin level in conjunctiva was lower in the SB203580 group and MMC group compared with the model group,and that of MMC group was significant lower than the SB203580 group (P＜0.05).Fluorescence real-time PCR showed that expressions of the ACTA2 mRNA,CTGF mRNA and COL1A2 mRNA were significantly different among the three groups( P＜0.01 ),with the highest expression in model group and the lowest expression in the MMC group ( P ＜ 0.05 ). Conclusions Fibrotic reaction after trabeculectomy can be suppressed by inhibiting p38 MAPK signal pathway.The mechanism of SB203580 is to reduce the synthesis of myofibroblasts transdiffercntiation and extracellular matrix.