兔后囊膜混浊模型的建立及其分子机制研究
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摘要
目的利用兔后囊膜混浊动物模型,明确后囊膜混浊(posterior capsule opacification,PCO)形成时间,进一步研究PCO发生及发展的分子机制以及胞外信号调节激酶(extracellular signal-reg-ulated kinase,ERK)信号传导通路的作用机制。
方法取63只兔行右眼晶状体囊外摘除术(extra capsular lens extraction,ECLE),在术后各时间点裂隙灯显微镜下观察PCO发生情况及形态,检测后囊膜上增生细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达以明确PCO形成最早期和最显著期;在以上两个时间点检测术眼(最早期为A组,最显著期为B组)和对照眼(C组)的后囊膜上的连接素43(connexin 43,Cx43)及Ⅳ型胶原的表达以明LECs的增殖程度、细胞之间及其与细胞外基质(extracellular matrix ,ECM)的相互作用及影响;运用westorn blot法和荧光定量聚合酶链反应(FQ-PCR)法测量基质金属蛋白酶-2(matrix metalloproteinases,MMP-2)、组织性金属蛋白酶抑制物-2(tissue inhibitor of metalloproteinase,TIMP-2)的基因及蛋白表达水平以明确两者在调节胶原的动态平衡中的作用;同法检测粘着斑激酶(focal adhesion kinase,FAK)、ERK1/2的基因及蛋白表达水平以了解在在PCO形成过程中ERK信号传导通路调控胶原分泌的作用机制。
结果裂隙灯显微镜下发现术后1个月PCO开始形成,术后3个月PCO最为显著;后囊膜PCNA阳性表达最早为术后2周,最显著为术后3个月;Cx43检测发现C组表达阴性,A组的阳性率为31.52±4.68%,B组的阳性率为54.34±3.65%,t=13.3193,P<0.05,差异有统计学意义。Ⅳ型胶原检测发现C组表达阴性,A组OD值为0.203±0.032,B组OD值为0.477±0.085,t=10.4506,P<0.05,差异有统计学意义,MMP-2, TIMP-2蛋白表达的检测发现C组无MMP-2, TIMP-2蛋白表达;A组MMP-2OD值为45.28±2.57,B组OD值为26.74±3.26,t=13.3985,P<0.05,差异有统计学意义,MMP-2蛋白表达下降。A组TIMP-2OD值为22.46±3.15,B组OD值为32.14±2.92,t=6.7610,P<0.05,差异有统计学意义,TIMP-2蛋白表达升高。基因水平的检测发现MMP-2A组Ct值为23.40±0.66,B组值为17.70±0.60(图13);二者t检验比较有显著性差异(t=14.9522,P<0.05),MMP-2mRNA表达下降;TIMP-2A组Ct值为13.40±0.66;B组Ct值为19.27±0.82;二者t检验比较有显著性差异(t=9.3808,P<0.05),TIMP-2mRNA表达增强。FAK、ERK1/2蛋白表达的检测发现C组无FAK、ERK1/2蛋白表达;A组FAKOD值为24.68±3.12,B组OD值为32.58±3.17,t=5.3284,P<0.05,差异有统计学意义,FAK蛋白表达升高。A组ERK1/2OD值为30.46±3.25,B组OD值为43.52±4.17 ,t=7.4108,P<0.05,差异有统计学意义,ERK1/2蛋白表达升高。基因水平的检测发现FAK A组Ct值为16.77±0.45,B组值为24.77±0.85(图13);二者t检验比较有显著性差异(t=14.3942,P<0.05),FAK mRNA表达增强;ERK1A组Ct值为14.73±0.40;B组Ct值为21.40±0.79;二者t检验比较有显著性差异(t=12.9641,P<0.05),ERK1mRNA表达增强。
结论兔ECLE手术后可成功建立后囊膜混浊模型;术后2周为PCO形成最早期,术后3月为PCO形成的最显著期;ERK信号传导通路在LECs调控ECM合成过程中起到重要作用:残留的LECs由于激活的ERK1/ 2产生异常的迁移,通过MAPK信号通路调控MMP-2及TIMP-2等因子的表达,使得ECM中的Ⅳ型胶原分泌增加,LECs黏附、增殖能力增强,促进了PCO的发生和发展。
0bjective We definituded the time point of forming posterior capsule opacification (PCO) using animal model in rabbits and then studied the mechanism of molecule about PCO and ERK signal pathway.
Methods We observed the complexion of PCO in every time point using microscope after the rabbits were undergone ECLE on the right eyes,and measured the PCNA of posterior capsule by immunohistochemical method, to definitude the time point of the earlyest and the markedest of PCO; To measure Cx43 by immunofluorescence method,to measure typeⅣcollagen by immunohistochemical method,to measure MMP-2, TIMP-2,FAK,ERK1/2 at the posterior capsule both surgery eye and comparison eye by westorn blot and FQ-PCR method in hereinbefore two time point.Group A is the earlyest of PCO,group B is the markedest of PCO,group C is comparison eye.
Results The formation of PCO was discovered after the operation at the 1st month by cranny light microscope,the markedness at the 3th month;PCNA at the posterior capsule was expressed after the operation at the 2nd week, the markedness at the 3th month; There were not TypeⅣcollagen,Cx43, MMPs-2,TIMPs-2,FAK, ERK1/2 at the posterior capsule in group C ,but there were TypeⅣcollagen,Cx43, MMPs-2, TIMPs-2, FAK, ERK1/2 at the posterior capsule in group A,B. There was remarkable difference between group A and group B.
Conclusions A model of PCO in rabbits can be successfully established by using ECLE; The formation of PCO was comfirmed after the operation at the 2nd week,the markedness at the 3th month;ERK signal tramsmit pathway played a key role in the process that LECs adjusted and controled ECM composing:the rudimental LECs engendered exceptional transference due to enabled ERK1/2,the expression of MMPs and TIMPs were adjusted and controled by MARK signal pathway,so as to the exudation of typeⅣcollagen increased in the ECM,the attaching and multiplication ability of LECs were swelled,promoting the happen and development of PCO.
方法取63只兔行右眼晶状体囊外摘除术(extra capsular lens extraction,ECLE),在术后各时间点裂隙灯显微镜下观察PCO发生情况及形态,检测后囊膜上增生细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达以明确PCO形成最早期和最显著期;在以上两个时间点检测术眼(最早期为A组,最显著期为B组)和对照眼(C组)的后囊膜上的连接素43(connexin 43,Cx43)及Ⅳ型胶原的表达以明LECs的增殖程度、细胞之间及其与细胞外基质(extracellular matrix ,ECM)的相互作用及影响;运用westorn blot法和荧光定量聚合酶链反应(FQ-PCR)法测量基质金属蛋白酶-2(matrix metalloproteinases,MMP-2)、组织性金属蛋白酶抑制物-2(tissue inhibitor of metalloproteinase,TIMP-2)的基因及蛋白表达水平以明确两者在调节胶原的动态平衡中的作用;同法检测粘着斑激酶(focal adhesion kinase,FAK)、ERK1/2的基因及蛋白表达水平以了解在在PCO形成过程中ERK信号传导通路调控胶原分泌的作用机制。
结果裂隙灯显微镜下发现术后1个月PCO开始形成,术后3个月PCO最为显著;后囊膜PCNA阳性表达最早为术后2周,最显著为术后3个月;Cx43检测发现C组表达阴性,A组的阳性率为31.52±4.68%,B组的阳性率为54.34±3.65%,t=13.3193,P<0.05,差异有统计学意义。Ⅳ型胶原检测发现C组表达阴性,A组OD值为0.203±0.032,B组OD值为0.477±0.085,t=10.4506,P<0.05,差异有统计学意义,MMP-2, TIMP-2蛋白表达的检测发现C组无MMP-2, TIMP-2蛋白表达;A组MMP-2OD值为45.28±2.57,B组OD值为26.74±3.26,t=13.3985,P<0.05,差异有统计学意义,MMP-2蛋白表达下降。A组TIMP-2OD值为22.46±3.15,B组OD值为32.14±2.92,t=6.7610,P<0.05,差异有统计学意义,TIMP-2蛋白表达升高。基因水平的检测发现MMP-2A组Ct值为23.40±0.66,B组值为17.70±0.60(图13);二者t检验比较有显著性差异(t=14.9522,P<0.05),MMP-2mRNA表达下降;TIMP-2A组Ct值为13.40±0.66;B组Ct值为19.27±0.82;二者t检验比较有显著性差异(t=9.3808,P<0.05),TIMP-2mRNA表达增强。FAK、ERK1/2蛋白表达的检测发现C组无FAK、ERK1/2蛋白表达;A组FAKOD值为24.68±3.12,B组OD值为32.58±3.17,t=5.3284,P<0.05,差异有统计学意义,FAK蛋白表达升高。A组ERK1/2OD值为30.46±3.25,B组OD值为43.52±4.17 ,t=7.4108,P<0.05,差异有统计学意义,ERK1/2蛋白表达升高。基因水平的检测发现FAK A组Ct值为16.77±0.45,B组值为24.77±0.85(图13);二者t检验比较有显著性差异(t=14.3942,P<0.05),FAK mRNA表达增强;ERK1A组Ct值为14.73±0.40;B组Ct值为21.40±0.79;二者t检验比较有显著性差异(t=12.9641,P<0.05),ERK1mRNA表达增强。
结论兔ECLE手术后可成功建立后囊膜混浊模型;术后2周为PCO形成最早期,术后3月为PCO形成的最显著期;ERK信号传导通路在LECs调控ECM合成过程中起到重要作用:残留的LECs由于激活的ERK1/ 2产生异常的迁移,通过MAPK信号通路调控MMP-2及TIMP-2等因子的表达,使得ECM中的Ⅳ型胶原分泌增加,LECs黏附、增殖能力增强,促进了PCO的发生和发展。
0bjective We definituded the time point of forming posterior capsule opacification (PCO) using animal model in rabbits and then studied the mechanism of molecule about PCO and ERK signal pathway.
Methods We observed the complexion of PCO in every time point using microscope after the rabbits were undergone ECLE on the right eyes,and measured the PCNA of posterior capsule by immunohistochemical method, to definitude the time point of the earlyest and the markedest of PCO; To measure Cx43 by immunofluorescence method,to measure typeⅣcollagen by immunohistochemical method,to measure MMP-2, TIMP-2,FAK,ERK1/2 at the posterior capsule both surgery eye and comparison eye by westorn blot and FQ-PCR method in hereinbefore two time point.Group A is the earlyest of PCO,group B is the markedest of PCO,group C is comparison eye.
Results The formation of PCO was discovered after the operation at the 1st month by cranny light microscope,the markedness at the 3th month;PCNA at the posterior capsule was expressed after the operation at the 2nd week, the markedness at the 3th month; There were not TypeⅣcollagen,Cx43, MMPs-2,TIMPs-2,FAK, ERK1/2 at the posterior capsule in group C ,but there were TypeⅣcollagen,Cx43, MMPs-2, TIMPs-2, FAK, ERK1/2 at the posterior capsule in group A,B. There was remarkable difference between group A and group B.
Conclusions A model of PCO in rabbits can be successfully established by using ECLE; The formation of PCO was comfirmed after the operation at the 2nd week,the markedness at the 3th month;ERK signal tramsmit pathway played a key role in the process that LECs adjusted and controled ECM composing:the rudimental LECs engendered exceptional transference due to enabled ERK1/2,the expression of MMPs and TIMPs were adjusted and controled by MARK signal pathway,so as to the exudation of typeⅣcollagen increased in the ECM,the attaching and multiplication ability of LECs were swelled,promoting the happen and development of PCO.
引文
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