眼科研究
眼科研究
안과연구
CHINESE OPHTHALMIC RESEARCH
2010年
2期
140-144
,共5页
郭斌%王应利%牛超%惠延年%范钦华
郭斌%王應利%牛超%惠延年%範欽華
곽빈%왕응리%우초%혜연년%범흠화
视网膜微血管内皮细胞%内皮素-1%一氧化氮%静态压力%青光眼
視網膜微血管內皮細胞%內皮素-1%一氧化氮%靜態壓力%青光眼
시망막미혈관내피세포%내피소-1%일양화담%정태압력%청광안
retinal microvascular endothelial cell%endothelin-1%nitric oxide%static pressure%glaucoma
目的 观察静态压力变化对大鼠视网膜微血管内皮细胞(RMECs)形态、增生活性及分泌血管活性物质内皮素-1(ET-1)和一氧化氮合酶(NOS)的影响.方法 体外培养并鉴定大鼠RMECs,分为A(1.33kPa)、B(2.67kPa)、C(5.33kPa)和D(10.67kPa)组和未加压对照组.用倒置相差显微镜观察细胞形态的变化,常规方法 进行细胞计数,锥虫蓝染色检测活细胞比例.用Griess硝酸还原酶法检测NO代谢产物NO_2~-/NO_3~-的含量,放射免疫法检测培养细胞的ET-1蛋白表达;用RT-PCR法半定量研究RMECs中ET-1、eNOS、iNOS mRNA的表达.结果 B、C、D组RMECs细胞核膨大,胞体拉长,可见少量悬浮细胞;静态压力促进RMECs增生(F=12.205,P＜0.01),C组和D组细胞数量高于对照组、A组和B组(P＜0.01);静态压力升高造成各组细胞损伤,拒染率降低(F=11.180,P＜0.01),B、C、D组细胞拒染率低于对照组(P＜0.05).B、C、D组ET-1浓度较对照组增加(P＜0.01).C组和D组RMECs培养液中NO_2~-/NO_3~-浓度高于对照组(P＜0.01).B、C、D组RMECs中ET-1 mRNA表达较对照组升高,差异有统计学意义(P＜0.01);C组和D组RMECs中eNOS mRNA和iNOS mRNA表达较对照组升高,差异有统计学意义(P＜0.01).结论 高静态压力可直接导致RMECs的结构损伤,高静态压力下RMECs合成ET-1、NO增多可能是高眼压眼底病变的机制之一.
目的 觀察靜態壓力變化對大鼠視網膜微血管內皮細胞(RMECs)形態、增生活性及分泌血管活性物質內皮素-1(ET-1)和一氧化氮閤酶(NOS)的影響.方法 體外培養併鑒定大鼠RMECs,分為A(1.33kPa)、B(2.67kPa)、C(5.33kPa)和D(10.67kPa)組和未加壓對照組.用倒置相差顯微鏡觀察細胞形態的變化,常規方法 進行細胞計數,錐蟲藍染色檢測活細胞比例.用Griess硝痠還原酶法檢測NO代謝產物NO_2~-/NO_3~-的含量,放射免疫法檢測培養細胞的ET-1蛋白錶達;用RT-PCR法半定量研究RMECs中ET-1、eNOS、iNOS mRNA的錶達.結果 B、C、D組RMECs細胞覈膨大,胞體拉長,可見少量懸浮細胞;靜態壓力促進RMECs增生(F=12.205,P＜0.01),C組和D組細胞數量高于對照組、A組和B組(P＜0.01);靜態壓力升高造成各組細胞損傷,拒染率降低(F=11.180,P＜0.01),B、C、D組細胞拒染率低于對照組(P＜0.05).B、C、D組ET-1濃度較對照組增加(P＜0.01).C組和D組RMECs培養液中NO_2~-/NO_3~-濃度高于對照組(P＜0.01).B、C、D組RMECs中ET-1 mRNA錶達較對照組升高,差異有統計學意義(P＜0.01);C組和D組RMECs中eNOS mRNA和iNOS mRNA錶達較對照組升高,差異有統計學意義(P＜0.01).結論 高靜態壓力可直接導緻RMECs的結構損傷,高靜態壓力下RMECs閤成ET-1、NO增多可能是高眼壓眼底病變的機製之一.
목적 관찰정태압력변화대대서시망막미혈관내피세포(RMECs)형태、증생활성급분비혈관활성물질내피소-1(ET-1)화일양화담합매(NOS)적영향.방법 체외배양병감정대서RMECs,분위A(1.33kPa)、B(2.67kPa)、C(5.33kPa)화D(10.67kPa)조화미가압대조조.용도치상차현미경관찰세포형태적변화,상규방법 진행세포계수,추충람염색검측활세포비례.용Griess초산환원매법검측NO대사산물NO_2~-/NO_3~-적함량,방사면역법검측배양세포적ET-1단백표체;용RT-PCR법반정량연구RMECs중ET-1、eNOS、iNOS mRNA적표체.결과 B、C、D조RMECs세포핵팽대,포체랍장,가견소량현부세포;정태압력촉진RMECs증생(F=12.205,P＜0.01),C조화D조세포수량고우대조조、A조화B조(P＜0.01);정태압력승고조성각조세포손상,거염솔강저(F=11.180,P＜0.01),B、C、D조세포거염솔저우대조조(P＜0.05).B、C、D조ET-1농도교대조조증가(P＜0.01).C조화D조RMECs배양액중NO_2~-/NO_3~-농도고우대조조(P＜0.01).B、C、D조RMECs중ET-1 mRNA표체교대조조승고,차이유통계학의의(P＜0.01);C조화D조RMECs중eNOS mRNA화iNOS mRNA표체교대조조승고,차이유통계학의의(P＜0.01).결론 고정태압력가직접도치RMECs적결구손상,고정태압력하RMECs합성ET-1、NO증다가능시고안압안저병변적궤제지일.
Background Researches have demonstrated that ocular hypertension induces the ischemia-reperfusion of retina and further leads to the degeneration of retinal ganglion cells,but its mechanism is beyond understanding.Objective The present study aims to observe the effects of static pressure on the morphology,proliferation activity and viability of cultured retinal microvascular endothelial cells (RMECs) and evaluate the expression of ET-1 and NO in these cells under variant static pressure.Methods RMECs were isolated from 30 healthy Wistar rats and cultured using explant culture method by Ⅷ factor antibody and PECAM-1 antibody.The static pressure of 1.33kPa,2.67kPa,5.33kPa and 10.67kPa was used in culture bottle respectively.The RMECs without static pressure were used as normal control group.The morphology of RMECs under the different static pressure was observed by inverted phase contrast microscopy,and the number of RMECs was counted using the counting plate.Cellular viability was studied by trypan blue staining.The changes of ET-1 and NO_2~-/NO_3~-,two metabolic products of NO,in the medium were detected by radioimmunoassay and Griess's nitrate reductase method.The expression of ET-1,eNOS and iNOS mRNA in RMECs was analyzed by semi-quantitative RT-PCR 24 hours after treatment of variant static pressure.Results Cultured RMECs sticked well at 24 hours and reached to confluence at 48 hours and showed the red fluorescence for Ⅷ factor antibody and PECAM-1 antibody.Enlargement of nuclei,extenders of cell bodies and suspension of RMECs in medium were observed.The number of RMECs was gradually increased.The cell viability was reduced with the raise of static pressure among these four groups(F=12.205,P＜0.01;F=11.180,P＜0.01).The static pressure increased the content of ET-1 released by RMECs in 2.67kPa,5.33kPa and 10.67kPa of static pressure groups,and concentrations of NO_2~-/NO_3~- in the medium showed a significant increase in 5.33kPa and 10.67kPa of static pressure groups compared with normal and 1.33kPa of static pressure groups(P＜0.01).The expressions of ET-1 mRNA,eNOS mRNA and iNOS mRNA were considerably enhanced in 5.33kPa and 10.67kPa of static pressure groups compared with normal control group(P＜0.01).Conclusion Raised static pressure causes the alteration of RMGCs structure and morphology.Static pressure could upregulate the expressions of ET-1,eNOS and iNOS mRNA in RMECs and increase the release of ET-1 and NO.This pathway might be one of pathologic mechanisms of retinal injury induced by high intraocular pressure.
