腺病毒介导p21抑制视网膜新生血管生成的实验研究
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摘要
目的：视网膜新生血管(retinal neovascularization, RNV)性疾病是目前主要的致盲眼病,而新生血管的生成依赖于血管内皮细胞的不断分裂增殖。p21是细胞周期中重要的负性调控因子,主要参与细胞周期中G1/S期调控点的调控,可使细胞阻滞于G1期,抑制细胞增殖。本研究通过建立氧诱导RNV小鼠模型并检测p21、CDK2及CyclinE mRNA和蛋白在视网膜组织中的表达,探讨p21在RNV生成过程中的作用及机制；进而应用腺病毒载体将p21基因转染至猴视网膜微血管内皮细胞及RNV模型小鼠视网膜组织,观察其对血管内皮细胞增殖、迁移及RNV的抑制作用及机制。
方法：(1)选取7日龄C57BL/6J小鼠,共96只,随机分为实验组与对照组,每组48只。实验组小鼠按照Smith法建立氧诱导RNV模型,分别于P12、P17、P22时行荧光视网膜铺片及组织病理学检查评估两组小鼠RNV的发生情况,行逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)及蛋白免疫印迹(Western blot. WB)检测p21、CDK2及Cyclin E mRNA和蛋白在两组小鼠视网膜组织中的表达。(2)体外培养猴微血管内皮细胞(RF/6A),取第3～5代处于对数生长期的RF/6A接种到6孔板内,细胞同步化后,分为磷酸盐缓冲液(phosphate buffered solution, PBS)组、腺病毒介导p21基因(adenovirus mediated delivery of p21,Ad-p21)组及腺病毒介导空基因(adenovirus mediated delivery of non-target control. Ad-NC)组,分别应用PBS、Ad-p21及Ad-NC转染RF/6A,培养48h,行流式细胞仪、Transwell检查及成管实验,行RT-PCR及WB检测p21、CDK2及Cyclin E mRNA和蛋白在RF/6A中的表达。(3)选取7日龄C57BL/6J小鼠,共80只,随机分为对照组、PBS组、Ad-p21组及Ad-NC组,每组20只。其中PBS组、Ad-p21组及Ad-NC组小鼠按照Smith法建立氧诱导RNV模型。P11时,非常组不做任何处理,PBS组、Ad-p21组及Ad-NC组玻璃体腔分别注入PBS、Ad-p21及Ad-NC1u1。P17及P22时行荧光视网膜铺片及组织病理学检查评估RNV的发生情况,P17时行RT-PCR及WB检测p21、CDK2及Cyclin E mRNA和蛋白在视网膜组织中的表达。
结果：(1)与对照组比较,P17时实验组小鼠荧光视网膜铺片显示大面积无灌注区及荧光素渗漏,视网膜切片可见大量突破内界膜的血管内皮细胞核。实验组小鼠视网膜组织中p21mRNA和蛋白表达较对照组明显下降,差异有统计学意义；而CDK2和Cyclin E mRNA及蛋白表达较对照组则明显升高,差异有统计学意义。(2)流示细胞仪结果显示Ad-p21组RF/6A细胞出现G0/G1期阻滞,G0/G1期细胞较PBS组及Ad-NC组比例明显增多,差异有统计学意义。Transwell结果显示较PBS组和Ad-NC组,Ad-p21组RF/6A细胞穿膜能力明显减弱,穿膜细胞数较少,差异有统计学意义。体外成管实验显示较PBS组和Ad-NC组,Ad-p21转染组内皮细胞管数较少,差异有统计学意义。Ad-p21组细胞中p21mRNA和蛋白表达较PBS组和Ad-NC组明显升高,差异有统计学意义；而CDK2和Cyclin E mRNA及蛋白表达较PBS组和Ad-NC组则明显降低,差异有统计学意义。(3)Ad-p21组视网膜无灌注区及新生血管较PBS组和Ad-NC组明显减少。Ad-p21组视网膜组织p21mRNA及蛋白表达较其余三组显著增高,差异有统计学意义,而CDK2和Cyclin E mRNA及蛋白表达较其余三组显著降低,差异有统计学意义。
结论：(1)p21表达降低可能是视网膜新生血管生成的发病机制之一。(2)Ad介导p21通过上调p21及下调CDK2、CyclinE的表达,发挥对细胞周期的调控作用,使RF/6A细胞停滞于G0/G1期并抑制其增生和迁移。(3)Ad介导p21可通过上调p21及下调CDK2、CyclinE的表达,抑制视网膜微血管内皮细胞的分裂、增生,从而发挥其抑制视网膜新生血管生成的作用。
Objective:The retinal neovascularization diseases, which result from pathological angiogenesis, are among the most common causes of blindness. The process of angiogenesis involves cellular and morphological changes, including endothelial cell proliferation, migration, and vascular tube formation. The p21WAF1/CIP1(p21) protein negatively regulates the cell cycle by binding to and inhibiting the activity of cyclin-dependent kinases (CDKs)-a family of proteins required for the transition from the G1-(interphase) to the S-(DNA synthesis) phases of the cell cycle. Inhibition of CDK2and cyclinE results in cell cycle arrest at the G1-S phase interface. In this study, firstly we established the mice model of oxygen-induced retinal neovascularization, and detected the expression of p21, CDK2and cylin E mRNA and protein, and discuss the role of p21in the pathogenesis of retinal neovascularization; secondly we transfected adenoviral vector-mediated delivery of p21to RF/6A and retina of the mice with retinal neovascularization, and discuss the inhibitory effect of adenoviral vector-mediated delivery of p21on RF/6A cell proliferation, migration and retinal neovascularization.
Methods:(1) Ninety-six C57BL/6J mice at the age of7days were divided into two groups (experimental and control group) randomly. The model of oxygen-induced retinal neovascularization was establishe according to Smith protocol in experimental group. Retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology at the age of12,17and22days respectively. The expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot at the same time.(2) RF/6A cells were cultured in vitro, and were divided into phosphate buffered solution (PBS) group, adenoviral vector-mediated delivery of p21(Ad-p21) group and adenoviral vector-mediated delivery of non-target control (Ad-NC) group. The cell cycle distribution was analyzed by flow cytometry. Matrigel was used in endothelial-cell tube formation. The migration were detected by transwell. The expression of p21, CDK2and cyclin E mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively.(3) Eighty C57BL/6J mice at the age of7days were divided into control, phosphate buffered solution (PBS), Ad-p21and Ad-negative control (Ad-NC) groups randomly. The oxygen-induced retinal neovasculaiton model was induced by Smith protocol in PBS, Ad-p21and Ad-NC groups. At the age of11days, the mice among PBS, Ad-p21and Ad-NC groups received intravitreal injection of1ul PBS, Ad-p21and Ad-NC respectively. At the age of17and22days, retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology. At the age of17days, the expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot respectively.
Results:(1) There were large areas of non-perfusion and fluorescein leakage in the retina of experimental group. The expression of p21mRNA and protein in experimental group were lower than that in control group, the difference between the two groups was significant. But the expression of CDK2mRNA and protein in experimental group were higher than that in control group, the difference between the two groups was significant.(2) The cell cycle distribution showed that the proportion of G0/G1cells in Ad-p21group was apprently higher than that in PBS and negative control group, the difference among these three groups was significant. Transwell results showed that the number of cells which passed the transwell in Ad-p21group was apprently less than that in PBS and negative control group. The number of endothelial-cell tubes in Ad-p21group was apprently less than that in PBS and negative control group. The expression of p21mRNA and protein in Ad-p21group were higher than that in PBS and negative control group. But the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were higher than that in PBS and negative control group.(3) Compared with PBS and Ad-NC groups, the retinal non-perfusion areas, neovasculartion and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly. The expression of p21mRNA and protein in Ad-p21group were higher than that among the other three groups; the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were lower than that among the other three groups.
Conclusions:(1) p21may play an important role in the development of retinal neovascularization. The decline of p21expression induced endothelium proliferation may be the main reason.(2) The p21mRNA and protein can stably express in RF/6A cells after Ad-p21transfection. Ad-p21can inhibit the proliferation and migration of RF/6A cells by increasing the expression of p21.(3) Ad-p21can inhabit the proliferation of retinal vascular endothelial cells, and then inhabit retinal neovasculaiton by upregulating the expression of p21and downregulating the expression of CDK2and cyclin E.
方法：(1)选取7日龄C57BL/6J小鼠,共96只,随机分为实验组与对照组,每组48只。实验组小鼠按照Smith法建立氧诱导RNV模型,分别于P12、P17、P22时行荧光视网膜铺片及组织病理学检查评估两组小鼠RNV的发生情况,行逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)及蛋白免疫印迹(Western blot. WB)检测p21、CDK2及Cyclin E mRNA和蛋白在两组小鼠视网膜组织中的表达。(2)体外培养猴微血管内皮细胞(RF/6A),取第3～5代处于对数生长期的RF/6A接种到6孔板内,细胞同步化后,分为磷酸盐缓冲液(phosphate buffered solution, PBS)组、腺病毒介导p21基因(adenovirus mediated delivery of p21,Ad-p21)组及腺病毒介导空基因(adenovirus mediated delivery of non-target control. Ad-NC)组,分别应用PBS、Ad-p21及Ad-NC转染RF/6A,培养48h,行流式细胞仪、Transwell检查及成管实验,行RT-PCR及WB检测p21、CDK2及Cyclin E mRNA和蛋白在RF/6A中的表达。(3)选取7日龄C57BL/6J小鼠,共80只,随机分为对照组、PBS组、Ad-p21组及Ad-NC组,每组20只。其中PBS组、Ad-p21组及Ad-NC组小鼠按照Smith法建立氧诱导RNV模型。P11时,非常组不做任何处理,PBS组、Ad-p21组及Ad-NC组玻璃体腔分别注入PBS、Ad-p21及Ad-NC1u1。P17及P22时行荧光视网膜铺片及组织病理学检查评估RNV的发生情况,P17时行RT-PCR及WB检测p21、CDK2及Cyclin E mRNA和蛋白在视网膜组织中的表达。
结果：(1)与对照组比较,P17时实验组小鼠荧光视网膜铺片显示大面积无灌注区及荧光素渗漏,视网膜切片可见大量突破内界膜的血管内皮细胞核。实验组小鼠视网膜组织中p21mRNA和蛋白表达较对照组明显下降,差异有统计学意义；而CDK2和Cyclin E mRNA及蛋白表达较对照组则明显升高,差异有统计学意义。(2)流示细胞仪结果显示Ad-p21组RF/6A细胞出现G0/G1期阻滞,G0/G1期细胞较PBS组及Ad-NC组比例明显增多,差异有统计学意义。Transwell结果显示较PBS组和Ad-NC组,Ad-p21组RF/6A细胞穿膜能力明显减弱,穿膜细胞数较少,差异有统计学意义。体外成管实验显示较PBS组和Ad-NC组,Ad-p21转染组内皮细胞管数较少,差异有统计学意义。Ad-p21组细胞中p21mRNA和蛋白表达较PBS组和Ad-NC组明显升高,差异有统计学意义；而CDK2和Cyclin E mRNA及蛋白表达较PBS组和Ad-NC组则明显降低,差异有统计学意义。(3)Ad-p21组视网膜无灌注区及新生血管较PBS组和Ad-NC组明显减少。Ad-p21组视网膜组织p21mRNA及蛋白表达较其余三组显著增高,差异有统计学意义,而CDK2和Cyclin E mRNA及蛋白表达较其余三组显著降低,差异有统计学意义。
结论：(1)p21表达降低可能是视网膜新生血管生成的发病机制之一。(2)Ad介导p21通过上调p21及下调CDK2、CyclinE的表达,发挥对细胞周期的调控作用,使RF/6A细胞停滞于G0/G1期并抑制其增生和迁移。(3)Ad介导p21可通过上调p21及下调CDK2、CyclinE的表达,抑制视网膜微血管内皮细胞的分裂、增生,从而发挥其抑制视网膜新生血管生成的作用。
Objective:The retinal neovascularization diseases, which result from pathological angiogenesis, are among the most common causes of blindness. The process of angiogenesis involves cellular and morphological changes, including endothelial cell proliferation, migration, and vascular tube formation. The p21WAF1/CIP1(p21) protein negatively regulates the cell cycle by binding to and inhibiting the activity of cyclin-dependent kinases (CDKs)-a family of proteins required for the transition from the G1-(interphase) to the S-(DNA synthesis) phases of the cell cycle. Inhibition of CDK2and cyclinE results in cell cycle arrest at the G1-S phase interface. In this study, firstly we established the mice model of oxygen-induced retinal neovascularization, and detected the expression of p21, CDK2and cylin E mRNA and protein, and discuss the role of p21in the pathogenesis of retinal neovascularization; secondly we transfected adenoviral vector-mediated delivery of p21to RF/6A and retina of the mice with retinal neovascularization, and discuss the inhibitory effect of adenoviral vector-mediated delivery of p21on RF/6A cell proliferation, migration and retinal neovascularization.
Methods:(1) Ninety-six C57BL/6J mice at the age of7days were divided into two groups (experimental and control group) randomly. The model of oxygen-induced retinal neovascularization was establishe according to Smith protocol in experimental group. Retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology at the age of12,17and22days respectively. The expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot at the same time.(2) RF/6A cells were cultured in vitro, and were divided into phosphate buffered solution (PBS) group, adenoviral vector-mediated delivery of p21(Ad-p21) group and adenoviral vector-mediated delivery of non-target control (Ad-NC) group. The cell cycle distribution was analyzed by flow cytometry. Matrigel was used in endothelial-cell tube formation. The migration were detected by transwell. The expression of p21, CDK2and cyclin E mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively.(3) Eighty C57BL/6J mice at the age of7days were divided into control, phosphate buffered solution (PBS), Ad-p21and Ad-negative control (Ad-NC) groups randomly. The oxygen-induced retinal neovasculaiton model was induced by Smith protocol in PBS, Ad-p21and Ad-NC groups. At the age of11days, the mice among PBS, Ad-p21and Ad-NC groups received intravitreal injection of1ul PBS, Ad-p21and Ad-NC respectively. At the age of17and22days, retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology. At the age of17days, the expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot respectively.
Results:(1) There were large areas of non-perfusion and fluorescein leakage in the retina of experimental group. The expression of p21mRNA and protein in experimental group were lower than that in control group, the difference between the two groups was significant. But the expression of CDK2mRNA and protein in experimental group were higher than that in control group, the difference between the two groups was significant.(2) The cell cycle distribution showed that the proportion of G0/G1cells in Ad-p21group was apprently higher than that in PBS and negative control group, the difference among these three groups was significant. Transwell results showed that the number of cells which passed the transwell in Ad-p21group was apprently less than that in PBS and negative control group. The number of endothelial-cell tubes in Ad-p21group was apprently less than that in PBS and negative control group. The expression of p21mRNA and protein in Ad-p21group were higher than that in PBS and negative control group. But the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were higher than that in PBS and negative control group.(3) Compared with PBS and Ad-NC groups, the retinal non-perfusion areas, neovasculartion and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly. The expression of p21mRNA and protein in Ad-p21group were higher than that among the other three groups; the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were lower than that among the other three groups.
Conclusions:(1) p21may play an important role in the development of retinal neovascularization. The decline of p21expression induced endothelium proliferation may be the main reason.(2) The p21mRNA and protein can stably express in RF/6A cells after Ad-p21transfection. Ad-p21can inhibit the proliferation and migration of RF/6A cells by increasing the expression of p21.(3) Ad-p21can inhabit the proliferation of retinal vascular endothelial cells, and then inhabit retinal neovasculaiton by upregulating the expression of p21and downregulating the expression of CDK2and cyclin E.
引文
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