组织蛋白酶B与血管内皮生长因子影响高氧诱导小鼠视网膜新生血管形成的实验研究
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摘要
目的探讨组织蛋白酶B(cathepsin B)与血管内皮生长因子(VEGF)在小鼠视网膜新生血管形成中的影响。方法选用清洁级C57BL/6J小鼠共44只,7～8日龄,雌雄不限。应用随机数字表法将其分为正常对照组和高氧处理组。其中,正常对照组11只小鼠(22只眼),高氧处理组33只小鼠(66只眼)。正常对照组小鼠在标准环境中生长;高氧处理组小鼠为建立视网膜新生血管动物模型,其环境除氧体积分数为(75±2)%,其他均相同,氧箱内饲养5 d后,将其返回到标准环境中。以视网膜表面出现新生血管簇、无灌注区,判定为造模成功。再应用随机数字表法将成模的小鼠随机分为模型对照组、实验对照组和实验组,每组11只小鼠(22只眼)。正常对照组和模型对照组不予任何药物干预,实验对照组和实验组分别给予玻璃体腔注射二甲基亚砜和溶于二甲基亚砜的CA-074 Me(cathepsin B抑制剂),继而在标准环境中饲养5 d。采用心腔注射荧光素标记葡聚糖,视网膜铺片法观察新生血管生成的情况;分别采用实时聚合酶链反应法和蛋白质印迹法检测小鼠视网膜组织中cathepsin B、VEGF信使核糖核酸(mRNA)相对表达量及蛋白的表达量。所有数据均以均数±标准差■表示。采用单因素方差分析比较各组总体差异,当差异有统计学意义时,再用SNK-q检验进行组间多重比较分析。结果在荧光显微镜下,实验组较模型对照组和实验对照组视网膜血管走行相对清晰,未见明显的缺血无灌注区。各组cathepsin B与VEGF mRNA及蛋白的表达量总体存在差异,差异有统计学意义(F=25.23,22.98,43.86,67.92;P<0.05)。模型对照组和实验对照组中cathepsin B与VEGF mRNA及蛋白的表达量均增高,而实验组中cathepsin B与VEGF的mRNA及蛋白的表达量则降低。实验组与正常对照组比较、实验对照组与模型对照组比较,差异均无统计学意义(P>0.05)。结论 CA-074 Me可抑制cathepsin B和VEGF的mRNA及蛋白的表达。cathepsin B和VEGF表达减少可抑制小鼠视网膜新生血管的形成,且cathepsin B与VEGF之间可能存在着双向调节的关系。
Objective The aim of this study was to investigate the correlation between cathepsin B and VEGF in the retinal neovascularization of mice. Methods 44 clean 7-day-old C57 BL/6 J mice were randomly divided into the control group(22 eyes of 11 mice) and hyperoxia treatment group(66 eyes of 33 mice). The mice in the control group were feed under the standard condition. Retinal neovascularization animal model was established in mice of the hyperoxic treatment group, which was placed in an airtight oxygen chamber with an oxygen volume fraction of(75±2)% for 5 days, and then transferred to the standard condition. After 5 days, the retinal surface was successfully modeled with new vascular clusters and non-perfusion areas. The model mice were randomly assigned into model control group, experimental control group and experimental group with 11 mice(22 eyes) in each group. The mice in the normal control group and the model control group were without any drug intervention. While the mice in the experimental control group and the experimental group received intravitreal injection of DMSO and CA-074 Me dissolved in DMSO( the inhibitor of cathepsin B),respectively. After 5 days for treatment,the neovessels were observed by FITC-dextran retinal angiography in 17-day-old mice. The mRNA expression levels of cathepsin B and VEGF were performed by real-time PCR; the protein expression levels were detected by Western blot. All data were presented with mean ± standard deviation. Univariate ANOVA was used to compare the overall difference among all groups. When the difference was statistically significant,SNK-q test was further used for multiple comparisons between each group. Results Under fluorescence microscope,the retinal vascular in the experimental group was relatively clear compared with the model control group and the experimental control group,and the obvious ischemia or perfusion area were not found. There was a significant difference in the total mean of each group( F = 25. 23,22. 98,43. 86,67. 92; P < 0. 05). Compared with the control group,the expression levels of mRNA and protein of cathepsin B and VEGF in the control group and the experimental control group were significantly increased; the expression levels in the experimental group were significantly decreased compared with the control group and the experimental control group. While there was not shown significant difference between the control group and the experimental group and between the control group and the experimental control group( P > 0. 05). Conclusions The CA-074 Me could inhibit the expression levels of mRNA and protein content of cathepsin B and VEGF; the reduction of cathepsin B and VEGF could be inhibited the expression of retinal neovascularization in mice; and thus there may be a twoway regulation mechanism between cathepsin B and VEGF.
Objective The aim of this study was to investigate the correlation between cathepsin B and VEGF in the retinal neovascularization of mice. Methods 44 clean 7-day-old C57 BL/6 J mice were randomly divided into the control group(22 eyes of 11 mice) and hyperoxia treatment group(66 eyes of 33 mice). The mice in the control group were feed under the standard condition. Retinal neovascularization animal model was established in mice of the hyperoxic treatment group, which was placed in an airtight oxygen chamber with an oxygen volume fraction of(75±2)% for 5 days, and then transferred to the standard condition. After 5 days, the retinal surface was successfully modeled with new vascular clusters and non-perfusion areas. The model mice were randomly assigned into model control group, experimental control group and experimental group with 11 mice(22 eyes) in each group. The mice in the normal control group and the model control group were without any drug intervention. While the mice in the experimental control group and the experimental group received intravitreal injection of DMSO and CA-074 Me dissolved in DMSO( the inhibitor of cathepsin B),respectively. After 5 days for treatment,the neovessels were observed by FITC-dextran retinal angiography in 17-day-old mice. The mRNA expression levels of cathepsin B and VEGF were performed by real-time PCR; the protein expression levels were detected by Western blot. All data were presented with mean ± standard deviation. Univariate ANOVA was used to compare the overall difference among all groups. When the difference was statistically significant,SNK-q test was further used for multiple comparisons between each group. Results Under fluorescence microscope,the retinal vascular in the experimental group was relatively clear compared with the model control group and the experimental control group,and the obvious ischemia or perfusion area were not found. There was a significant difference in the total mean of each group( F = 25. 23,22. 98,43. 86,67. 92; P < 0. 05). Compared with the control group,the expression levels of mRNA and protein of cathepsin B and VEGF in the control group and the experimental control group were significantly increased; the expression levels in the experimental group were significantly decreased compared with the control group and the experimental control group. While there was not shown significant difference between the control group and the experimental group and between the control group and the experimental control group( P > 0. 05). Conclusions The CA-074 Me could inhibit the expression levels of mRNA and protein content of cathepsin B and VEGF; the reduction of cathepsin B and VEGF could be inhibited the expression of retinal neovascularization in mice; and thus there may be a twoway regulation mechanism between cathepsin B and VEGF.
引文
[1] Rubio RG,Adamis AP.Ocular angiogenesis:vascular endothelial growth factor and other factors[J].Dev Ophthalmol,2016,55:28-37.
[2] Turk V,Stoka V,Vasiljeva O,et al.Cysteine cathepsins:from structure,function and regulation to new frontiers[J].Biochim Biophys Acta,2012,1824(1):68-88.
[3] Smith LEH,Wesoloiuski E,McLellan A,et al.Oxygen-induced retinopathy in the mouse[J].Invest Ophthalmol Vis Sci,1994,35(1):101-111.
[4] 张南文,吴伟芳,林剑鸣.不同浓度水合氯醛在两种雄性小鼠麻醉中的应用[J].临床麻醉学杂志,2014,30(2):171-173.
[5] 贺荣华,周国宏,孔丽,等.酪氨酸激酶抑制剂对高氧诱导的视网膜新生血管的抑制作用[J].中华实验眼科杂志,2018,36(1):29-33.
[6] 易健明,屈武斌,张成岗.实时荧光定量PCR的数据分析方法[J].生物技术通讯,2015,26(1):140-145.
[7] 王文娟,周国宏.组织蛋白酶B和血管内皮生长因子在高氧诱导的视网膜新生血管中的作用[J].中华实验眼科杂志,2017,36(10):873-877.
[8] Campochiaro PA.Molecular pathogenesis of retinal and choroidal vascular diseases[J].Prog Retin Eye Res,2015,49:67-81.
[9] 郭炜杭,黄巧冰.新生血管成熟不足机制的研究进展[J].广东医学,2017,38(17):2718-2721.
[10] Seghezzi G,Patel S,Ren CJ,et al.Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries:an autocrine mechanism contributing to angiogenesis[J].J Cell Biol,1998,141(7):1659-1673.
[11] Goldman CK,Kim J,Wong WL,et al.Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells:a model of glioblastoma multiforme pathophysiology.[J].Mol Biol Cell,1993,4(1):121-133.
[12] He X,Cheng R,Benyajati S,et al.PEDF and its roles in physiological and pathological conditions:implication in diabetic and hypoxia-induced angiogenic diseases[J].Clin Sci,2015,128(11):805-823.
[13] 毕超,刘廷,董晓光.细胞外基质相关分子在眼底新生血管形成中的作用[J].中华实验眼科杂志,2012,30(4):371-375.
[14] Bishop PN.The role of extracellular matrix in retinal vascular development and preretinal neovascularization[J].Exp Eye Res,2015,133:30-36.
[15] Marchand M,Monnot C,Muller L,et al.Extracellular matrix scaffolding in angiogenesis and capillary homeostasis[J].Semin Cell Dev Biol,2019,89:147-156.
[16] Benders KE,van Weeren PR,Badylak SF,et al.Extracellular matrix scaffolds for cartilage and bone regeneration[J].Trends Biotechnol,2013,31(3):169-176.
[17] Lohler J,Timpl R,Jaenisch R.Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death[J].Cell,1984,38(2):597-607.
[18] Kalluri R.Basement membranes:structure,assembly and role in tumour angiogenesis[J].Nat Rev Cancer,2003,3(6):422-433.
[19] 李祥春,张开光.组织蛋白酶B与胃癌研究进展[J].安徽医药,2010,14(9):166-167.
[20] Chen WN,Chen JY,Jiao BY,et al.Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion[J].J Virol,2012,86(24):13533-13541.
[21] Ruan J,Zheng H,Rong X,et al.Over-expression of cathepsin B in hepatocellular carcinomas predicts poor prognosis of HCC patients[J].Mol Cancer,2016,15:17.
[22] Buhler A ,Berger S,Bengsch F,et al.Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice[J].Exp Eye Res,2013,115:73-78.
[23] 周国宏,于文贞,黎晓新.视网膜新生血管形成中Cathepsin B的变化[J].眼科研究,2007,25(10):745-748.
[24] 周国宏,孔丽.Cathepsin B-RNAi-lentivirus抑制小鼠视网膜新生血管形成的研究[J].中华实验眼科杂志,2013,31(5):452-455.
[25] Suzuki Y,Matsumoto M,Katoh C,et al.Medical treatment for experimental retinal vein occlusion-thrombolytic effect of nasaruplase[J].Nippon Ganka Gakkai zasshi,1996,100(1):27-33.
[26] Takei K,Sato T,Nonoyama T,et al.A new model of transient complete obstruction of retinal vessels induced by endothelin-1 injection into the posterior vitreous body in rabbits[J].Graefes Arch Clin Exp Ophthalmol,1993,231(8):476-481.
[27] Matsushima M,Yamada H,Yamamoto C,et al.Expression of basic fibroblast growth factor and fibroblast growth factor receptor 1 in the experimental retinal vein occlusion model[J].Nippon Ganka Gakkai zasshi,1997,101(7):564-570.
[28] 陈晓隆,李芳.缬沙坦抑制糖尿病大鼠视网膜内皮素-1表达的实验研究[J/CD].中华眼科医学杂志(电子版),2017,7(2):72-77.
[29] Pierce EA,Avery RL,Foley ED,et al.Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization[J].Proc Natl Acad Sci,1995,92(3):905-909.
[30] Wang F,Bai Y,Yu W,et al.Anti-angiogenic effect of KH902 on retinal neovascularization[J].Graefes Arch Clin Exp Ophthalmol,2013,251(9):2131-2139.
[31] Liang X,Zhou H,Ding Y,et al.TMP prevents retinal neovascularization and imparts neuroprotection in an oxygen-induced retinopathy model[J].Invest Ophthalmol Vis Sci,2012,53(4):2157-2169.
[32] Montaser M,Lalmanach G,Mach L.CA-074,but not its methyl ester CA-074Me,is a selective inhibitor of cathepsin B within living cells[J].Biol Chem,2002,383(7-8):1305-1308.
[33] 周国宏,黎晓新.组织蛋白酶B抑制剂对视网膜血管内皮细胞系生物学行为的影响[J].山西医药杂志,2007,36(6):519-520.
[34] 周国宏,于文贞,黎晓新.组织蛋白酶B抑制剂干预鼠视网膜新生血管形成的研究[J].中华眼科杂志,2008,44(3):207-211.
[35] Chang SH ,Kanasaki K,Gocheva V,et al.VEGF a induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules,causing basement membrane degradation and mother vessel formation[J].Cancer Res,2009,69(10):4537-4544.
[36] Zhang W,Xiong Z,Wei T,et al.Nuclear factor 90 promotes angiogenesis by regulating HIF-1alpha/VEGF-A expression through the PI3K/Akt signaling pathway in human cervical cancer[J].Cell Death Dis,2018,9(3):276.
[37] Wang T ,Liu J,Xiao XQ.Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3,ERK and AKT signaling pathways[J].Arch Pharm Res,2014,38(2):282-289.
[38] Lei Z,Duan H,Zhao T,et al.PARK2 inhibits osteosarcoma cell growth through the JAK2/STAT3/VEGF signaling pathway[J].Cell Death Dis,2018,9(3):375.
[39] Malla RR,Gopinath S,Gondi CS,et al.Cathepsin B and uPAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma[J].Cancer Gene Ther,2011,18(6):419-434.
[2] Turk V,Stoka V,Vasiljeva O,et al.Cysteine cathepsins:from structure,function and regulation to new frontiers[J].Biochim Biophys Acta,2012,1824(1):68-88.
[3] Smith LEH,Wesoloiuski E,McLellan A,et al.Oxygen-induced retinopathy in the mouse[J].Invest Ophthalmol Vis Sci,1994,35(1):101-111.
[4] 张南文,吴伟芳,林剑鸣.不同浓度水合氯醛在两种雄性小鼠麻醉中的应用[J].临床麻醉学杂志,2014,30(2):171-173.
[5] 贺荣华,周国宏,孔丽,等.酪氨酸激酶抑制剂对高氧诱导的视网膜新生血管的抑制作用[J].中华实验眼科杂志,2018,36(1):29-33.
[6] 易健明,屈武斌,张成岗.实时荧光定量PCR的数据分析方法[J].生物技术通讯,2015,26(1):140-145.
[7] 王文娟,周国宏.组织蛋白酶B和血管内皮生长因子在高氧诱导的视网膜新生血管中的作用[J].中华实验眼科杂志,2017,36(10):873-877.
[8] Campochiaro PA.Molecular pathogenesis of retinal and choroidal vascular diseases[J].Prog Retin Eye Res,2015,49:67-81.
[9] 郭炜杭,黄巧冰.新生血管成熟不足机制的研究进展[J].广东医学,2017,38(17):2718-2721.
[10] Seghezzi G,Patel S,Ren CJ,et al.Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries:an autocrine mechanism contributing to angiogenesis[J].J Cell Biol,1998,141(7):1659-1673.
[11] Goldman CK,Kim J,Wong WL,et al.Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells:a model of glioblastoma multiforme pathophysiology.[J].Mol Biol Cell,1993,4(1):121-133.
[12] He X,Cheng R,Benyajati S,et al.PEDF and its roles in physiological and pathological conditions:implication in diabetic and hypoxia-induced angiogenic diseases[J].Clin Sci,2015,128(11):805-823.
[13] 毕超,刘廷,董晓光.细胞外基质相关分子在眼底新生血管形成中的作用[J].中华实验眼科杂志,2012,30(4):371-375.
[14] Bishop PN.The role of extracellular matrix in retinal vascular development and preretinal neovascularization[J].Exp Eye Res,2015,133:30-36.
[15] Marchand M,Monnot C,Muller L,et al.Extracellular matrix scaffolding in angiogenesis and capillary homeostasis[J].Semin Cell Dev Biol,2019,89:147-156.
[16] Benders KE,van Weeren PR,Badylak SF,et al.Extracellular matrix scaffolds for cartilage and bone regeneration[J].Trends Biotechnol,2013,31(3):169-176.
[17] Lohler J,Timpl R,Jaenisch R.Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death[J].Cell,1984,38(2):597-607.
[18] Kalluri R.Basement membranes:structure,assembly and role in tumour angiogenesis[J].Nat Rev Cancer,2003,3(6):422-433.
[19] 李祥春,张开光.组织蛋白酶B与胃癌研究进展[J].安徽医药,2010,14(9):166-167.
[20] Chen WN,Chen JY,Jiao BY,et al.Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion[J].J Virol,2012,86(24):13533-13541.
[21] Ruan J,Zheng H,Rong X,et al.Over-expression of cathepsin B in hepatocellular carcinomas predicts poor prognosis of HCC patients[J].Mol Cancer,2016,15:17.
[22] Buhler A ,Berger S,Bengsch F,et al.Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice[J].Exp Eye Res,2013,115:73-78.
[23] 周国宏,于文贞,黎晓新.视网膜新生血管形成中Cathepsin B的变化[J].眼科研究,2007,25(10):745-748.
[24] 周国宏,孔丽.Cathepsin B-RNAi-lentivirus抑制小鼠视网膜新生血管形成的研究[J].中华实验眼科杂志,2013,31(5):452-455.
[25] Suzuki Y,Matsumoto M,Katoh C,et al.Medical treatment for experimental retinal vein occlusion-thrombolytic effect of nasaruplase[J].Nippon Ganka Gakkai zasshi,1996,100(1):27-33.
[26] Takei K,Sato T,Nonoyama T,et al.A new model of transient complete obstruction of retinal vessels induced by endothelin-1 injection into the posterior vitreous body in rabbits[J].Graefes Arch Clin Exp Ophthalmol,1993,231(8):476-481.
[27] Matsushima M,Yamada H,Yamamoto C,et al.Expression of basic fibroblast growth factor and fibroblast growth factor receptor 1 in the experimental retinal vein occlusion model[J].Nippon Ganka Gakkai zasshi,1997,101(7):564-570.
[28] 陈晓隆,李芳.缬沙坦抑制糖尿病大鼠视网膜内皮素-1表达的实验研究[J/CD].中华眼科医学杂志(电子版),2017,7(2):72-77.
[29] Pierce EA,Avery RL,Foley ED,et al.Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization[J].Proc Natl Acad Sci,1995,92(3):905-909.
[30] Wang F,Bai Y,Yu W,et al.Anti-angiogenic effect of KH902 on retinal neovascularization[J].Graefes Arch Clin Exp Ophthalmol,2013,251(9):2131-2139.
[31] Liang X,Zhou H,Ding Y,et al.TMP prevents retinal neovascularization and imparts neuroprotection in an oxygen-induced retinopathy model[J].Invest Ophthalmol Vis Sci,2012,53(4):2157-2169.
[32] Montaser M,Lalmanach G,Mach L.CA-074,but not its methyl ester CA-074Me,is a selective inhibitor of cathepsin B within living cells[J].Biol Chem,2002,383(7-8):1305-1308.
[33] 周国宏,黎晓新.组织蛋白酶B抑制剂对视网膜血管内皮细胞系生物学行为的影响[J].山西医药杂志,2007,36(6):519-520.
[34] 周国宏,于文贞,黎晓新.组织蛋白酶B抑制剂干预鼠视网膜新生血管形成的研究[J].中华眼科杂志,2008,44(3):207-211.
[35] Chang SH ,Kanasaki K,Gocheva V,et al.VEGF a induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules,causing basement membrane degradation and mother vessel formation[J].Cancer Res,2009,69(10):4537-4544.
[36] Zhang W,Xiong Z,Wei T,et al.Nuclear factor 90 promotes angiogenesis by regulating HIF-1alpha/VEGF-A expression through the PI3K/Akt signaling pathway in human cervical cancer[J].Cell Death Dis,2018,9(3):276.
[37] Wang T ,Liu J,Xiao XQ.Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3,ERK and AKT signaling pathways[J].Arch Pharm Res,2014,38(2):282-289.
[38] Lei Z,Duan H,Zhao T,et al.PARK2 inhibits osteosarcoma cell growth through the JAK2/STAT3/VEGF signaling pathway[J].Cell Death Dis,2018,9(3):375.
[39] Malla RR,Gopinath S,Gondi CS,et al.Cathepsin B and uPAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma[J].Cancer Gene Ther,2011,18(6):419-434.