中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
4期
334-339
,共6页
糖尿病/并发症%视网膜%糖尿病视网膜病变%枸杞多糖%血管内皮生长因子
糖尿病/併髮癥%視網膜%糖尿病視網膜病變%枸杞多糖%血管內皮生長因子
당뇨병/병발증%시망막%당뇨병시망막병변%구기다당%혈관내피생장인자
Diabetes mellitus/complication%Retina/diabetic retinopathy%Lycium barbarum polysaccharide%Vascular endothelial growth factor
糖尿病视网膜病变(DR)的主要病理基础是缺氧和炎症引起的新生血管形成,导致血-视网膜屏障(BRB)的破坏,而血管内皮生长因子(VEGF)是主要的血管生成因子.研究证实,枸杞多糖(LBP)具有保护细胞抗氧化损伤的作用,但其在眼科疾病的应用研究较少. 目的 观察LBP对不同时期糖尿病大鼠视网膜的病理改变及VEGF表达的影响. 方法 取SPF级健康雄性SD大鼠117只,以随机数字表法将动物随机分为正常对照组、糖尿病组和LBP组.糖尿病组和LBP组大鼠一次性腹腔内注射55 mg/kg链脲佐菌素(STZ)柠檬酸缓冲液诱导1型糖尿病大鼠模型,正常对照组注射等体积的柠檬酸钠-柠檬酸缓冲液.造模成功后LBP组大鼠每日以250 mg/kg LBP定时定量灌胃,分别于成模后4、10、16周取各组大鼠行伊文思蓝(EB)染色视网膜铺片,动态观察视网膜血管的形态改变;以45 mg/kg EB由大鼠右颈静脉缓慢注入,循环2h后将质量分数1％多聚甲醛由左心室灌注,循环3 min后摘除眼球并分离视网膜,用标准曲线法检测视网膜中EB质量分数.采用免疫组织化学法和实时荧光定量PCR(real-time PCR)法分别检测视网膜中VEGF蛋白及其mRNA的表达. 结果 EB视网膜铺片显示,造模后4、10、16周,LBP组大鼠视网膜血管的走行、管径及渗漏等形态学改变均明显轻于糖尿病组大鼠.造模后4、10、16周,LBP组大鼠视网膜中EB的质量分数分别为(12.17±1.55)、(16.46±1.60)和(19.55± 1.49) mg/g,均明显低于同时期糖尿病组大鼠的(15.76±1.90)、(21.61±2.05)和(26.30±2.28) mg/g,差异均有统计学意义(P＜0.05).免疫组织化学染色结果显示,造模后4、10、16周,大鼠视网膜中VEGF蛋白的表达以视网膜神经节细胞(RGCs)层为主,LBP组大鼠各时间点VEGF蛋白的染色明显弱于糖尿病组.免疫组织化学法检测表明,造模后4、10、16周LBP组大鼠VEGF蛋白在视网膜中的表达量分别为0.234±0.011、0.331±0.023和0.536±0.031,均明显低于同期糖尿病组大鼠的0.281±0.018、0.533±0.055和0.765±0.075,差异均有统计学意义(P＜0.05).Real-time PCR显示,造模后4、10、16周LBP组VEGF mRNA的相对表达量分别为0.157±0.013、0.505±0.114和1.577±0.074,均低于同期糖尿病组的0.235±0.209、1.043±0.084和2.446±0.061,差异均有统计学意义(P＜0.05). 结论 LBP可以减轻糖尿病造成的视网膜血管形态改变,减少血管壁的渗漏,从而保护BRB功能.
糖尿病視網膜病變(DR)的主要病理基礎是缺氧和炎癥引起的新生血管形成,導緻血-視網膜屏障(BRB)的破壞,而血管內皮生長因子(VEGF)是主要的血管生成因子.研究證實,枸杞多糖(LBP)具有保護細胞抗氧化損傷的作用,但其在眼科疾病的應用研究較少. 目的 觀察LBP對不同時期糖尿病大鼠視網膜的病理改變及VEGF錶達的影響. 方法 取SPF級健康雄性SD大鼠117隻,以隨機數字錶法將動物隨機分為正常對照組、糖尿病組和LBP組.糖尿病組和LBP組大鼠一次性腹腔內註射55 mg/kg鏈脲佐菌素(STZ)檸檬痠緩遲液誘導1型糖尿病大鼠模型,正常對照組註射等體積的檸檬痠鈉-檸檬痠緩遲液.造模成功後LBP組大鼠每日以250 mg/kg LBP定時定量灌胃,分彆于成模後4、10、16週取各組大鼠行伊文思藍(EB)染色視網膜鋪片,動態觀察視網膜血管的形態改變;以45 mg/kg EB由大鼠右頸靜脈緩慢註入,循環2h後將質量分數1％多聚甲醛由左心室灌註,循環3 min後摘除眼毬併分離視網膜,用標準麯線法檢測視網膜中EB質量分數.採用免疫組織化學法和實時熒光定量PCR(real-time PCR)法分彆檢測視網膜中VEGF蛋白及其mRNA的錶達. 結果 EB視網膜鋪片顯示,造模後4、10、16週,LBP組大鼠視網膜血管的走行、管徑及滲漏等形態學改變均明顯輕于糖尿病組大鼠.造模後4、10、16週,LBP組大鼠視網膜中EB的質量分數分彆為(12.17±1.55)、(16.46±1.60)和(19.55± 1.49) mg/g,均明顯低于同時期糖尿病組大鼠的(15.76±1.90)、(21.61±2.05)和(26.30±2.28) mg/g,差異均有統計學意義(P＜0.05).免疫組織化學染色結果顯示,造模後4、10、16週,大鼠視網膜中VEGF蛋白的錶達以視網膜神經節細胞(RGCs)層為主,LBP組大鼠各時間點VEGF蛋白的染色明顯弱于糖尿病組.免疫組織化學法檢測錶明,造模後4、10、16週LBP組大鼠VEGF蛋白在視網膜中的錶達量分彆為0.234±0.011、0.331±0.023和0.536±0.031,均明顯低于同期糖尿病組大鼠的0.281±0.018、0.533±0.055和0.765±0.075,差異均有統計學意義(P＜0.05).Real-time PCR顯示,造模後4、10、16週LBP組VEGF mRNA的相對錶達量分彆為0.157±0.013、0.505±0.114和1.577±0.074,均低于同期糖尿病組的0.235±0.209、1.043±0.084和2.446±0.061,差異均有統計學意義(P＜0.05). 結論 LBP可以減輕糖尿病造成的視網膜血管形態改變,減少血管壁的滲漏,從而保護BRB功能.
당뇨병시망막병변(DR)적주요병리기출시결양화염증인기적신생혈관형성,도치혈-시망막병장(BRB)적파배,이혈관내피생장인자(VEGF)시주요적혈관생성인자.연구증실,구기다당(LBP)구유보호세포항양화손상적작용,단기재안과질병적응용연구교소. 목적 관찰LBP대불동시기당뇨병대서시망막적병리개변급VEGF표체적영향. 방법 취SPF급건강웅성SD대서117지,이수궤수자표법장동물수궤분위정상대조조、당뇨병조화LBP조.당뇨병조화LBP조대서일차성복강내주사55 mg/kg련뇨좌균소(STZ)저몽산완충액유도1형당뇨병대서모형,정상대조조주사등체적적저몽산납-저몽산완충액.조모성공후LBP조대서매일이250 mg/kg LBP정시정량관위,분별우성모후4、10、16주취각조대서행이문사람(EB)염색시망막포편,동태관찰시망막혈관적형태개변;이45 mg/kg EB유대서우경정맥완만주입,순배2h후장질량분수1％다취갑철유좌심실관주,순배3 min후적제안구병분리시망막,용표준곡선법검측시망막중EB질량분수.채용면역조직화학법화실시형광정량PCR(real-time PCR)법분별검측시망막중VEGF단백급기mRNA적표체. 결과 EB시망막포편현시,조모후4、10、16주,LBP조대서시망막혈관적주행、관경급삼루등형태학개변균명현경우당뇨병조대서.조모후4、10、16주,LBP조대서시망막중EB적질량분수분별위(12.17±1.55)、(16.46±1.60)화(19.55± 1.49) mg/g,균명현저우동시기당뇨병조대서적(15.76±1.90)、(21.61±2.05)화(26.30±2.28) mg/g,차이균유통계학의의(P＜0.05).면역조직화학염색결과현시,조모후4、10、16주,대서시망막중VEGF단백적표체이시망막신경절세포(RGCs)층위주,LBP조대서각시간점VEGF단백적염색명현약우당뇨병조.면역조직화학법검측표명,조모후4、10、16주LBP조대서VEGF단백재시망막중적표체량분별위0.234±0.011、0.331±0.023화0.536±0.031,균명현저우동기당뇨병조대서적0.281±0.018、0.533±0.055화0.765±0.075,차이균유통계학의의(P＜0.05).Real-time PCR현시,조모후4、10、16주LBP조VEGF mRNA적상대표체량분별위0.157±0.013、0.505±0.114화1.577±0.074,균저우동기당뇨병조적0.235±0.209、1.043±0.084화2.446±0.061,차이균유통계학의의(P＜0.05). 결론 LBP가이감경당뇨병조성적시망막혈관형태개변,감소혈관벽적삼루,종이보호BRB공능.
Background The primary pathological basis of diabetic retinopathy (DR) is new blood formation due to anoxia and inflammation,which results in breakdown of blood-retinal barrier (BRB).Vascular endothelial growth factor (VEGF) is a key factor promoting neovascularization.Researches determined that lycium barbarum polysaccharides (LBP) can protect cells against oxidative damage.However,the study of LBP in ophthalmology is lack.Objective This study was to investigate the effects of LBP on the dynamic pathological change of retinal vessels and expression of VEGF in retina of diabetic rats.Methods One hundred and seventeen SPF male SD rats were randomly divided into the normal control group,the diabetic mellitus (DM) group and the LBP group according to random number table.Type 1 DM models were induced by intraperitoneal injection of streptozotocin (STZ,55 mg/kg) in the rats of the DM group and the LBP group,and then 250 mg/kg LBP was intragastically administered in the rats of the LBP group.The morphological change of retinal vessels was dynamically observed by retinal stretched preparation with Evans blue (EB) in 4,10 and 16 weeks after modeling.E B (45 mg/kg) was slowly injection via jugular vein,and 1％ polyoxymethylene was infused into the left ventricule.The eyeballs were extracted and retina were isolated.EB content in the retinas (mg/g) was calculated using retinal stretched preparation method at the time points mentioned above.Expressions of VEGF protein and mRNA in the retinas were detected by immunohistochemistry and real-time quantitative PCR at various time points,respectively.Results Retinal stretched preparation with EB exhibited that the abnormal degree in the shape,diameter of vessels and leakage of the retinal blood vessels were significantly slighter in the LBP group than those of the DM group in 4,10,16 weeks after modeling.At 4,10,16 weeks,EB content in the retinas was (12.17±1.55),(16.46±1.60) and (19.55±1.49) mg/g,which was significantly lower than (15.76± 1.90),(21.61 ±2.05) and (26.30±2.28) mg/g of the DM group (P＜0.05).Immunochemistry showed that the expression of VEGF protein primarily located at retinal ganglion cells (RGCs) layer.The staining intensity for VEGF protein was weaker in the LBP group than that of the DM group.The expression levels of VEGF protein (A value) in the LBP group were 0.234±0.011,0.331±0.023 and 0.536±0.031at various time points,with significant decline in comparison with 0.281±0.018,0.533±0.055 and 0.765±0.075 of the DM group (all at P＜0.05).Real-time quantitative PCR revealed that the expression levels of VEGF mRNA were 0.157±0.013,0.505 ±0.114 and 1.577±0.074 in the LBP group at various time points,which were significantly lower than 0.235±0.209,1.043±0.084 and 2.446±0.061 of the DM group (all at P＜0.05).Conclusions LBP can alleviate the DM-induced retinal vasculopathy,lessen the leakage of vessels well,and further protect the BRB.
