CAJ | 학술논문

目的观察探讨糖尿病(DM)大鼠视网膜组织中脯氨酰羟化酶(PHD-2)的表达及意义.方法雄性Wistar大鼠108只,随机分为DM模型组和正常对照组,分别为60、48只.对DM模型组大鼠进行一次性腹腔注射1％链脲霉素枸橼酸(STZ)溶液建模,正常对照组腹腔注射等量的构橼酸缓冲液.造模后1、3、6个月,荧光显微镜下观察大鼠视网膜血管分布形态.造模后1～6个月,采用伊凡思蓝(EB)灌注视网膜铺片检测视网膜组织内EB浓度；免疫组织化学染色法观察大鼠视网膜PHD-2阳性染色的分布特点；蛋白质免疫印迹法(Western blot)检测大鼠视网膜中PHD-2、缺氧诱导因子-1α(HIF-1α)及血管内皮生长因子(VEGF)的表达.结果荧光显微镜观察结果显示,正常对照组大鼠视网膜血管走行规则,浅深层血管形态清晰；DM模型组大鼠视网膜血管渗漏持续存在.造模后1～6个月,DM模型组大鼠视网膜血管外组织内EB含量较正常对照组明显增加,差异有统计学意义(t=3.810,2.722,2.845,2.342,2.456,3.823；P＜0.05).免疫组织化学染色结果显示,DM模型组及正常对照组大鼠视网膜中均可见PHD-2阳性染色,主要分布于视网膜内核层、神经节细胞层及血管壁.Western blot检测结果显示,DM模型组大鼠视网膜中PHD-2表达在造模后1、2个月时较正常对照组下降(t=16.230,16.390;P＜0.05)；HIF-1a表达在造模后1、2、3个月时较正常对照组明显升高(t=27.073,36.709,10.176; P＜0.05)；VEGF表达在造模后1～6个月均较正常对照组升高(t=13.547,31.984,21.897,8.912,9.019,14.046；P＜0.05).结论 PHD-2在DM大鼠视网膜组织中有丰富表达.PHD-2可能在糖尿病视网膜病变发生发展中有一定的调控作用,其作用途径及机制与VEGF作用通路有关.
목적 관찰탐토당뇨병(DM)대서시망막조직중포안선간화매(PHD-2)적표체급의의.방법 웅성Wistar대서108지,수궤분위DM모형조화정상대조조,분별위60、48지.대DM모형조대서진행일차성복강주사1％련뇨매소구연산(STZ)용액건모,정상대조조복강주사등량적구연산완충액.조모후1、3、6개월,형광현미경하관찰대서시망막혈관분포형태.조모후1～6개월,채용이범사람(EB)관주시망막포편검측시망막조직내EB농도；면역조직화학염색법관찰대서시망막PHD-2양성염색적분포특점；단백질면역인적법(Western blot)검측대서시망막중PHD-2、결양유도인자-1α(HIF-1α)급혈관내피생장인자(VEGF)적표체.결과 형광현미경관찰결과현시,정상대조조대서시망막혈관주행규칙,천심층혈관형태청석；DM모형조대서시망막혈관삼루지속존재.조모후1～6개월,DM모형조대서시망막혈관외조직내EB함량교정상대조조명현증가,차이유통계학의의(t=3.810,2.722,2.845,2.342,2.456,3.823；P＜0.05).면역조직화학염색결과현시,DM모형조급정상대조조대서시망막중균가견PHD-2양성염색,주요분포우시망막내핵층、신경절세포층급혈관벽.Western blot검측결과현시,DM모형조대서시망막중PHD-2표체재조모후1、2개월시교정상대조조하강(t=16.230,16.390;P＜0.05)；HIF-1a표체재조모후1、2、3개월시교정상대조조명현승고(t=27.073,36.709,10.176; P＜0.05)；VEGF표체재조모후1～6개월균교정상대조조승고(t=13.547,31.984,21.897,8.912,9.019,14.046；P＜0.05).결론 PHD-2재DM대서시망막조직중유봉부표체.PHD-2가능재당뇨병시망막병변발생발전중유일정적조공작용,기작용도경급궤제여VEGF작용통로유관.
Objective To investigate the expression and prolyl hydroxylase (PHD)-2 in retina of diabetic rats.Methods Wistar rats were randomly divided into the control group (n=48) and the diabetes group (n=60).The rats in diabetes group were induced with streptozotocin (STZ) injection creating a diabetic retinopathy model.The same volume of citric acid buffer was injected into the rats in the control group.Fluorescence microscope was used to observe the retinal vaseulature at one,three and six months after injection.Evans blue perfusion was used to detect the blood-retinal barrier (BRB) permeability.Immunohistochemical staining was used to observe the distribution of PHD-2 positive staining.Western blot was used to measure the protein expression of PHD-2,hypoxia inducible factor(HIF)-1α and vascular endothelial growth factor (VEGF) every month from one to six months after injection.Results The vascularization was normal and form was clear in retina of rats in control group.The retinal blood vessels of rats in diabetes group showed significantly increased fluorescence.Compared with the control group,the BRB permeability was significantly increased in diabetes group (P＜0.05).Abundant expression of PHD-2protein was detected in the inner layers of retina in control and diabetes group.Compared with the control group,the PHD-2 expression was decreased in diabetes group at one and two months after iujection (t=16.230,16.390;P＜0.05).The HIF-1α expression was significantly increased in diabetes group at one,two and three months after injection (t=27.073,36.709,10.176; P＜0.05).The VEGF expression was significantly increased in diabetes group every month from one to six months after injection (t =13.547,31.984,21.897,8.912,9.019,14.046; P＜0.05).Conclusions There is abundant expression of PHD-2 in the inner layer of retina in diabetic rats.PHD-2 may play an important role in diabetic retinopathy,which is correlated with VEGF.