中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2015年
2期
157-161
,共5页
糖尿病视网膜病变/病理生理学%RNA,小分子干扰%受体,G-蛋白偶联%血管内皮生长因子类%p38丝裂原活化蛋白激酶类%动物实验
糖尿病視網膜病變/病理生理學%RNA,小分子榦擾%受體,G-蛋白偶聯%血管內皮生長因子類%p38絲裂原活化蛋白激酶類%動物實驗
당뇨병시망막병변/병리생이학%RNA,소분자간우%수체,G-단백우련%혈관내피생장인자류%p38사렬원활화단백격매류%동물실험
Diabetic retinopathy/physiopathology%RNA,small interfering%Receptors,G-protein-coupled%Vascular endothelial growth factors%p38 Mitogen-activated protein kinases%Animal experimentation
目的 观察G蛋白偶联受体91(GPR91)对早期糖尿病大鼠血视网膜屏障(BRB)的影响及可能的作用机制.方法 构建GPR91小发夹RNA(shRNA)慢病毒载体pGCSIL-GFP-shGPR91及相应的慢病毒空载体pGCSIL-GFP-shScrambled.健康雄性Sprague-Dawley大鼠60只,随机分为对照组(A组)、糖尿病链脲佐菌素(STZ)组(B组)、空病毒LV.shScrambled组(C组)、病毒LV.shGPR91组(D组),每组均为15只大鼠.大鼠STZ腹腔注射2周后,C组大鼠玻璃体腔注射浓度为1×108 TU/ml的空载体病毒1μl;D组大鼠玻璃体腔注射浓度为1×108 TU/ml的pGCSIL-GFP-shGPR91慢病毒1 μl.注射后12周,免疫组织化学染色和蛋白免疫印迹法(Western blot)检测各组大鼠视网膜中GPR91及丝裂原活化蛋白激酶(MAPK)的表达和激活情况.苏木精-伊红染色和伊凡思蓝(EB)渗漏试验观察大鼠视网膜微血管结构改变和渗漏情况.酶联免疫吸附试验(ELISA)检测视网膜中血管内皮生长因子(VEGF)表达情况.结果 免疫组织化学染色可见GPR91主要表达于大鼠视网膜神经节细胞层;Western blot检测结果显示,D组GPR91表达较C组显著降低,差异有统计学意义(F=39.31,P＜0.01).光学显微镜观察发现,B、C组大鼠视网膜内层毛细血管较A组大鼠视网膜内层毛细血管明显扩张,D组大鼠视网膜内层扩张的毛细血管显著改善.EB渗漏试验结果显示,D组大鼠视网膜EB渗漏量较C组下降(33.8±4.11)％,差异有统计学意义(F=30.35,P＜0.05).ELISA检测结果显示,B、C组大鼠视网膜VEGF蛋白表达较A组明显增加;D组大鼠视网膜VEGF蛋白表达较C组显著下降,差异有统计学意义(F=253.15,P＜0.05).Westernblot检测结果显示,B组大鼠视网膜细胞外信号调节激酶(ERK) 1/2、c-jun氨基末端激酶、p38 MAPK通路均激活,B组各比值与A组比较,差异有统计学意义(q=6.38、2.94、3.45,P＜0.05).D组中p-ERK1/2与t-ERK1/2的比值较C组显著降低,差异有统计学意义(F=22.50,P＜0.05).结论 GPR91受体shRNA玻璃体腔注射可改善早期糖尿病大鼠视网膜BRB损伤;GPRg1受体可能通过激活ERK1/2信号通路参与调控早期糖尿病视网膜中VEGF的表达及BRB的损伤.
目的 觀察G蛋白偶聯受體91(GPR91)對早期糖尿病大鼠血視網膜屏障(BRB)的影響及可能的作用機製.方法 構建GPR91小髮夾RNA(shRNA)慢病毒載體pGCSIL-GFP-shGPR91及相應的慢病毒空載體pGCSIL-GFP-shScrambled.健康雄性Sprague-Dawley大鼠60隻,隨機分為對照組(A組)、糖尿病鏈脲佐菌素(STZ)組(B組)、空病毒LV.shScrambled組(C組)、病毒LV.shGPR91組(D組),每組均為15隻大鼠.大鼠STZ腹腔註射2週後,C組大鼠玻璃體腔註射濃度為1×108 TU/ml的空載體病毒1μl;D組大鼠玻璃體腔註射濃度為1×108 TU/ml的pGCSIL-GFP-shGPR91慢病毒1 μl.註射後12週,免疫組織化學染色和蛋白免疫印跡法(Western blot)檢測各組大鼠視網膜中GPR91及絲裂原活化蛋白激酶(MAPK)的錶達和激活情況.囌木精-伊紅染色和伊凡思藍(EB)滲漏試驗觀察大鼠視網膜微血管結構改變和滲漏情況.酶聯免疫吸附試驗(ELISA)檢測視網膜中血管內皮生長因子(VEGF)錶達情況.結果 免疫組織化學染色可見GPR91主要錶達于大鼠視網膜神經節細胞層;Western blot檢測結果顯示,D組GPR91錶達較C組顯著降低,差異有統計學意義(F=39.31,P＜0.01).光學顯微鏡觀察髮現,B、C組大鼠視網膜內層毛細血管較A組大鼠視網膜內層毛細血管明顯擴張,D組大鼠視網膜內層擴張的毛細血管顯著改善.EB滲漏試驗結果顯示,D組大鼠視網膜EB滲漏量較C組下降(33.8±4.11)％,差異有統計學意義(F=30.35,P＜0.05).ELISA檢測結果顯示,B、C組大鼠視網膜VEGF蛋白錶達較A組明顯增加;D組大鼠視網膜VEGF蛋白錶達較C組顯著下降,差異有統計學意義(F=253.15,P＜0.05).Westernblot檢測結果顯示,B組大鼠視網膜細胞外信號調節激酶(ERK) 1/2、c-jun氨基末耑激酶、p38 MAPK通路均激活,B組各比值與A組比較,差異有統計學意義(q=6.38、2.94、3.45,P＜0.05).D組中p-ERK1/2與t-ERK1/2的比值較C組顯著降低,差異有統計學意義(F=22.50,P＜0.05).結論 GPR91受體shRNA玻璃體腔註射可改善早期糖尿病大鼠視網膜BRB損傷;GPRg1受體可能通過激活ERK1/2信號通路參與調控早期糖尿病視網膜中VEGF的錶達及BRB的損傷.
목적 관찰G단백우련수체91(GPR91)대조기당뇨병대서혈시망막병장(BRB)적영향급가능적작용궤제.방법 구건GPR91소발협RNA(shRNA)만병독재체pGCSIL-GFP-shGPR91급상응적만병독공재체pGCSIL-GFP-shScrambled.건강웅성Sprague-Dawley대서60지,수궤분위대조조(A조)、당뇨병련뇨좌균소(STZ)조(B조)、공병독LV.shScrambled조(C조)、병독LV.shGPR91조(D조),매조균위15지대서.대서STZ복강주사2주후,C조대서파리체강주사농도위1×108 TU/ml적공재체병독1μl;D조대서파리체강주사농도위1×108 TU/ml적pGCSIL-GFP-shGPR91만병독1 μl.주사후12주,면역조직화학염색화단백면역인적법(Western blot)검측각조대서시망막중GPR91급사렬원활화단백격매(MAPK)적표체화격활정황.소목정-이홍염색화이범사람(EB)삼루시험관찰대서시망막미혈관결구개변화삼루정황.매련면역흡부시험(ELISA)검측시망막중혈관내피생장인자(VEGF)표체정황.결과 면역조직화학염색가견GPR91주요표체우대서시망막신경절세포층;Western blot검측결과현시,D조GPR91표체교C조현저강저,차이유통계학의의(F=39.31,P＜0.01).광학현미경관찰발현,B、C조대서시망막내층모세혈관교A조대서시망막내층모세혈관명현확장,D조대서시망막내층확장적모세혈관현저개선.EB삼루시험결과현시,D조대서시망막EB삼루량교C조하강(33.8±4.11)％,차이유통계학의의(F=30.35,P＜0.05).ELISA검측결과현시,B、C조대서시망막VEGF단백표체교A조명현증가;D조대서시망막VEGF단백표체교C조현저하강,차이유통계학의의(F=253.15,P＜0.05).Westernblot검측결과현시,B조대서시망막세포외신호조절격매(ERK) 1/2、c-jun안기말단격매、p38 MAPK통로균격활,B조각비치여A조비교,차이유통계학의의(q=6.38、2.94、3.45,P＜0.05).D조중p-ERK1/2여t-ERK1/2적비치교C조현저강저,차이유통계학의의(F=22.50,P＜0.05).결론 GPR91수체shRNA파리체강주사가개선조기당뇨병대서시망막BRB손상;GPRg1수체가능통과격활ERK1/2신호통로삼여조공조기당뇨병시망막중VEGF적표체급BRB적손상.
Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats.Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed.Healthy male Sprague-Dawley (SD) rats were selected in this study.The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A,n=15),the rats received injections of an equal volume of 0.1％ citrate buffer;(2) streptozocin (STZ) group (Group B,n=15),the rats received injections of STZ;(3) LV.shScrambled group (Group C,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 10s TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes;(4) LV.shGPRg1 group (Group D,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles.At 12 weeks after intravitreal injection,immunohistochemistry and Western blot were used to assess the expression of GPRg1,p-extracellular signal-regulated kinase(ERK)1/2,t-ERK1/2,p-Jun N-terminal kinase (JNK),t-JNK,p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK.Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel.Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF.Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer.Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31,P＜0.01).HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina,while the telangiectatic vessels attenuated in Group D.It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)％ compared with Group C and there was significant difference (F =30.35,P＜0.05).The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15,P＜0.05).The results of Western blot indicated that compared with Group A,the expressions of p-ERK1/2,p-JNK and p-p38 MAPK were significantly upregulated (q=6.38,2.94,3.45;P＜0.05).Meanwhile,the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50,P＜0.05).Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats.GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
