中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2011年
3期
245-249
,共5页
孟丽珠%陈松%刘艳%王玉川%林锦镛%韩梅
孟麗珠%陳鬆%劉豔%王玉川%林錦鏞%韓梅
맹려주%진송%류염%왕옥천%림금용%한매
糖尿病视网膜病变/病理生理学%视网膜新生血管化%缺氧诱导因子1,α亚基%血管内皮生长因子类%RNA干扰%糖尿病,实验性
糖尿病視網膜病變/病理生理學%視網膜新生血管化%缺氧誘導因子1,α亞基%血管內皮生長因子類%RNA榦擾%糖尿病,實驗性
당뇨병시망막병변/병리생이학%시망막신생혈관화%결양유도인자1,α아기%혈관내피생장인자류%RNA간우%당뇨병,실험성
Diabetic retinopathy/pathophysiology%Retinal neovaseularization%Hypoxia-inducible factor 1,alpha subunit%Vascular endothelial growth factors%RNA Interference%Diabetes mellitus,experimental
目的 观察缺氧诱导因子-1α(HIF-1α)特异性小干扰RNA(siRNA)对糖尿病大鼠视网膜组织中血管内皮生长因子(VEGF)mRNA表达的抑制作用.方法 随机对照研究.以pSilencer 2.1-U6neo为质粒载体,构建HIF-1α siRNA重组质粒.雄性Sprague-Dawley大鼠54只,随机分为正常对照组和实验组,分别为15、39只大鼠.实验组大鼠尾静脉注射链尿佐菌素(STZ)诱导建立糖尿病动物模型,再分为糖尿病模型组、空载体组和基因治疗组,分别为15、12、12只大鼠.脂质体Lipofectamine TM 2000介导,空载体组和基因治疗组分别转染pSilencer空载体质粒和HIF-1αsiRNA重组质粒,糖尿病模型组和正常组对照组不做转染.采用实时逆转录(RT)-聚合酶链式反应(PCR)检测各组大鼠VEGF mRNA的表达.分别于干扰后24、48、72 h,1周时计算VEGF mRNA的抑制效率.采用单因素方差分析和LSD-t检验进行统计学分析.结果 HIF-1α siRNA重组质粒经酶切、测序鉴定,确定为目的 序列.实时RT-PCR检测结果显示,正常对照组仅见弱的VEGF mRNA表达,糖尿病模型组及空载体组表达明显上调,基因治疗组表达下调;各时间点空载体组与糖尿病模型组比较,差异无统计学意义(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980),基因治疗组较糖尿病模型组及空载体组表达下调,差异有统计学意义(t=8.768、13.695、11.285、8.253,t=9.437、13.554、11.436、8.228;P值均=0.000);VEGF mRNA抑制率24、48、72 h和1周时分别为32.76%、43.60%、47.70%、50.86%.结论 HIF-1α siRNA重组质粒能有效抑制糖尿病大鼠视网膜中VEGFmRNA的表达.
目的 觀察缺氧誘導因子-1α(HIF-1α)特異性小榦擾RNA(siRNA)對糖尿病大鼠視網膜組織中血管內皮生長因子(VEGF)mRNA錶達的抑製作用.方法 隨機對照研究.以pSilencer 2.1-U6neo為質粒載體,構建HIF-1α siRNA重組質粒.雄性Sprague-Dawley大鼠54隻,隨機分為正常對照組和實驗組,分彆為15、39隻大鼠.實驗組大鼠尾靜脈註射鏈尿佐菌素(STZ)誘導建立糖尿病動物模型,再分為糖尿病模型組、空載體組和基因治療組,分彆為15、12、12隻大鼠.脂質體Lipofectamine TM 2000介導,空載體組和基因治療組分彆轉染pSilencer空載體質粒和HIF-1αsiRNA重組質粒,糖尿病模型組和正常組對照組不做轉染.採用實時逆轉錄(RT)-聚閤酶鏈式反應(PCR)檢測各組大鼠VEGF mRNA的錶達.分彆于榦擾後24、48、72 h,1週時計算VEGF mRNA的抑製效率.採用單因素方差分析和LSD-t檢驗進行統計學分析.結果 HIF-1α siRNA重組質粒經酶切、測序鑒定,確定為目的 序列.實時RT-PCR檢測結果顯示,正常對照組僅見弱的VEGF mRNA錶達,糖尿病模型組及空載體組錶達明顯上調,基因治療組錶達下調;各時間點空載體組與糖尿病模型組比較,差異無統計學意義(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980),基因治療組較糖尿病模型組及空載體組錶達下調,差異有統計學意義(t=8.768、13.695、11.285、8.253,t=9.437、13.554、11.436、8.228;P值均=0.000);VEGF mRNA抑製率24、48、72 h和1週時分彆為32.76%、43.60%、47.70%、50.86%.結論 HIF-1α siRNA重組質粒能有效抑製糖尿病大鼠視網膜中VEGFmRNA的錶達.
목적 관찰결양유도인자-1α(HIF-1α)특이성소간우RNA(siRNA)대당뇨병대서시망막조직중혈관내피생장인자(VEGF)mRNA표체적억제작용.방법 수궤대조연구.이pSilencer 2.1-U6neo위질립재체,구건HIF-1α siRNA중조질립.웅성Sprague-Dawley대서54지,수궤분위정상대조조화실험조,분별위15、39지대서.실험조대서미정맥주사련뇨좌균소(STZ)유도건립당뇨병동물모형,재분위당뇨병모형조、공재체조화기인치료조,분별위15、12、12지대서.지질체Lipofectamine TM 2000개도,공재체조화기인치료조분별전염pSilencer공재체질립화HIF-1αsiRNA중조질립,당뇨병모형조화정상조대조조불주전염.채용실시역전록(RT)-취합매련식반응(PCR)검측각조대서VEGF mRNA적표체.분별우간우후24、48、72 h,1주시계산VEGF mRNA적억제효솔.채용단인소방차분석화LSD-t검험진행통계학분석.결과 HIF-1α siRNA중조질립경매절、측서감정,학정위목적 서렬.실시RT-PCR검측결과현시,정상대조조부견약적VEGF mRNA표체,당뇨병모형조급공재체조표체명현상조,기인치료조표체하조;각시간점공재체조여당뇨병모형조비교,차이무통계학의의(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980),기인치료조교당뇨병모형조급공재체조표체하조,차이유통계학의의(t=8.768、13.695、11.285、8.253,t=9.437、13.554、11.436、8.228;P치균=0.000);VEGF mRNA억제솔24、48、72 h화1주시분별위32.76%、43.60%、47.70%、50.86%.결론 HIF-1α siRNA중조질립능유효억제당뇨병대서시망막중VEGFmRNA적표체.
Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.
