中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2012年
6期
358-362
,共5页
杨晓光%张文辉%黄晓艳%杨舒广%谢小强%叶贞志%白青青%周晓光
楊曉光%張文輝%黃曉豔%楊舒廣%謝小彊%葉貞誌%白青青%週曉光
양효광%장문휘%황효염%양서엄%사소강%협정지%백청청%주효광
RNA,小分子干扰%缺氧诱导因子1,α亚基%缺氧%视网膜血管%内皮细胞%细胞增殖
RNA,小分子榦擾%缺氧誘導因子1,α亞基%缺氧%視網膜血管%內皮細胞%細胞增殖
RNA,소분자간우%결양유도인자1,α아기%결양%시망막혈관%내피세포%세포증식
RNA,small interfering%Hypoxia-inducible factor 1,alpha subunit%Anoxia%Retinal vessels%Endothelial cells%Cell proliferation
目的 探讨缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF- 1α)在早产儿视网膜病发病中的作用,为其基因治疗寻找新的靶点.方法 将化学合成的小分子干扰RNA( smallinterference RNA,siRNA)用脂质体介导法转染大鼠视网膜血管内皮细胞,转染的细胞在含化学缺氧剂——二氯化钴的培养基中培养.培养8h后,应用荧光定量逆转录-聚合酶链反应和Western印迹技术检测转染后细胞HIF-1α mRNA和蛋白的表达量.培养24 h后应用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法检测细胞的增殖活性.采用两独立样本t检验比较各转染组与阴性对照组之间的差异.结果 成功将siRNA转染至大鼠视网膜血管内皮细胞,荧光定量逆转录-聚合酶链反应技术检测显示,针对靶基因HIF-1α设计的4条平行干扰序列siRNA均能不同程度抑制HIF-1α的转录表达,siRNA1、siRNA2和siRNA4组HIF-1α mRNA的相对表达水平分别为0.1620±0.0147、0.2034±0.0251和0.3049±0.0165,分别降至空白对照组(1.0000±0.0344)的16.20%、20.34%和30.49%,与阴性对照组(0.8334±0.0242)之间差异有统计学意义(t分别为16.786、8.953和4.087,P均<0.05).Western印迹技术显示,siRNA1和siRNA2转染的大鼠视网膜血管内皮细胞HIF-1α蛋白表达水平分别为0.4956±0.0421和0.6544±1.0032,明显低于空白对照组(3.5105±0.4084)和阴性对照组(3.4019±1.0677),差异均有统计学意义(t分别为6.861、2.893、4.567和5.072,P均<0.05).siRNA1和siRNA2组细胞增殖抑制率分别为(49.5±2.9)%和(67.4±1.2)%,明显高于阴性对照组[(15.7±1.5)%],差异有统计学意义(t分别为2.786和6.904,P<0.05).结论 化学合成的HIF-1α siRNA能有效抑制大鼠视网膜血管内皮细胞在缺氧条件下HIF-1α mRNA及蛋白的表达,从而降低细胞增殖活性.
目的 探討缺氧誘導因子-1α(hypoxia-inducible factor-1α,HIF- 1α)在早產兒視網膜病髮病中的作用,為其基因治療尋找新的靶點.方法 將化學閤成的小分子榦擾RNA( smallinterference RNA,siRNA)用脂質體介導法轉染大鼠視網膜血管內皮細胞,轉染的細胞在含化學缺氧劑——二氯化鈷的培養基中培養.培養8h後,應用熒光定量逆轉錄-聚閤酶鏈反應和Western印跡技術檢測轉染後細胞HIF-1α mRNA和蛋白的錶達量.培養24 h後應用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽比色法檢測細胞的增殖活性.採用兩獨立樣本t檢驗比較各轉染組與陰性對照組之間的差異.結果 成功將siRNA轉染至大鼠視網膜血管內皮細胞,熒光定量逆轉錄-聚閤酶鏈反應技術檢測顯示,針對靶基因HIF-1α設計的4條平行榦擾序列siRNA均能不同程度抑製HIF-1α的轉錄錶達,siRNA1、siRNA2和siRNA4組HIF-1α mRNA的相對錶達水平分彆為0.1620±0.0147、0.2034±0.0251和0.3049±0.0165,分彆降至空白對照組(1.0000±0.0344)的16.20%、20.34%和30.49%,與陰性對照組(0.8334±0.0242)之間差異有統計學意義(t分彆為16.786、8.953和4.087,P均<0.05).Western印跡技術顯示,siRNA1和siRNA2轉染的大鼠視網膜血管內皮細胞HIF-1α蛋白錶達水平分彆為0.4956±0.0421和0.6544±1.0032,明顯低于空白對照組(3.5105±0.4084)和陰性對照組(3.4019±1.0677),差異均有統計學意義(t分彆為6.861、2.893、4.567和5.072,P均<0.05).siRNA1和siRNA2組細胞增殖抑製率分彆為(49.5±2.9)%和(67.4±1.2)%,明顯高于陰性對照組[(15.7±1.5)%],差異有統計學意義(t分彆為2.786和6.904,P<0.05).結論 化學閤成的HIF-1α siRNA能有效抑製大鼠視網膜血管內皮細胞在缺氧條件下HIF-1α mRNA及蛋白的錶達,從而降低細胞增殖活性.
목적 탐토결양유도인자-1α(hypoxia-inducible factor-1α,HIF- 1α)재조산인시망막병발병중적작용,위기기인치료심조신적파점.방법 장화학합성적소분자간우RNA( smallinterference RNA,siRNA)용지질체개도법전염대서시망막혈관내피세포,전염적세포재함화학결양제——이록화고적배양기중배양.배양8h후,응용형광정량역전록-취합매련반응화Western인적기술검측전염후세포HIF-1α mRNA화단백적표체량.배양24 h후응용3-(4,5-이갑기새서-2)-2,5-이분기사담서추염비색법검측세포적증식활성.채용량독립양본t검험비교각전염조여음성대조조지간적차이.결과 성공장siRNA전염지대서시망막혈관내피세포,형광정량역전록-취합매련반응기술검측현시,침대파기인HIF-1α설계적4조평행간우서렬siRNA균능불동정도억제HIF-1α적전록표체,siRNA1、siRNA2화siRNA4조HIF-1α mRNA적상대표체수평분별위0.1620±0.0147、0.2034±0.0251화0.3049±0.0165,분별강지공백대조조(1.0000±0.0344)적16.20%、20.34%화30.49%,여음성대조조(0.8334±0.0242)지간차이유통계학의의(t분별위16.786、8.953화4.087,P균<0.05).Western인적기술현시,siRNA1화siRNA2전염적대서시망막혈관내피세포HIF-1α단백표체수평분별위0.4956±0.0421화0.6544±1.0032,명현저우공백대조조(3.5105±0.4084)화음성대조조(3.4019±1.0677),차이균유통계학의의(t분별위6.861、2.893、4.567화5.072,P균<0.05).siRNA1화siRNA2조세포증식억제솔분별위(49.5±2.9)%화(67.4±1.2)%,명현고우음성대조조[(15.7±1.5)%],차이유통계학의의(t분별위2.786화6.904,P<0.05).결론 화학합성적HIF-1α siRNA능유효억제대서시망막혈관내피세포재결양조건하HIF-1α mRNA급단백적표체,종이강저세포증식활성.
Objective To investigate the effects of hypoxia-inducible factor-1α (HIF-1α)expression on pathogenesis of retinopathy of prematurity (ROP) and to find new target for gene therapy.Methods After liposome-mediated small interference RNA (siRNA) transfection into rat retinal endothelial cells,the cells were cultured in medium with CoCl2-induced hypoxic condition.Expression of HIF-1α mRNA was determined by fluorenscence quantitative reverse transcription-polymerase chain reaction(RT-PCR),HIF-1α protein expression was detected by Western Blot after cocultured for 8 hours.Cell proliferation was measured with 3-(4,5)-dimethylthiazol (-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay after cocultured for 24 hours.Difference between groups was compared with independent samples t test.Results Rat retinal vascular endothelial cells were successfully transfected with siRNA.Fluorescence quantitative RT-PCR results showed that at 48 hours of transfection,the expression of HIF-1α mRNA in the interference group of siRNA1,siRNA2 and siRNA4 were 0.1620 ± 0.0147,0.2034 ± 0.0251 and 0.3049 ± 0.0165,which were 16.20%,20.34 % and 30.49% of blank control group (1.0000±0.0344),and were lower than that of negative control group (0.8334±0.0242) (t=16.786,8.953 and 4.087,P<0.05 respectively).Western Blot results showed that HIF-1α protein expression was significantly inhibited by siRNA1(0.4956 ± 0.0421 ) and siRNA2 (0.6544 ± 1.0032) comparing with blank control group (3.5105 ±0.4084) and negative control group (3.4019 ± 1.0677) (t =6.861,2.893,4.567 and 5.072,P<0.05 respectively).As for cellular proliferation activity,(49.5±2.9) % and (67.4±1.2) % of cells growth inhibition were observed after transfection with siRNA1 and siRNA2,which were higher than those of negative control group [(15.7±1.5) % ] (t=2.786 and 6.904,P<0.05).Conclusions The synthetic HIF-1α siRNA could effectively inhibit the expression of HIF-1α gene and reduce cell proliferation in rat retinal endothelial cells under hypoxic condition.RNA interference technology targeting HIF-1α might become a new strategy for gene therapy of ROP.