中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2012年
6期
513-518
,共6页
血管内皮生长因子A%血管内皮生长因子受体1%血管内皮生长因子受体2%结膜成纤维细胞%细胞,培养的%荧光抗体技术,间接
血管內皮生長因子A%血管內皮生長因子受體1%血管內皮生長因子受體2%結膜成纖維細胞%細胞,培養的%熒光抗體技術,間接
혈관내피생장인자A%혈관내피생장인자수체1%혈관내피생장인자수체2%결막성섬유세포%세포,배양적%형광항체기술,간접
Vascular endothelial growth factor A%Vascular endothelial growth factor receptor-1%Vascular endothelial growth factor receptor-2%Conjunctival%Fibroblasts%Cells, cultured%Fluorescent antibody technique,indirect
目的 观察培养的大鼠结膜成纤维细胞中是否表达血管内皮生长因子(VEGF)及其受体(VEGFR).方法 实验研究.健康Sprague Dawley大鼠5只,分别施行麻醉处死后,剪取球结膜组织,以组织块法接种并培养成纤维细胞.通过荧光倒置显微镜观察细胞培养情况,以间接免疫荧光法检测波形蛋白、角蛋白表达水平,对所培养的成纤维细胞进行鉴定;设计VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)3种基因的RNA引物序列,以Trizol法提取成纤维细胞中mRNA,对目的片断进行逆转录PCR扩增及凝胶电泳.扩增产物进行回收及测序,同时采用间接免疫荧光法检测VEGFR-1的表达情况.利用荧光定量PCR法观察培养的成纤维细胞中3种基因的熔解曲线和扩增曲线,同时采用间接免疫荧光法检测VEGFR-1的表达情况.结果 大鼠结膜成纤维细胞中波形蛋白表达阳性,角蛋白表达阴性.逆转录PCR扩增后分别显示VEGF164、Flt-1、Flk-1条带,测序结果符合VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)基因序列.荧光定量PCR法检测结果显示上述基因均有陡直的扩增曲线,未见杂峰,指数扩增期均在30个循环内.免疫荧光法检测VECFR-1蛋白表达阳性.结论 大鼠结膜成纤维细胞存在VEGF及其受体的基因表达,有VEGFR-1的蛋白表达,抗VEGF类药物可能对大鼠的结膜成纤维细胞产生影响.
目的 觀察培養的大鼠結膜成纖維細胞中是否錶達血管內皮生長因子(VEGF)及其受體(VEGFR).方法 實驗研究.健康Sprague Dawley大鼠5隻,分彆施行痳醉處死後,剪取毬結膜組織,以組織塊法接種併培養成纖維細胞.通過熒光倒置顯微鏡觀察細胞培養情況,以間接免疫熒光法檢測波形蛋白、角蛋白錶達水平,對所培養的成纖維細胞進行鑒定;設計VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)3種基因的RNA引物序列,以Trizol法提取成纖維細胞中mRNA,對目的片斷進行逆轉錄PCR擴增及凝膠電泳.擴增產物進行迴收及測序,同時採用間接免疫熒光法檢測VEGFR-1的錶達情況.利用熒光定量PCR法觀察培養的成纖維細胞中3種基因的鎔解麯線和擴增麯線,同時採用間接免疫熒光法檢測VEGFR-1的錶達情況.結果 大鼠結膜成纖維細胞中波形蛋白錶達暘性,角蛋白錶達陰性.逆轉錄PCR擴增後分彆顯示VEGF164、Flt-1、Flk-1條帶,測序結果符閤VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)基因序列.熒光定量PCR法檢測結果顯示上述基因均有陡直的擴增麯線,未見雜峰,指數擴增期均在30箇循環內.免疫熒光法檢測VECFR-1蛋白錶達暘性.結論 大鼠結膜成纖維細胞存在VEGF及其受體的基因錶達,有VEGFR-1的蛋白錶達,抗VEGF類藥物可能對大鼠的結膜成纖維細胞產生影響.
목적 관찰배양적대서결막성섬유세포중시부표체혈관내피생장인자(VEGF)급기수체(VEGFR).방법 실험연구.건강Sprague Dawley대서5지,분별시행마취처사후,전취구결막조직,이조직괴법접충병배양성섬유세포.통과형광도치현미경관찰세포배양정황,이간접면역형광법검측파형단백、각단백표체수평,대소배양적성섬유세포진행감정;설계VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)3충기인적RNA인물서렬,이Trizol법제취성섬유세포중mRNA,대목적편단진행역전록PCR확증급응효전영.확증산물진행회수급측서,동시채용간접면역형광법검측VEGFR-1적표체정황.이용형광정량PCR법관찰배양적성섬유세포중3충기인적용해곡선화확증곡선,동시채용간접면역형광법검측VEGFR-1적표체정황.결과 대서결막성섬유세포중파형단백표체양성,각단백표체음성.역전록PCR확증후분별현시VEGF164、Flt-1、Flk-1조대,측서결과부합VEGF164、VEGFR-1(Flt-1)、VEGFR-2( Flk-1)기인서렬.형광정량PCR법검측결과현시상술기인균유두직적확증곡선,미견잡봉,지수확증기균재30개순배내.면역형광법검측VECFR-1단백표체양성.결론 대서결막성섬유세포존재VEGF급기수체적기인표체,유VEGFR-1적단백표체,항VEGF류약물가능대대서적결막성섬유세포산생영향.
Objective To detect the expressions of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFR) in cultured rat conjunctival fibroblasts. Methods Experimental study.Gonjunctiva was obtained from each eye of five Sprague Dawley(SD) rats under local anesthesia.Tissues were minced and grown in culture flask containing Dulbecco's Modified Eagle's medium (DMEM).The fibroblasts were identified by observing cell morphology,and the expression of staining of cytokeratin and vimentin by inverted microscopy and indirect immunofluorescence technique.mRNA primers were designed for VEGF164,VEGFR-1 ( Flt-1 ),and VEGFR-2 ( Flk-1 ).mRNA was extracted by Trizol method.cDNA was synthesized under the action of RNA reverse transcriptase.Ladder-like pattern of DNA fragmentation appeared upon 2％ agarose gel electrophoresis. The amplification curves and dissociation curves of VEGF164,VEGFR-1 (Flt-1),and VEGFR-2 (Flk-1) were detected by realtime polymerase chain reaction (PCR).The expression of VEGFR-1 on fibroblast was observed by immunofluorescence technique.Results The fluorescent staining was negative in cytokeratin of fibroblasts,but was positive in vimentin of fibroblasts.The mRNA expressions of VEGF164,VEGFR-1 ( Flt-1 ) and VEGFR-2( Flk-1 ) were detected in fibroblast and verified by gene sequencing.The peaks of the dissociation curves were all steep and specific.The fluorescent staining of VEGFR-1 was positive. Conclusions Our results demonstrate that the expression of VEGF,VEGF receptors,and VEGFR-1 cytokine in rat conjunctival fibroblasts suggests that anti-VEGF compounds may exert a direct influence to the growth of rat conjunctival fibroblast.
