[Zhonghua yan ke za zhi] Chinese journal of ophthalmology · 2012
[Expressions of vascular endothelial growth factor and its receptors in rat conjunctival fibroblasts].
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Cheng Gang-wei, Zhao Jia-liang, Ma Jian-min
Аннотация
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. Conjunctiva 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 VEGF(164), 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 VEGF(164), 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 VEGF(164), 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.
METHODS: Experimental study. Conjunctiva 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 VEGF(164), 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 VEGF(164), 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 VEGF(164), 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.