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RNAi体外沉默淋巴管内皮祖细胞VEGFR—3基因表达(2)

时间:2014-10-22 17:07 点击:
VEGFR-3(261bp) A B A:VEGFR-3siRNA/PEI转染LEPCs48h各组VEGFR-3mRNA的表达。B:统计学结果siRNA a组、siRNA b组、siRNA c组与空白转染组和阴性转染组比较有统计学意义,#P0.05,*P0.01,siRNAa组与siRNAb组、si

  VEGFR-3(261bp)

  A

  B

  A:VEGFR-3siRNA/PEI转染LEPCs48h各组VEGFR-3mRNA的表达。B:统计学结果siRNA a组、siRNA b组、siRNA c组与空白转染组和阴性转染组比较有统计学意义,#P<0.05,*P<0.01,siRNAa组与siRNAb组、siRNAc组比有统计学意义P<0.05

  图1 半定量RTPCR结果分析

  2.2.2 Western blot检测结果分析 转染各组细胞VEGFR-3蛋白表达结果显示,空白与阴性转染组之间无差异siRNA a组、siRNA b组、siRNA c组与空白转染组和阴性转染组比较均有显著差异,其中siRNA a组抑制效率最高(图2)。此结果在蛋白水平上符合半定量RT-PCR的实验结果。

  空白 阴性 a b c

  VEGFR-3165KD

  β-actin 43KD

  C

  D

  C:VEGFR-3siRNA/PEI转染LEPCs72h各组VEGFR-3蛋白的表达。D:统计学结果siRNA a组、siRNA b组、siRNA c组与空白转染组和阴性转染组比较有统计学意义,#P<0.05,*P<0.01,siRNAa组与siRNAb组、siRNAc组比有统计学意义,P<0.05

  图2 Westem blot 检测结果

  3 讨论

  在肿瘤转移过程中,肿瘤细胞可经新生的淋巴管转移[11],抑制肿瘤淋巴管新生对肿瘤治疗至关重要。LEPCs参与肿瘤淋巴管新生已成为研究热点[12]。LEPCs可迁移和定居到肿瘤,参与肿瘤淋巴管新生[3,13-14]。关于LEPCs参与淋巴管新生的机制尚未完全明确。本实验在体外将VEGFR-3siRNA转染入LEPCs。裸siRNA分子易降解且靶向性差,目前多采用借助载体给药,阳离子聚合物具有较低的免疫原性和细胞毒性多被应用于研究[15]。PEI是一种被广泛用于DNA和siRNA等的阳离子聚合物载体,通过形成纳米粒包裹siRNA后给药[16]。

  本研究采用分子量为25KDa的PEI为载体介导VEGFR-3siRNA的转染,成功转染VEGFR-3siRNA进入LEPCs内,用RT-PCR和Western blot检测mRNA和蛋白质表达,证实了VEGFR-3siRNA对靶基因VEGFR-3的沉默效果。说明通过利用RNAi技术能够在体外靶向抑制LEPCs VEGFR-3的表达,进而干扰VEGF-C/VEGFR-3信号途径,抑制其分化和淋巴管新生。如果以体外实验结果作为基础,将VEGFR-3siRNA导入LEPCs并靶向于肿瘤,可能会抑制淋巴管新生引起的肿瘤转移。

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