Rb94对人肺腺癌裸鼠移植瘤的抑制作用及SphK表达影响的研究*

Inhibitory Action of the Retinoblastoma 94 Gene on Human Lung Adenocarcinoma and Its Effect on the Expression of SphK in a Mouse Xenograft Model

    摘要
  • 摘要: 目的：Rb94基因转染裸鼠肺腺癌皮下移植瘤，观察Rb94基因对人肺腺癌裸鼠移植瘤的抑制作用及对鞘氨醇激酶（sphingosine kinase ，SphK）表达的影响。方法：收集一定数量的对数生长期A549 细胞，生理盐水重悬后每只裸鼠右后肢皮下接种5 × 106/0.1mL A549 细胞悬液，待瘤体生长至一定体积后随机分为4 组，分别为对照组、空载体组、脂质体组、转染Rb94基因组；每组裸鼠瘤体内分别注射PBS 、质粒pIRES 、脂质体（lipofectamine）、质粒pIRES-Rb94，观察肿瘤生长情况并检测抑瘤率；裸鼠脱颈处死后，采用Western bolt法检测瘤组织中Rb94基因表达情况，并采用实时RT-PCR 和免疫荧光法（immunofluorescence，IF ）观察其对裸鼠肺腺癌皮下移植瘤中SphK表达的影响。结果：对照组及转染组裸鼠在接种部位均有移植瘤生成；与空白对照组、脂质体组、空载体组相比，转染Rb94 基因组的裸鼠移植瘤的生长速度明显减慢，肿瘤的体积较小，重量较轻，有显著性差异（P<0.05），而3 个对照组之间，无显著性差异（P>0.05）；空白对照组、空载体组、脂质体组抑瘤率分别为0、11.78% 、7.86% ，而转染Rb94基因组平均抑瘤率高达51.99% ，肿瘤生长明显抑制；SphK1 在mRNA 和蛋白水平表达下调而SphK2 表达上调。结论：Rb94基因成功转染裸鼠肺腺癌皮下移植瘤；Rb94基因抑制裸鼠皮下肿瘤生长；Rb94基因抑制SphK1 的表达，增强SphK2 的表达。

     

    Abstract: Objective: To construct an animal model of pIRES-Rb94transfection of the A549 adenocarcinoma cells to investigate the inhibitory action and effect on the expression of SphK in a mouse xenograft model. Methods:Cell suspen-sions at 5 × 10 6/0.1mL were subcutaneously injected into flanks of every nude mouse. Then the inoculated mice were divid-ed into4 groups as follows: with PBS; with lipofectamine; with pIRES empty vector; and with pIRES-Rb 94. The inhibitory rate was observed and the expression of the Rb 94gene was detected by Western blot. Then its effect on SphK expression was determined by RT-PCR and immunofluorescence. Results: The growth of tumor graft was obviously inhibited in the treatment group. The inhibitory ratio of the transplanted tumor was 51.99% in the pIRES-Rb94group, higher than that in the control group ( 0), lipofectamine group (7.86% ) and pIRES group (11.78% ), with significant differences between pIRES-Rb94group and the other three groups (P<0.01). The expression of SphK 1 was down-regulated and the expression of SphK 2 was up-regulated. Conclusion:Mouse xenograft model of lung adenocarcinoma was successfully constructed us-ing eukaryotic expression plasmid containing pIRES-Rb 94gene. Rb94can effectively inhibit the growth of lung adenocarci -noma and the expression of SphK1 but can enhance SphK2 expression.

     

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