Ad-IL-24-OSM双基因腺病毒载体的构建及其对人黑色素瘤细胞生长抑制作用的体内外实验研究
发布时间：2018-10-12 20:59
【摘要】：目的： 构建携带白细胞介素24（Interleukin-24,IL-24）基因和抑瘤素M（OSM）基因的双基因共表达腺病毒载体（Ad-IL-24-OSM）并开展对A375人黑色素瘤细胞体内外抗肿瘤效应的实验研究。 方法： 在本科室已成功构建pTrack-CMV-poly-A-promoter转移质粒的基础上，在多克隆酶切位点的Bgl II、Sal I之间和Not I、Xho I之间分别插入IL-24和OSM基因片段，构建pAdTrack-CMV-IL-24-polyA-promoter-OSM重组转移载体，经同源重组、包装和扩增获得Ad-IL-24-OSM双基因共表达重组腺病毒子，体外检测Ad-IL-24-OSM重组腺病毒子的效价，感染A375黑色素瘤细胞后，RT-PCR、westernblot法检测IL-24、OSM在A375人黑色素瘤细胞中的转录和表达；荧光显微镜观察感染后的A375细胞的形态学改变； MTT法检测Ad-IL-24-OSM对A375黑色素瘤细胞的生长抑制作用；流式细胞术(FCM)检测重组病毒子对A375黑色素瘤细胞的周期生长抑制和促凋亡效应；半定量RT-PCR法检测A375黑色素瘤细胞中的p21、p53、Bax、Bcl-2mRNA的表达水平。western blot检测Caspase-3凋亡相关蛋白的表达；通过建立A375人黑色素瘤细胞移植瘤瘤动物模型,用Ad-IL-24-OSM重组病毒子进行实验干预，检测瘤体重量，HE染色法检测瘤块组织切片中的细胞核形态的变化；免疫组化检测移植瘤块切片中P21、P53、bax、bcl-2、CD34、COX2、Caspase-3的表达情况。 结果： 1. PCR和双酶切分析结果显示成功构建了Ad-IL-24-OSM双基因共表达腺病毒载体。 2. RT-PCR和Western blot检测证实腺病毒介导的IL-24和OSM双基因均可在A375人黑色素瘤细胞中稳定转录及表达。 3. Ad-IL-24-OSM双基因共表达腺病毒载体能显著抑制A375细胞的生长和诱导细胞凋亡，并显著优于Ad-IL-24、Ad-OSM单基因组（P0.05）。 4.体内实验表明Ad-IL-24-OSM腺病毒载体可以显著抑制裸鼠人黑色素瘤移植瘤的生长，且均具有抑瘤增效的相加作用（Q=1.02）。 5.细胞凋亡分子机制检测发现：Ad-IL-24-OSM能明显上调A375人黑色素瘤细胞P21、P53、Bax、Caspase-3和下调Cox-2、Bcl-2、CD34等细胞周期和凋亡相关蛋白的表达;且其效应均较Ad-IL-24、Ad-OSM更为显著。 结论： Ad-IL-24-OSM双基因腺病毒共表达载体构建成功，在体内外均可明显抑制A375细胞的生长，诱导其凋亡，其机制可能是与上调p21、p53、bax和下调Cox-2、Bcl-2、CD34的基因表达水平，，并通过激活Caspase-3凋亡途径诱导A375细胞凋亡和抑制肿瘤血管形成有关。
[Abstract]:Aim: to construct an adenovirus vector (Ad-IL-24-OSM) carrying interleukin-24 (Interleukin-24,IL-24) gene and tumor suppressor M (OSM) gene and to investigate the anti-tumor effect of A375 human melanoma cells in vitro and in vivo. Methods: on the basis of the successful construction of pTrack-CMV-poly-A-promoter transfer plasmid in our department, the IL-24 and OSM gene fragments were inserted between Bgl II,Sal I and Not Iho I, respectively, and the pAdTrack-CMV-IL-24-polyA-promoter-OSM recombinant transfer vector was constructed by homologous recombination. The recombinant adenovirons of Ad-IL-24-OSM were obtained by packaging and amplification. The titers of Ad-IL-24-OSM recombinant adenovirus were detected in vitro. After A375 melanoma cells were infected, the transcription and expression of IL-24,OSM in A375 human melanoma cells were detected by RT-PCR,westernblot assay. The morphological changes of A375 cells were observed by fluorescence microscope, and the growth inhibition of A375 melanoma cells by Ad-IL-24-OSM was detected by MTT assay. Flow cytometry (FCM) was used to detect the cell cycle growth inhibition and apoptosis of A375 melanoma cells induced by recombinant virion, and the expression level of p21 p53 BaxBcl-2 mRNA in A375 melanoma cells was detected by semi-quantitative RT-PCR. The expression of Caspase-3 apoptosis-related protein was detected by. Western blot. By establishing the animal model of A375 human melanoma cell transplantation tumor, the weight of tumor was detected by Ad-IL-24-OSM recombinant virus, and the nuclear morphology in the tissue section of tumor was detected by HE staining. Immunohistochemistry was used to detect the expression of COX-2 Caspase-3 in the sections of transplanted tumors. Results: 1. The results of PCR and double enzyme digestion showed that the adenovirus vector of Ad-IL-24-OSM double gene expression was successfully constructed. 2. 2. RT-PCR and Western blot assays confirmed that both IL-24 and OSM genes mediated by adenovirus could be stably transcribed and expressed in A375 human melanoma cells. Ad-IL-24-OSM double gene co-expression adenovirus vector could significantly inhibit the growth of A375 cells and induce apoptosis, and was significantly superior to Ad-IL-24,Ad-OSM single genome (P0.05). In vivo experiments showed that Ad-IL-24-OSM adenovirus vector could significantly inhibit the growth of human melanoma xenografts in nude mice and had a synergistic additive effect (Q1. 02). 5. The molecular mechanism of apoptosis showed that Ad-IL-24-OSM could significantly up-regulate the expression of cell cycle and apoptosis-related protein such as Cox-2,Bcl-2,CD34 in human melanoma cell line P21P53nBaxCaspase-3 and down-regulate the expression of apoptosis-related protein, and its effect was more significant than that of Ad-IL-24,Ad-OSM. Conclusion: Ad-IL-24-OSM double gene adenovirus coexpression vector was successfully constructed, which could inhibit the growth and induce apoptosis of A375 cells in vitro and in vivo. The mechanism may be the up-regulation of p21, p53, Bax and down-regulation of Cox-2,Bcl-2,CD34 gene expression. The apoptosis of A375 cells was induced by activating Caspase-3 apoptosis pathway and inhibiting tumor angiogenesis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5
本文编号：2267577
[Abstract]:Aim: to construct an adenovirus vector (Ad-IL-24-OSM) carrying interleukin-24 (Interleukin-24,IL-24) gene and tumor suppressor M (OSM) gene and to investigate the anti-tumor effect of A375 human melanoma cells in vitro and in vivo. Methods: on the basis of the successful construction of pTrack-CMV-poly-A-promoter transfer plasmid in our department, the IL-24 and OSM gene fragments were inserted between Bgl II,Sal I and Not Iho I, respectively, and the pAdTrack-CMV-IL-24-polyA-promoter-OSM recombinant transfer vector was constructed by homologous recombination. The recombinant adenovirons of Ad-IL-24-OSM were obtained by packaging and amplification. The titers of Ad-IL-24-OSM recombinant adenovirus were detected in vitro. After A375 melanoma cells were infected, the transcription and expression of IL-24,OSM in A375 human melanoma cells were detected by RT-PCR,westernblot assay. The morphological changes of A375 cells were observed by fluorescence microscope, and the growth inhibition of A375 melanoma cells by Ad-IL-24-OSM was detected by MTT assay. Flow cytometry (FCM) was used to detect the cell cycle growth inhibition and apoptosis of A375 melanoma cells induced by recombinant virion, and the expression level of p21 p53 BaxBcl-2 mRNA in A375 melanoma cells was detected by semi-quantitative RT-PCR. The expression of Caspase-3 apoptosis-related protein was detected by. Western blot. By establishing the animal model of A375 human melanoma cell transplantation tumor, the weight of tumor was detected by Ad-IL-24-OSM recombinant virus, and the nuclear morphology in the tissue section of tumor was detected by HE staining. Immunohistochemistry was used to detect the expression of COX-2 Caspase-3 in the sections of transplanted tumors. Results: 1. The results of PCR and double enzyme digestion showed that the adenovirus vector of Ad-IL-24-OSM double gene expression was successfully constructed. 2. 2. RT-PCR and Western blot assays confirmed that both IL-24 and OSM genes mediated by adenovirus could be stably transcribed and expressed in A375 human melanoma cells. Ad-IL-24-OSM double gene co-expression adenovirus vector could significantly inhibit the growth of A375 cells and induce apoptosis, and was significantly superior to Ad-IL-24,Ad-OSM single genome (P0.05). In vivo experiments showed that Ad-IL-24-OSM adenovirus vector could significantly inhibit the growth of human melanoma xenografts in nude mice and had a synergistic additive effect (Q1. 02). 5. The molecular mechanism of apoptosis showed that Ad-IL-24-OSM could significantly up-regulate the expression of cell cycle and apoptosis-related protein such as Cox-2,Bcl-2,CD34 in human melanoma cell line P21P53nBaxCaspase-3 and down-regulate the expression of apoptosis-related protein, and its effect was more significant than that of Ad-IL-24,Ad-OSM. Conclusion: Ad-IL-24-OSM double gene adenovirus coexpression vector was successfully constructed, which could inhibit the growth and induce apoptosis of A375 cells in vitro and in vivo. The mechanism may be the up-regulation of p21, p53, Bax and down-regulation of Cox-2,Bcl-2,CD34 gene expression. The apoptosis of A375 cells was induced by activating Caspase-3 apoptosis pathway and inhibiting tumor angiogenesis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5
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