【摘要】：目的： 構建攜帶白細胞介素24（Interleukin-24,IL-24）基因和抑瘤素M（OSM）基因的雙基因共表達腺病毒載體（Ad-IL-24-OSM）并開展對A375人黑色素瘤細胞體內(nèi)外抗腫瘤效應的實驗研究。 方法： 在本科室已成功構建pTrack-CMV-poly-A-promoter轉(zhuǎn)移質(zhì)粒的基礎上，在多克隆酶切位點的Bgl II、Sal I之間和Not I、Xho I之間分別插入IL-24和OSM基因片段，構建pAdTrack-CMV-IL-24-polyA-promoter-OSM重組轉(zhuǎn)移載體，經(jīng)同源重組、包裝和擴增獲得Ad-IL-24-OSM雙基因共表達重組腺病毒子，體外檢測Ad-IL-24-OSM重組腺病毒子的效價，感染A375黑色素瘤細胞后，RT-PCR、westernblot法檢測IL-24、OSM在A375人黑色素瘤細胞中的轉(zhuǎn)錄和表達；熒光顯微鏡觀察感染后的A375細胞的形態(tài)學改變； MTT法檢測Ad-IL-24-OSM對A375黑色素瘤細胞的生長抑制作用；流式細胞術(FCM)檢測重組病毒子對A375黑色素瘤細胞的周期生長抑制和促凋亡效應；半定量RT-PCR法檢測A375黑色素瘤細胞中的p21、p53、Bax、Bcl-2mRNA的表達水平。western blot檢測Caspase-3凋亡相關蛋白的表達；通過建立A375人黑色素瘤細胞移植瘤瘤動物模型,用Ad-IL-24-OSM重組病毒子進行實驗干預，檢測瘤體重量，HE染色法檢測瘤塊組織切片中的細胞核形態(tài)的變化；免疫組化檢測移植瘤塊切片中P21、P53、bax、bcl-2、CD34、COX2、Caspase-3的表達情況。 結果： 1. PCR和雙酶切分析結果顯示成功構建了Ad-IL-24-OSM雙基因共表達腺病毒載體。 2. RT-PCR和Western blot檢測證實腺病毒介導的IL-24和OSM雙基因均可在A375人黑色素瘤細胞中穩(wěn)定轉(zhuǎn)錄及表達。 3. Ad-IL-24-OSM雙基因共表達腺病毒載體能顯著抑制A375細胞的生長和誘導細胞凋亡，并顯著優(yōu)于Ad-IL-24、Ad-OSM單基因組（P0.05）。 4.體內(nèi)實驗表明Ad-IL-24-OSM腺病毒載體可以顯著抑制裸鼠人黑色素瘤移植瘤的生長，且均具有抑瘤增效的相加作用（Q=1.02）。 5.細胞凋亡分子機制檢測發(fā)現(xiàn)：Ad-IL-24-OSM能明顯上調(diào)A375人黑色素瘤細胞P21、P53、Bax、Caspase-3和下調(diào)Cox-2、Bcl-2、CD34等細胞周期和凋亡相關蛋白的表達;且其效應均較Ad-IL-24、Ad-OSM更為顯著。 結論： Ad-IL-24-OSM雙基因腺病毒共表達載體構建成功，在體內(nèi)外均可明顯抑制A375細胞的生長，誘導其凋亡，其機制可能是與上調(diào)p21、p53、bax和下調(diào)Cox-2、Bcl-2、CD34的基因表達水平，，并通過激活Caspase-3凋亡途徑誘導A375細胞凋亡和抑制腫瘤血管形成有關。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.5

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