【作者】 单云波；

【导师】 杨吉成；

【作者基本信息】 苏州大学 ， 药理学， 2009， 硕士

【摘要】 目的:构建LIF和IL-24双基因共表达腺病毒载体(Ad-LIF-IL-24),研究其对胶质瘤细胞的抑癌增效和分子机制。方法:分别以pORF-mbcl-2α质粒为模板,PCR分别扩增出polyA和promoter基因片段,再以此为模板,经SOE-PCR扩增polyA+promoter(SalI、NotI)融合片段,并突变其中的PacⅠ酶切位点。再以pcDNA3.0-LIF质粒和pAdTrack-CMV-IL-24质粒为模板,PCR扩增出LIF(BglII、SalI)和IL-24(XhoI、XbaI)目的片段,并与突变后polyA+promoter(简称polyA+promoter△)依次亚克隆至pAdTrack-CMV转移质粒构建pAdTrack-CMV-LIF-polyA+promoter△-IL-24双基因共表达重组转移质粒。将构建正确的pAdTrack-CMV-LIF-polyA+promoter△-IL-24重组转移质粒经PmeI酶切后与pAdEasy-1腺病毒骨架质粒在BJ5183大肠杆菌中同源重组,得到的同源重组质粒pAdEasy-1-pAdTrack-CMV-LIF-polyA+promoter△-IL-24(简称为pAd-LIF- polyA+promoter△-IL-24)经PacI酶切后再用脂质体转染QBI-293A细胞,经多轮感染和扩增后获得Ad-LIF-polyA+promoter△-IL-24(简称Ad-LIF-IL-24)双基因共表达重组腺病毒载体。将Ad-LIF-IL-24体外感染肿瘤细胞SMMC-7721、U251、HT29、A549、HL-60、K562和正常细胞WI-38,筛选出Ad-LIF-IL-24最敏感肿瘤细胞和最佳感染剂量。MTT法和FCM检测Ad-LIF-IL-24对敏感肿瘤细胞的生长抑制和诱导凋亡的增效作用,Hochest染色进一步检测肿瘤细胞凋亡的核形态特征,用RT-PCR检测与细胞凋亡和肿瘤血管形成相关基因bcl-2、bax、ICE、p53和HIF-1α的转录,Western blotting检测肿瘤细胞中的细胞凋亡执行蛋白Cleaved Caspase3的表达。结果:成功构建了双启动子介导的LIF和IL-24双基因共表达腺病毒载体Ad-LIF-IL-24。Ad-LIF-IL-24均能高效感染实体瘤细胞和正常人胚肺二倍体成纤维细胞WI-38,但对白血病细胞感染效率很低,并且对U251胶质瘤细胞抑制作用最为明显,最佳感染剂量为100MOI。腺病毒介导的LIF和IL-24双基因共表达在体外能明显抑制U251胶质瘤细胞生长和诱导凋亡,并呈现叠加效应。分子机制检测结果表明:Ad-LIF-IL-24能明显上调U251胶质瘤细胞ICE、bax、p53和下调bcl-2、HIF-1α等基因的转录,并激活Caspase3剪切成Cleaved Caspase3片段。结论:成功构建的LIF和IL-24双基因共表达腺病毒载体Ad-LIF-IL-24体外能明显抑制U251胶质瘤细胞生长和诱导其凋亡,并呈现叠加作用。Ad-LIF-IL-24可通过上调U251细胞中ICE、bax、p53和下调bcl-2等细胞凋亡相关基因促使Caspase3激活以及下凋与肿瘤血管形成密切相关的HIF-1α基因等途径抑制U251胶质瘤细胞的生长和诱导其凋亡。更多还原

【Abstract】 Objective: To construct a recombinant adenoviral vector co-expressing leukaemia inhibitory factor (LIF) and interleukin-24 (IL-24) and study its enhanced anti-tumor effects on glioma cells and its mechanism.Methods: The polyA+promoter fusion fragments, LIF, and IL-24 fragments were amplified by PCR using pORF-mbcl-2a, pcDNA3.0-LIF, and pAdTrack-CMV-IL-24 plasmids as templates, respectively, and PacI enzyme site in polyA+promoter was mutated(polyA+promoter△). Then the polyA+promoter△(SalI、NotI), LIF (BglII、SalI), and IL-24 (XhoI、XbaI) fragments were subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-LIF-polyA+promoter△-IL-24 and identified by PCR, double endonuclease digestion, and DNA sequencing. The pAdTrack-CMV- LIF-polyA+promoter△-IL-24 transfer vector linearized with PmeI digestion and pAdEasy-1 backbone vector were further cotransfected into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy- 1-pAdTrack-CMV-LIF-polyA+promoter△-IL-24(pAd-LIF-polyA+promoter△- IL-24) homologous recombinant plasmids purified from the above BJ5183 cells were transfected into the bacterial DH5αcells competent cells to abundantly amplify pAd-LIF-polyA+promoter△-IL-24 plasmids. Then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by Lipofectaimne2000, leading to formation of the recombinant adenoviruses Ad-LIF-polyA+promoter△-IL-24(Ad-LIF-IL-24) co-expressing LIF and IL-24. The most sensitive cell and the best MOI of Ad-LIF-IL-24 was chosen by Ad-LIF-IL-24 infecting SMMC-7721, U251, HT29, A549, HL-60, K562 and WI-38 in vitro. The effect of enhanced growth-suppressing and apoptosis-inducing by Ad-LIF-IL-24 on the tumor cells in vitro were assessed by MTT assay and FCM, and the apoptosis of the cell nucleus were detected by Hochest staining. The expression of apoptosis- and angiogenesis-related genes (bcl-2、bax、ICE、p53 and HIF-1α) and the Cleaved Caspase3 in U251 glioma cells was determined by RT-PCR and Western blotting, respectively.Results: The adenoviral vector co-expressing LIF and IL-24, Ad-LIF- polyA+promoter△-IL-24, was successfully constructed. More than 95% of theses tumor cells (SMMC-7721, U251, HT29, and A549) and WI-38 cells were infected by Ad-LIF-IL-24 at MOI of 50, whereas HL-60 and K562 cells were hardly infected. The growth of U251 glioma cells was significantly inhibited by Ad-LIF-IL-24 at MOI of 100. Ad-LIF-IL-24- mediated LIF and IL-24 co-expression significantly inhibited U251 glioma cells growth, induced cells apoptosis, exhibiting additive effect. Ad-LIF-IL-24 significantly up-regulated the expression of ICE、bax、p53, and Cleaved Caspase-3 and down-regulated the expression of bcl-2 and HIF-1α.Conclusion: The adenoviral vector co-expressing LIF and IL-24 (Ad-LIF-IL-24) was successfully constructed. Ad-LIF-IL-24 had significant additive effect in suppressing U251 glioma cells growth and inducing cells apoptosis in vitro. Ad-LIF-IL -24 can up-regulate the expression of ICE、bax、p53 and down-regulate Bcl-2 resulting activation of Caspase-3 and down-regulate the expression of angiogenesis-related gene HIF-1αto exhibit significantly anti-tumor effect on U251 glioma cells in vitro .更多还原