RGD修饰的腺病毒介导LIF和IL-24双基因共表达对人白血病细胞体内外的抑癌增效作用

发布时间：2018-01-19 04:35

本文关键词： RGD 腺病毒 LIF IL-24 白血病细胞生长抑制　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:构建RGD修饰的LIF和/或IL-24单、双基因腺病毒表达载体,研究其对人白血病MEG01细胞及裸鼠移植瘤体内外的抑制作用及分子机制。方法:将本实验室构建的LIF和/或IL-24重组转移质粒与RGD修饰的腺病毒骨架质粒pAd(RGD)同源重组,经QBI-293A细胞包装获得RGD修饰的Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24重组腺病毒载体。在293A细胞中扩增并检测病毒的效价。感染白血病K562、MEG01细胞,荧光显微镜和流式细胞仪检测经RGD修饰后的腺病毒载体对白血病细胞的感染效率。体外实验分为Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24腺病毒载体组和PBS对照组、Ad.RGD空病毒载体组。各实验组感染MEG01细胞后,Western blot检测目的基因LIF、IL-24的表达,通过CCK-8法检测LIF和/或IL-24基因表达对MEG01细胞增殖的影响,PE-AnexinV/7-AAD双染后,经流式细胞仪检测携带LIF和/或IL-24基因的腺病毒对MEG01细胞凋亡的影响。PI染色,流式细胞仪检测各实验组细胞周期的变化。Transwell法检测LIF和/或IL-24基因表达对MEG01细胞侵袭能力的影响。Western blot检测细胞凋亡相关蛋白Bcl-2、Bax、Bad、P53的表达,Real-time PCR检测细胞周期及侵袭相关基因P21、E2F1、MMP-2/9的表达。体内实验,建立人白血病MEG01细胞裸鼠移植瘤模型,实验组在瘤体内注射腺病毒Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24进行治疗,对照组注射PBS和Ad.RGD空病毒,隔日一次,共注射5次。通过观察并测量瘤体体积、重量,研究LIF和/或IL-24基因表达对裸鼠移植瘤的生长抑制作用。移植瘤取出后,石蜡包埋切片,免疫组化检测相关因子Bcl-2、Bax、Caspase3、P53、E2F1的表达。结果:成功构建RGD修饰的腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24。经RGD修饰后,对白血病细胞MEG01的感染效率从30%提升到90%,K562细胞的感染效率从15%提升到50%左右。Western blot证实了腺病毒载体携带LIF和/或IL-24基因能够在MEG01细胞中成功表达,体外实验结果表明:与PBS组和空病毒组相比,Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24实验处理组均能够明显抑制MEG01细胞的增殖和诱导细胞凋亡,且Ad.RGD-LIF-IL24双基因共表达组对细胞抑制作用更显著;周期结果显示,各实验组均能产生G2/M期阻滞作用;侵袭实验结果表明LIF和/或IL-24单、双基因表达均能够抑制MEG01细胞的侵袭转移,且双基因共表达的抑制作用更显著。体内实验,成功建立裸鼠人白血病MEG01细胞移植瘤模型,瘤体内注射携带目的基因LIF和/或IL-24的腺病毒治疗后,移植瘤的生长受到明显的抑制,且双基因共表达组抑瘤作用更显著。分子机制结果表明:Ad.RGD-LIF-IL24双基因共表达能够明显上调Bax、P53、Caspase3、P21和下调Bcl-xl、Bcl-2、E2F1、MMP-2/9等细胞凋亡、周期及侵袭相关基因的表达,且其效应较Ad.RGD-LIF或Ad.RGD-IL24单基因组更为显著。结论:1、成功构建RGD修饰的腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24。RGD修饰后,能够显著提升腺病毒载体对MEG01和K562细胞的感染效率。2、腺病毒载体携带LIF和/或IL-24单/双基因表达在体外、体内均能够显著抑制白血病MEG01细胞的生长和诱导细胞发生凋亡,且Ad.RGD-LIF-IL24双基因共表达组具有抑癌增效作用。3、腺病毒载体携带LIF和/或IL-24单/双基因在MEG01细胞中表达均能明显上调Bax、P53、P21和下调Bcl-2、E2F1等凋亡和周期相关因子的表达,从而诱导细胞凋亡并产生G2/M期阻滞;下调MMP-2/9的表达从而抑制MEG01细胞的侵袭;且Ad.RGD-LIF-IL24双基因共表达组对凋亡、周期相关基因的调控作用更显著,这可能是双基因共表达发挥抑瘤增效作用的重要分子机制。
[Abstract]:Objective: to construct the RGD modified LIF and / or IL-24 single and double gene adenovirus expression vector of human leukemia MEG01 cells and xenografts in vivo inhibitory effect and molecular mechanism. Methods: the laboratory construction of adenovirus LIF and / or IL-24 recombinant transfer plasmid and RGD modified plasmid pAd (RGD) by homologous recombination, QBI-293A packaging cell RGD modified Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24. The titer of recombinant adenovirus vector in 293A cells to amplify and detect the virus infection. Leukemia K562, MEG01 cell, fluorescence microscopy and flow cytometry. The infection efficiency of adenovirus vector RGD modified on leukemia cells in vitro experiments were divided into Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 adenovirus vector group and PBS control group, Ad.RGD empty vector group. The experimental group MEG01 cells infected with Western blot detection LIF gene, the expression of IL-24 expression on the proliferation of MEG01 cell by detecting gene LIF and / or IL-24 CCK-8 method, PE-AnexinV/7-AAD staining, flow cytometry analysis with LIF and / or IL-24 gene of adenovirus.PI on apoptosis in MEG01 staining,.Transwell method was used to detect LIF gene changes and / or flow cytometry was used to detect IL-24 of each experimental group. Cell cycle detection of apoptosis related protein Bcl-2.Western blot, effects on the invasion ability of MEG01 cells to Bax, Bad, P53 expression, Real-time PCR detection of cell cycle and invasion related genes P21, E2F1, MMP-2/9 expression. In vivo, the establishment of the nude mouse model of leukemia MEG01 cells in the experimental group, intratumoral injection of adenovirus Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 treatment, control group were injected with PBS and Ad.RGD virus, once every other day, a total of 5 injections. Through observation and measurement The tumor volume, weight, LIF and / or IL-24 expression inhibits tumor growth in nude mice. Tumor removed, paraffin section, immunohistochemical detection of factor Bcl-2, Bax, Caspase3, P53, E2F1. Results: the expression of adenovirus vector Ad.RGD-LIF was successfully constructed by RGD modified Ad.RGD-IL24. And Ad.RGD-LIF-IL24. modified by RGD infection efficiency on leukemia MEG01 cells increased from 30% to 90%, the infection efficiency of K562 cells increased from 15% to 50%.Western blot confirmed the adenovirus vector carrying LIF and / or IL-24 gene can be successfully expressed in MEG01 cells in vitro, the experimental results show that compared with the PBS group, and the empty virus group Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 treatments could significantly inhibit the proliferation of MEG01 cells and induce cell apoptosis, and Ad.RGD-LIF-IL24 genes were more inhibitory effect on cells The cycle; results show that all the experimental groups can produce G2/M phase arrest; invasion experiments show that LIF and / or IL-24 single and double gene expression were able to inhibit MEG01 cell invasion and metastasis, the more significant inhibition and double gene co expression. In vivo, successfully established in nude mice of human leukemia MEG01 cells model, intratumoral injection of adenovirus carrying LIF gene and / or IL-24 after transplantation tumor growth was inhibited, and the double gene co expression inhibition effect was more significant. The results show that the molecular mechanism of co expression of Ad.RGD-LIF-IL24 gene increased the expression of Bax, P53, Caspase3, P21 and down-regulation of Bcl-xl. Bcl-2, E2F1, MMP-2/9, apoptosis, cycle and expression of invasion related genes, and its effect is Ad.RGD-LIF or Ad.RGD-IL24 single genome is more significant. Conclusion: 1, adenovirus vector Ad.RGD-LIF was successfully constructed by RGD modified Ad.R. GD-IL24, Ad.RGD-LIF-IL24.RGD modification can significantly improve the infection efficiency of adenovirus vector.2 of MEG01 and K562 cells, LIF and / or IL-24 single / double gene expression in vitro and in vivo adenovirus, can inhibit leukemia MEG01 cell growth and induce cell apoptosis, and Ad.RGD-LIF-IL24 double gene co expression group with tumor effect of synergism of.3 cancer, adenovirus vector carrying LIF and / or IL-24 single / double gene expression in MEG01 cells was significantly up-regulated the expression of Bax, P53, P21 and down-regulation of Bcl-2 expression of E2F1, apoptosis and cycle related factors, from induced apoptosis and G2/M phase arrest; downregulation of MMP-2/9 inhibits MEG01 cell invasion; and Ad.RGD-LIF-IL24 double gene co expression group more significant role on the apoptosis, cycle related genes, which may be the co expression of tumor suppressor molecules play an important synergistic effect Mechanism.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7;R450

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