IL-24在髓系白血病生物疗法基础研究中的应用
发布时间：2018-07-26 17:22
【摘要】：第一部分含IL-24的培养体系对CIK细胞生物学活性的影响目的:观察IL-24对CIK细胞生物学活性的影响,探寻增强CIK细胞毒活性的有效方法。方法:分离人外周血单个核细胞,用含有或不含IL-24的培养体系体外诱导培养CIK细胞,观察细胞增殖情况,FCM和ELISA法分析CIK表型及分泌细胞因子能力的差别,溶血试验和FCM法评价CIK细胞产生颗粒酶等毒性颗粒和IFN-γ的能力,将培养获得的CIK细胞和白血病细胞共培养,MTT法和FCM法检测各组CIK细胞对白血病细胞的细胞毒活性。结果:成功诱导培养获得CIK细胞,CIK细胞呈集落样生长,增殖速度较快,未添加IL-24的对照组CIK细胞的增殖率高于添加IL-24培养组。与对照组相比,培养体系中添加IL-24后CIK细胞中CD3+、CD4+和CD3+CD56+细胞的比例无明显改变,而CD3+CD8+、CD8+CD62L+和CD8+CCR7+细胞的数量呈一定程度增加,同时CD4+CD25+CD127-细胞比例有所下降。IL-24可上调CIK细胞表面CD107a和CD54分子以及胞内颗粒酶B的表达,此外,IL-24还可使CIK细胞分泌TNF-α和IFN-γ的能力显著增强。诱导培养的各组CIK细胞均能不同程度诱导K562、K562/A02及新鲜分离的原代白血病细胞凋亡,IL-24组的CIK细胞比常规方法培养的CIK细胞的肿瘤杀伤活性更强。结论:成功从健康人外周血单个核细胞中诱导培养了CIK细胞,所获得的CIK细胞具有正常的生物学功能。IL-24可通过增加CD3+CD8+细胞和中央型记忆T细胞(CD8+TCM)的数量,上调CIK细胞表面粘附分子、CD107a和胞内颗粒酶等毒性颗粒的表达,以及促进CIK细胞分泌Th1型细胞因子,减少CIK细胞中Treg调节性T细胞的比例等途径来改变CIK细胞的生物学特性,增强CIK细胞的抗肿瘤作用。第二部分il-24基因修饰的树突细胞促进cik细胞对白血病细胞的杀伤作用目的:研究cik细胞与il-24基因修饰的同源树突状细胞共培养后对白血病细胞的杀伤作用及其作用机理。方法:从外周血单个核细胞中常规诱导培养dc和cik细胞,同时构建重组腺病毒载体advgfp/il-24(ad-il-24),将il-24基因通过重组病毒导入已负载肿瘤抗原的dc,所构建的细胞称为dc-il-24,rt-pcr和elisa法检测dc-il-24中il-24基因的表达。重组病毒感染72h后在培养体系中加入il-10,fcm和elisa法分别检测dc表型和细胞因子分泌能力的变化,将各组dc和cik细胞混合培养,fcm法检测与dc共培养的cik细胞对白血病细胞杀伤活性的变化。结果:成功从健康人外周血单个核细胞中诱导培养了dc和cik细胞,重组腺病毒ad-il-24能有效将il-24基因导入dc,il-24可上调dc表面cd80、cd83、hla-dr、cd40、cxcr4分子的表达。基因转染后的dc分泌il-12、tnf-α的能力显著提高。il-10能够抑制dc表型成熟,促使其向单核-巨噬细胞方向分化,并降低dc分泌il-12、tnf-α的能力,但il-10对已转入il-24基因的dc并无明显抑制作用。cik细胞与dc-il-24共培养后对白血病细胞的细胞毒活性明显增强,且这种作用不受il-10的抑制。结论:通过重组病毒将il-24基因导入dc后,il-24基因的表达能有效活化dc,促进dc表型成熟,分泌th1型细胞因子,进而增强共培养的cik细胞对肿瘤细胞的细胞毒作用,且这种转基因dc能抵抗il-10的免疫抑制作用。第三部分rgd修饰的il-24重组腺病毒载体对髓系白血病目的:构建rgd修饰的il-24重组腺病毒载体,观察它对白血病细胞的抑制效应及其作用机制。方法:构建重组腺病毒ad.rgd-il-24,观察它对髓系白血病细胞thp-1、k562、k562/a02、meg-01的感染效率,瑞氏-姬姆萨染色和fcm检测白血病细胞的分化情况。mtt法检测重组腺病毒对白血病细胞生长的影响,流式细胞仪检测il-24基因表达对白血病细胞周期和凋亡的影响。real-timepcr和westernblot法检测转染il-24基因后细胞凋亡相关基因grp78/bip、gadd153、gadd34、gadd45α、bax、bcl-2和mcl-1在thp-1细胞中的表达,分光光度法检测caspase-3活性的变化。结果:我们成功构建并获得rgd修饰的重组腺病毒载体ad.rgd-il-24,ad.rgd-il-24对meg-01等髓系白血病细胞的感染效率较高。异位过表达il-24能通过细胞周期阻滞来抑制靶细胞生长,并对髓系白血病细胞有一定诱导分化的作用。ad.rgd-il-24重组腺病毒能显著诱导thp-1细胞凋亡,但不能明显诱导k562和k562/a02细胞凋亡。ad.rgd-il-24能明显上调thp-1细胞grp78/bip、gadd153、gadd34、gadd45α和bax基因的表达,并下调bcl-2和mcl-1基因的表达,同时增强caspase-3的活性。结论:rgd修饰的重组腺病毒载体ad.rgd-il-24对某些种类的髓系白血病细胞具有较高的基因转导能力,内源性il-24的过表达可明显抑制白血病细胞生长,并一定程度地诱导分化。ad.rgd-il-24能通过调节凋亡相关蛋白的表达,诱导thp-1细胞凋亡。第四部分il-24基因表达对髓系白血病细胞的免疫调变作用目的:观察il-24基因表达对髓系白血病细胞免疫原性的调变作用。方法:为观察il-24对髓系白血病细胞的免疫调变作用,fcm检测il-24对白血病细胞表面cd80、cd86、hla-abc、hla-dr、mica/b、cd137、cd137l、cd200等免疫分子表达的影响,elisa检测ad.rgd-il-24对白血病细胞分泌vegf、tnf-α、il-6和il-8细胞因子的影响,细胞毒试验检测白血病细胞感染重组病毒后对cik细胞毒敏感性的变化。体内成瘤试验观察转基因白血病细胞在裸鼠体内的生长和成瘤情况,免疫组织化学法检测vegf、cd31、cd34、collageniv、cd147、mt1-mmp、mmp-2和mmp-9等因子的表达。结果:髓系白血病细胞低表达上述分子,而IL-24可影响部分免疫分子的表达,并对白血病细胞分泌某些与免疫相关的细胞因子具有一定的调节作用。IL-24基因修饰可提高白血病细胞对免疫杀伤细胞的敏感性,并抑制裸鼠白血病细胞移植瘤的生长。分子机制检测结果显示:Ad.RGD-IL-24重组腺病毒能明显下调与肿瘤血管生成密切相关的蛋白分子VEGF、CD31、CD34和collagen IV的表达,同时下调肿瘤侵袭相关分子CD147和基质金属蛋白酶MT1-MMP、MMP-2和MMP-9的表达。结论:IL-24对髓系白血病细胞的免疫原性具有调变作用,能增加白血病细胞对CIK细胞体外杀伤作用的敏感性,还能通过抑制肿瘤血管生成和降低其侵袭性,抑制裸鼠白血病细胞移植瘤的生长。
[Abstract]:The first part is the effect of IL-24 culture system on the biological activity of CIK cells: To observe the effect of IL-24 on the biological activity of CIK cells and to explore the effective methods to enhance the cytotoxic activity of CIK. Methods: to isolate human peripheral blood mononuclear cells and to induce the culture of CIK cells in vitro by the culture system containing or without IL-24 and to observe the proliferation of the cells in vitro. FCM and ELISA methods were used to analyze the difference of CIK phenotype and the ability to secrete cytokine. Hemolytic test and FCM method were used to evaluate the ability of CIK cells to produce granzyme and IFN- gamma. The cultured CIK cells and leukemic cells were co cultured. MTT and FCM methods were used to detect the cytotoxicity of CIK cells to leukemia cells. In induction culture, CIK cells were obtained, CIK cells were colony like growth, and the proliferation rate was faster. The proliferation rate of CIK cells in the control group without IL-24 was higher than that in the IL-24 culture group. Compared with the control group, the proportion of CD3+, CD4+ and CD3+CD56+ cells in CIK cells in the culture system was not significantly changed, while CD3+CD8+, CD8+CD62L+ and thinner cells were not changed. The number of cells increased to a certain extent, while the proportion of CD4+CD25+CD127- cells decreased by.IL-24. The expression of CD107a and CD54 molecules on the surface of CIK cells and the expression of intracellular granzyme B were up-regulated. In addition, IL-24 can also significantly enhance the ability of CIK cells to secrete TNF- and IFN- gamma. 2 and fresh isolated primary leukemia cells apoptosis, the CIK cells in group IL-24 were more potent than those of conventional methods. Conclusion: CIK cells were successfully cultured from the peripheral blood mononuclear cells of healthy human, and the obtained CIK cells have normal biological function.IL-24 by increasing CD3+CD8+ cells and medium. The number of central memory T cells (CD8+TCM), up - regulation of the expression of CIK cell surface adhesion molecules, CD107a and intracellular granzyme and other toxic particles, as well as promoting the secretion of Th1 type cytokines in CIK cells and reducing the proportion of Treg regulatory T cells in CIK cells to change the biological characteristics of CIK cells and enhance the anti-tumor effect of CIK cells. The two part of IL-24 gene modified dendritic cells promote the killing effect of CIK cells on leukemic cells: To study the killing effect and mechanism of CIK cells and IL-24 modified homologous dendritic cells in co culture of leukemia cells. Methods: the normal induction and culture of DC and CIK cells from peripheral blood mononuclear cells The recombinant adenovirus vector advgfp/il-24 (ad-il-24) was constructed to transfer the IL-24 gene into the DC loaded with tumor antigen through the recombinant virus. The constructed cells were called dc-il-24, RT-PCR and ELISA to detect the expression of IL-24 gene in dc-il-24. The recombinant virus was infected with 72h and added into the IL-10, FCM, and cells respectively in the culture system. The changes in factor secreting capacity were mixed with DC and CIK cells in each group. FCM assay was used to detect the changes in the cytotoxic activity of CIK cells co cultured with DC. Results: DC and CIK cells were successfully induced from the peripheral blood mononuclear cells of the healthy human peripheral blood. The recombinant adenovirus ad-il-24 could effectively import the IL-24 gene into DC, IL-24 can increase the DC table. The expression of CD80, CD83, HLA-DR, CD40, CXCR4 molecules. The DC secreted IL-12 after gene transfection, the ability of tnf- a to significantly increase the.Il-10 can inhibit the maturation of the DC phenotype, promote its differentiation into the mononuclear macrophage direction, and reduce the DC secretion of IL-12, and the ability to inhibit alpha. After -24 co culture, the cytotoxic activity of leukemic cells was significantly enhanced, and this effect was not inhibited by IL-10. Conclusion: the expression of IL-24 gene can effectively activate DC, promote the maturation of the DC phenotype and secrete Th1 type cytokines, and then enhance the cytotoxic activity of the co cultured CIK cells to the tumor cells after the recombinant virus has introduced the IL-24 gene into DC. Use, and this transgenic DC can resist the immunosuppressive effect of IL-10. Third RGD modified IL-24 recombinant adenovirus vector to myeloid leukemia Objective: to construct RGD modified IL-24 recombinant adenovirus vector, to observe its inhibitory effect on leukemic cells and its mechanism. Infection efficiency of leukemic cells THP-1, K562, k562/a02, MEG-01, Rayleigh Giemsa staining and FCM detection of leukemia cell differentiation by.Mtt method to detect the effect of recombinant adenovirus on the growth of leukemia cells. Flow cytometry was used to detect the influence of IL-24 gene expression on the cycle and apoptosis of leukemia cells.Real-timepcr and Westernblot method The expression of apoptosis related genes grp78/bip, gadd153, gadd34, GADD45, Bax, Bcl-2 and Mcl-1 in THP-1 cells was measured after transfection of IL-24 gene. The changes of caspase-3 activity were detected by spectrophotometry. Results: we successfully constructed and obtained RGD modified adenovirus vector ad.rgd-il-24. The infection efficiency is high. Ectopic overexpression IL-24 can inhibit the growth of target cells through cell cycle arrest and induce differentiation of myeloid leukemia cells..ad.rgd-il-24 recombinant adenovirus can significantly induce apoptosis of THP-1 cells, but the apoptosis of K562 and k562/a02 cells can not be obviously induced to up regulate the GRP of THP-1 cells. The expression of 78/bip, gadd153, gadd34, GADD45 alpha and Bax genes, and down regulation of the expression of Bcl-2 and Mcl-1 genes and enhancing the activity of Caspase-3. Conclusion: RGD modified recombinant adenovirus vector ad.rgd-il-24 has a high gene transduction energy for some kinds of myeloid leukemia cells, and the endogenous IL-24 overexpression can inhibit leukemic thinning. Cell growth, and to a certain extent, induced differentiation.Ad.rgd-il-24 can induce apoptosis related proteins by regulating the expression of apoptosis related proteins. Fourth part of the expression of IL-24 gene expression on myeloid leukemia cell immunogenicity purpose: To observe the modulation effect of IL-24 gene expression on the immunogenicity of myeloid leukemia cells. Method: To observe IL-24 The immune modulation of myeloid leukemia cells. FCM detected the effect of IL-24 on the expression of CD80, CD86, HLA-ABC, HLA-DR, mica/b, CD137, CD137L, CD200 and other immune molecules on the surface of leukemia cells. Changes in virus sensitivity of CIK cells after recombinant virus. In vivo tumorigenesis test was used to observe the growth and tumor formation of transgenic leukemia cells in nude mice. Immunohistochemical method was used to detect the expression of VEGF, CD31, CD34, collageniv, CD147, MT1-MMP, MMP-2 and MMP-9. The expression of some immune molecules and the regulation of some immune related cytokines secreted by leukemic cells,.IL-24 gene modification can improve the sensitivity of leukemic cells to immune killer cells and inhibit the growth of xenografts in nude mice. Molecular mechanism detection results show that the recombinant Ad.RGD-IL-24 is reorganized. Adenovirus can obviously reduce the expression of protein molecules VEGF, CD31, CD34 and collagen IV, which are closely related to tumor angiogenesis, and down regulate the expression of CD147 and matrix metalloproteinase MT1-MMP, MMP-2 and MMP-9 in tumor invasion related molecules. Conclusion: IL-24 has an modulating effect on the immunogenicity of myeloid leukemia cells, which can increase leukemic thinning. The sensitivity of cells to killing CIK cells in vitro can also inhibit the growth of transplanted tumor cells in nude mice by inhibiting tumor angiogenesis and reducing their invasiveness.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.7
，
本文编号：2146733
[Abstract]:The first part is the effect of IL-24 culture system on the biological activity of CIK cells: To observe the effect of IL-24 on the biological activity of CIK cells and to explore the effective methods to enhance the cytotoxic activity of CIK. Methods: to isolate human peripheral blood mononuclear cells and to induce the culture of CIK cells in vitro by the culture system containing or without IL-24 and to observe the proliferation of the cells in vitro. FCM and ELISA methods were used to analyze the difference of CIK phenotype and the ability to secrete cytokine. Hemolytic test and FCM method were used to evaluate the ability of CIK cells to produce granzyme and IFN- gamma. The cultured CIK cells and leukemic cells were co cultured. MTT and FCM methods were used to detect the cytotoxicity of CIK cells to leukemia cells. In induction culture, CIK cells were obtained, CIK cells were colony like growth, and the proliferation rate was faster. The proliferation rate of CIK cells in the control group without IL-24 was higher than that in the IL-24 culture group. Compared with the control group, the proportion of CD3+, CD4+ and CD3+CD56+ cells in CIK cells in the culture system was not significantly changed, while CD3+CD8+, CD8+CD62L+ and thinner cells were not changed. The number of cells increased to a certain extent, while the proportion of CD4+CD25+CD127- cells decreased by.IL-24. The expression of CD107a and CD54 molecules on the surface of CIK cells and the expression of intracellular granzyme B were up-regulated. In addition, IL-24 can also significantly enhance the ability of CIK cells to secrete TNF- and IFN- gamma. 2 and fresh isolated primary leukemia cells apoptosis, the CIK cells in group IL-24 were more potent than those of conventional methods. Conclusion: CIK cells were successfully cultured from the peripheral blood mononuclear cells of healthy human, and the obtained CIK cells have normal biological function.IL-24 by increasing CD3+CD8+ cells and medium. The number of central memory T cells (CD8+TCM), up - regulation of the expression of CIK cell surface adhesion molecules, CD107a and intracellular granzyme and other toxic particles, as well as promoting the secretion of Th1 type cytokines in CIK cells and reducing the proportion of Treg regulatory T cells in CIK cells to change the biological characteristics of CIK cells and enhance the anti-tumor effect of CIK cells. The two part of IL-24 gene modified dendritic cells promote the killing effect of CIK cells on leukemic cells: To study the killing effect and mechanism of CIK cells and IL-24 modified homologous dendritic cells in co culture of leukemia cells. Methods: the normal induction and culture of DC and CIK cells from peripheral blood mononuclear cells The recombinant adenovirus vector advgfp/il-24 (ad-il-24) was constructed to transfer the IL-24 gene into the DC loaded with tumor antigen through the recombinant virus. The constructed cells were called dc-il-24, RT-PCR and ELISA to detect the expression of IL-24 gene in dc-il-24. The recombinant virus was infected with 72h and added into the IL-10, FCM, and cells respectively in the culture system. The changes in factor secreting capacity were mixed with DC and CIK cells in each group. FCM assay was used to detect the changes in the cytotoxic activity of CIK cells co cultured with DC. Results: DC and CIK cells were successfully induced from the peripheral blood mononuclear cells of the healthy human peripheral blood. The recombinant adenovirus ad-il-24 could effectively import the IL-24 gene into DC, IL-24 can increase the DC table. The expression of CD80, CD83, HLA-DR, CD40, CXCR4 molecules. The DC secreted IL-12 after gene transfection, the ability of tnf- a to significantly increase the.Il-10 can inhibit the maturation of the DC phenotype, promote its differentiation into the mononuclear macrophage direction, and reduce the DC secretion of IL-12, and the ability to inhibit alpha. After -24 co culture, the cytotoxic activity of leukemic cells was significantly enhanced, and this effect was not inhibited by IL-10. Conclusion: the expression of IL-24 gene can effectively activate DC, promote the maturation of the DC phenotype and secrete Th1 type cytokines, and then enhance the cytotoxic activity of the co cultured CIK cells to the tumor cells after the recombinant virus has introduced the IL-24 gene into DC. Use, and this transgenic DC can resist the immunosuppressive effect of IL-10. Third RGD modified IL-24 recombinant adenovirus vector to myeloid leukemia Objective: to construct RGD modified IL-24 recombinant adenovirus vector, to observe its inhibitory effect on leukemic cells and its mechanism. Infection efficiency of leukemic cells THP-1, K562, k562/a02, MEG-01, Rayleigh Giemsa staining and FCM detection of leukemia cell differentiation by.Mtt method to detect the effect of recombinant adenovirus on the growth of leukemia cells. Flow cytometry was used to detect the influence of IL-24 gene expression on the cycle and apoptosis of leukemia cells.Real-timepcr and Westernblot method The expression of apoptosis related genes grp78/bip, gadd153, gadd34, GADD45, Bax, Bcl-2 and Mcl-1 in THP-1 cells was measured after transfection of IL-24 gene. The changes of caspase-3 activity were detected by spectrophotometry. Results: we successfully constructed and obtained RGD modified adenovirus vector ad.rgd-il-24. The infection efficiency is high. Ectopic overexpression IL-24 can inhibit the growth of target cells through cell cycle arrest and induce differentiation of myeloid leukemia cells..ad.rgd-il-24 recombinant adenovirus can significantly induce apoptosis of THP-1 cells, but the apoptosis of K562 and k562/a02 cells can not be obviously induced to up regulate the GRP of THP-1 cells. The expression of 78/bip, gadd153, gadd34, GADD45 alpha and Bax genes, and down regulation of the expression of Bcl-2 and Mcl-1 genes and enhancing the activity of Caspase-3. Conclusion: RGD modified recombinant adenovirus vector ad.rgd-il-24 has a high gene transduction energy for some kinds of myeloid leukemia cells, and the endogenous IL-24 overexpression can inhibit leukemic thinning. Cell growth, and to a certain extent, induced differentiation.Ad.rgd-il-24 can induce apoptosis related proteins by regulating the expression of apoptosis related proteins. Fourth part of the expression of IL-24 gene expression on myeloid leukemia cell immunogenicity purpose: To observe the modulation effect of IL-24 gene expression on the immunogenicity of myeloid leukemia cells. Method: To observe IL-24 The immune modulation of myeloid leukemia cells. FCM detected the effect of IL-24 on the expression of CD80, CD86, HLA-ABC, HLA-DR, mica/b, CD137, CD137L, CD200 and other immune molecules on the surface of leukemia cells. Changes in virus sensitivity of CIK cells after recombinant virus. In vivo tumorigenesis test was used to observe the growth and tumor formation of transgenic leukemia cells in nude mice. Immunohistochemical method was used to detect the expression of VEGF, CD31, CD34, collageniv, CD147, MT1-MMP, MMP-2 and MMP-9. The expression of some immune molecules and the regulation of some immune related cytokines secreted by leukemic cells,.IL-24 gene modification can improve the sensitivity of leukemic cells to immune killer cells and inhibit the growth of xenografts in nude mice. Molecular mechanism detection results show that the recombinant Ad.RGD-IL-24 is reorganized. Adenovirus can obviously reduce the expression of protein molecules VEGF, CD31, CD34 and collagen IV, which are closely related to tumor angiogenesis, and down regulate the expression of CD147 and matrix metalloproteinase MT1-MMP, MMP-2 and MMP-9 in tumor invasion related molecules. Conclusion: IL-24 has an modulating effect on the immunogenicity of myeloid leukemia cells, which can increase leukemic thinning. The sensitivity of cells to killing CIK cells in vitro can also inhibit the growth of transplanted tumor cells in nude mice by inhibiting tumor angiogenesis and reducing their invasiveness.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.7
，
本文编号：2146733
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