IL-24在髓系白血病生物療法基礎(chǔ)研究中的應用
發(fā)布時間：2018-07-26 17:22
【摘要】：第一部分含IL-24的培養(yǎng)體系對CIK細胞生物學活性的影響目的:觀察IL-24對CIK細胞生物學活性的影響,探尋增強CIK細胞毒活性的有效方法。方法:分離人外周血單個核細胞,用含有或不含IL-24的培養(yǎng)體系體外誘導培養(yǎng)CIK細胞,觀察細胞增殖情況,FCM和ELISA法分析CIK表型及分泌細胞因子能力的差別,溶血試驗和FCM法評價CIK細胞產(chǎn)生顆粒酶等毒性顆粒和IFN-γ的能力,將培養(yǎng)獲得的CIK細胞和白血病細胞共培養(yǎng),MTT法和FCM法檢測各組CIK細胞對白血病細胞的細胞毒活性。結(jié)果:成功誘導培養(yǎng)獲得CIK細胞,CIK細胞呈集落樣生長,增殖速度較快,未添加IL-24的對照組CIK細胞的增殖率高于添加IL-24培養(yǎng)組。與對照組相比,培養(yǎng)體系中添加IL-24后CIK細胞中CD3+、CD4+和CD3+CD56+細胞的比例無明顯改變,而CD3+CD8+、CD8+CD62L+和CD8+CCR7+細胞的數(shù)量呈一定程度增加,同時CD4+CD25+CD127-細胞比例有所下降。IL-24可上調(diào)CIK細胞表面CD107a和CD54分子以及胞內(nèi)顆粒酶B的表達,此外,IL-24還可使CIK細胞分泌TNF-α和IFN-γ的能力顯著增強。誘導培養(yǎng)的各組CIK細胞均能不同程度誘導K562、K562/A02及新鮮分離的原代白血病細胞凋亡,IL-24組的CIK細胞比常規(guī)方法培養(yǎng)的CIK細胞的腫瘤殺傷活性更強。結(jié)論:成功從健康人外周血單個核細胞中誘導培養(yǎng)了CIK細胞,所獲得的CIK細胞具有正常的生物學功能。IL-24可通過增加CD3+CD8+細胞和中央型記憶T細胞(CD8+TCM)的數(shù)量,上調(diào)CIK細胞表面粘附分子、CD107a和胞內(nèi)顆粒酶等毒性顆粒的表達,以及促進CIK細胞分泌Th1型細胞因子,減少CIK細胞中Treg調(diào)節(jié)性T細胞的比例等途徑來改變CIK細胞的生物學特性,增強CIK細胞的抗腫瘤作用。第二部分il-24基因修飾的樹突細胞促進cik細胞對白血病細胞的殺傷作用目的:研究cik細胞與il-24基因修飾的同源樹突狀細胞共培養(yǎng)后對白血病細胞的殺傷作用及其作用機理。方法:從外周血單個核細胞中常規(guī)誘導培養(yǎng)dc和cik細胞,同時構(gòu)建重組腺病毒載體advgfp/il-24(ad-il-24),將il-24基因通過重組病毒導入已負載腫瘤抗原的dc,所構(gòu)建的細胞稱為dc-il-24,rt-pcr和elisa法檢測dc-il-24中il-24基因的表達。重組病毒感染72h后在培養(yǎng)體系中加入il-10,fcm和elisa法分別檢測dc表型和細胞因子分泌能力的變化,將各組dc和cik細胞混合培養(yǎng),fcm法檢測與dc共培養(yǎng)的cik細胞對白血病細胞殺傷活性的變化。結(jié)果:成功從健康人外周血單個核細胞中誘導培養(yǎng)了dc和cik細胞,重組腺病毒ad-il-24能有效將il-24基因?qū)雂c,il-24可上調(diào)dc表面cd80、cd83、hla-dr、cd40、cxcr4分子的表達�；蜣D(zhuǎn)染后的dc分泌il-12、tnf-α的能力顯著提高。il-10能夠抑制dc表型成熟,促使其向單核-巨噬細胞方向分化,并降低dc分泌il-12、tnf-α的能力,但il-10對已轉(zhuǎn)入il-24基因的dc并無明顯抑制作用。cik細胞與dc-il-24共培養(yǎng)后對白血病細胞的細胞毒活性明顯增強,且這種作用不受il-10的抑制。結(jié)論:通過重組病毒將il-24基因?qū)雂c后,il-24基因的表達能有效活化dc,促進dc表型成熟,分泌th1型細胞因子,進而增強共培養(yǎng)的cik細胞對腫瘤細胞的細胞毒作用,且這種轉(zhuǎn)基因dc能抵抗il-10的免疫抑制作用。第三部分rgd修飾的il-24重組腺病毒載體對髓系白血病目的:構(gòu)建rgd修飾的il-24重組腺病毒載體,觀察它對白血病細胞的抑制效應及其作用機制。方法:構(gòu)建重組腺病毒ad.rgd-il-24,觀察它對髓系白血病細胞thp-1、k562、k562/a02、meg-01的感染效率,瑞氏-姬姆薩染色和fcm檢測白血病細胞的分化情況。mtt法檢測重組腺病毒對白血病細胞生長的影響,流式細胞儀檢測il-24基因表達對白血病細胞周期和凋亡的影響。real-timepcr和westernblot法檢測轉(zhuǎn)染il-24基因后細胞凋亡相關(guān)基因grp78/bip、gadd153、gadd34、gadd45α、bax、bcl-2和mcl-1在thp-1細胞中的表達,分光光度法檢測caspase-3活性的變化。結(jié)果:我們成功構(gòu)建并獲得rgd修飾的重組腺病毒載體ad.rgd-il-24,ad.rgd-il-24對meg-01等髓系白血病細胞的感染效率較高。異位過表達il-24能通過細胞周期阻滯來抑制靶細胞生長,并對髓系白血病細胞有一定誘導分化的作用。ad.rgd-il-24重組腺病毒能顯著誘導thp-1細胞凋亡,但不能明顯誘導k562和k562/a02細胞凋亡。ad.rgd-il-24能明顯上調(diào)thp-1細胞grp78/bip、gadd153、gadd34、gadd45α和bax基因的表達,并下調(diào)bcl-2和mcl-1基因的表達,同時增強caspase-3的活性。結(jié)論:rgd修飾的重組腺病毒載體ad.rgd-il-24對某些種類的髓系白血病細胞具有較高的基因轉(zhuǎn)導能力,內(nèi)源性il-24的過表達可明顯抑制白血病細胞生長,并一定程度地誘導分化。ad.rgd-il-24能通過調(diào)節(jié)凋亡相關(guān)蛋白的表達,誘導thp-1細胞凋亡。第四部分il-24基因表達對髓系白血病細胞的免疫調(diào)變作用目的:觀察il-24基因表達對髓系白血病細胞免疫原性的調(diào)變作用。方法:為觀察il-24對髓系白血病細胞的免疫調(diào)變作用,fcm檢測il-24對白血病細胞表面cd80、cd86、hla-abc、hla-dr、mica/b、cd137、cd137l、cd200等免疫分子表達的影響,elisa檢測ad.rgd-il-24對白血病細胞分泌vegf、tnf-α、il-6和il-8細胞因子的影響,細胞毒試驗檢測白血病細胞感染重組病毒后對cik細胞毒敏感性的變化。體內(nèi)成瘤試驗觀察轉(zhuǎn)基因白血病細胞在裸鼠體內(nèi)的生長和成瘤情況,免疫組織化學法檢測vegf、cd31、cd34、collageniv、cd147、mt1-mmp、mmp-2和mmp-9等因子的表達。結(jié)果:髓系白血病細胞低表達上述分子,而IL-24可影響部分免疫分子的表達,并對白血病細胞分泌某些與免疫相關(guān)的細胞因子具有一定的調(diào)節(jié)作用。IL-24基因修飾可提高白血病細胞對免疫殺傷細胞的敏感性,并抑制裸鼠白血病細胞移植瘤的生長。分子機制檢測結(jié)果顯示:Ad.RGD-IL-24重組腺病毒能明顯下調(diào)與腫瘤血管生成密切相關(guān)的蛋白分子VEGF、CD31、CD34和collagen IV的表達,同時下調(diào)腫瘤侵襲相關(guān)分子CD147和基質(zhì)金屬蛋白酶MT1-MMP、MMP-2和MMP-9的表達。結(jié)論:IL-24對髓系白血病細胞的免疫原性具有調(diào)變作用,能增加白血病細胞對CIK細胞體外殺傷作用的敏感性,還能通過抑制腫瘤血管生成和降低其侵襲性,抑制裸鼠白血病細胞移植瘤的生長。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.7
，
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.7
，
本文編號：2146733
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