【摘要】：第一部分含IL-24的培養(yǎng)體系對(duì)CIK細(xì)胞生物學(xué)活性的影響目的:觀察IL-24對(duì)CIK細(xì)胞生物學(xué)活性的影響,探尋增強(qiáng)CIK細(xì)胞毒活性的有效方法。方法:分離人外周血單個(gè)核細(xì)胞,用含有或不含IL-24的培養(yǎng)體系體外誘導(dǎo)培養(yǎng)CIK細(xì)胞,觀察細(xì)胞增殖情況,FCM和ELISA法分析CIK表型及分泌細(xì)胞因子能力的差別,溶血試驗(yàn)和FCM法評(píng)價(jià)CIK細(xì)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(xì)胞和白血病細(xì)胞共培養(yǎng),MTT法和FCM法檢測(cè)各組CIK細(xì)胞對(duì)白血病細(xì)胞的細(xì)胞毒活性。結(jié)果:成功誘導(dǎo)培養(yǎng)獲得CIK細(xì)胞,CIK細(xì)胞呈集落樣生長(zhǎng),增殖速度較快,未添加IL-24的對(duì)照組CIK細(xì)胞的增殖率高于添加IL-24培養(yǎng)組。與對(duì)照組相比,培養(yǎng)體系中添加IL-24后CIK細(xì)胞中CD3+、CD4+和CD3+CD56+細(xì)胞的比例無(wú)明顯改變,而CD3+CD8+、CD8+CD62L+和CD8+CCR7+細(xì)胞的數(shù)量呈一定程度增加,同時(shí)CD4+CD25+CD127-細(xì)胞比例有所下降。IL-24可上調(diào)CIK細(xì)胞表面CD107a和CD54分子以及胞內(nèi)顆粒酶B的表達(dá),此外,IL-24還可使CIK細(xì)胞分泌TNF-α和IFN-γ的能力顯著增強(qiáng)。誘導(dǎo)培養(yǎng)的各組CIK細(xì)胞均能不同程度誘導(dǎo)K562、K562/A02及新鮮分離的原代白血病細(xì)胞凋亡,IL-24組的CIK細(xì)胞比常規(guī)方法培養(yǎng)的CIK細(xì)胞的腫瘤殺傷活性更強(qiáng)。結(jié)論:成功從健康人外周血單個(gè)核細(xì)胞中誘導(dǎo)培養(yǎng)了CIK細(xì)胞,所獲得的CIK細(xì)胞具有正常的生物學(xué)功能。IL-24可通過(guò)增加CD3+CD8+細(xì)胞和中央型記憶T細(xì)胞(CD8+TCM)的數(shù)量,上調(diào)CIK細(xì)胞表面粘附分子、CD107a和胞內(nèi)顆粒酶等毒性顆粒的表達(dá),以及促進(jìn)CIK細(xì)胞分泌Th1型細(xì)胞因子,減少CIK細(xì)胞中Treg調(diào)節(jié)性T細(xì)胞的比例等途徑來(lái)改變CIK細(xì)胞的生物學(xué)特性,增強(qiáng)CIK細(xì)胞的抗腫瘤作用。第二部分il-24基因修飾的樹(shù)突細(xì)胞促進(jìn)cik細(xì)胞對(duì)白血病細(xì)胞的殺傷作用目的:研究cik細(xì)胞與il-24基因修飾的同源樹(shù)突狀細(xì)胞共培養(yǎng)后對(duì)白血病細(xì)胞的殺傷作用及其作用機(jī)理。方法:從外周血單個(gè)核細(xì)胞中常規(guī)誘導(dǎo)培養(yǎng)dc和cik細(xì)胞,同時(shí)構(gòu)建重組腺病毒載體advgfp/il-24(ad-il-24),將il-24基因通過(guò)重組病毒導(dǎo)入已負(fù)載腫瘤抗原的dc,所構(gòu)建的細(xì)胞稱為dc-il-24,rt-pcr和elisa法檢測(cè)dc-il-24中il-24基因的表達(dá)。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(cè)dc表型和細(xì)胞因子分泌能力的變化,將各組dc和cik細(xì)胞混合培養(yǎng),fcm法檢測(cè)與dc共培養(yǎng)的cik細(xì)胞對(duì)白血病細(xì)胞殺傷活性的變化。結(jié)果:成功從健康人外周血單個(gè)核細(xì)胞中誘導(dǎo)培養(yǎng)了dc和cik細(xì)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(dá)�；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(xì)胞方向分化,并降低dc分泌il-12、tnf-α的能力,但il-10對(duì)已轉(zhuǎn)入il-24基因的dc并無(wú)明顯抑制作用。cik細(xì)胞與dc-il-24共培養(yǎng)后對(duì)白血病細(xì)胞的細(xì)胞毒活性明顯增強(qiáng),且這種作用不受il-10的抑制。結(jié)論:通過(guò)重組病毒將il-24基因?qū)雂c后,il-24基因的表達(dá)能有效活化dc,促進(jìn)dc表型成熟,分泌th1型細(xì)胞因子,進(jìn)而增強(qiáng)共培養(yǎng)的cik細(xì)胞對(duì)腫瘤細(xì)胞的細(xì)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(duì)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(duì)白血病細(xì)胞的抑制效應(yīng)及其作用機(jī)制。方法:構(gòu)建重組腺病毒ad.rgd-il-24,觀察它對(duì)髓系白血病細(xì)胞thp-1、k562、k562/a02、meg-01的感染效率,瑞氏-姬姆薩染色和fcm檢測(cè)白血病細(xì)胞的分化情況。mtt法檢測(cè)重組腺病毒對(duì)白血病細(xì)胞生長(zhǎng)的影響,流式細(xì)胞儀檢測(cè)il-24基因表達(dá)對(duì)白血病細(xì)胞周期和凋亡的影響。real-timepcr和westernblot法檢測(cè)轉(zhuǎn)染il-24基因后細(xì)胞凋亡相關(guān)基因grp78/bip、gadd153、gadd34、gadd45α、bax、bcl-2和mcl-1在thp-1細(xì)胞中的表達(dá),分光光度法檢測(cè)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(duì)meg-01等髓系白血病細(xì)胞的感染效率較高。異位過(guò)表達(dá)il-24能通過(guò)細(xì)胞周期阻滯來(lái)抑制靶細(xì)胞生長(zhǎng),并對(duì)髓系白血病細(xì)胞有一定誘導(dǎo)分化的作用。ad.rgd-il-24重組腺病毒能顯著誘導(dǎo)thp-1細(xì)胞凋亡,但不能明顯誘導(dǎo)k562和k562/a02細(xì)胞凋亡。ad.rgd-il-24能明顯上調(diào)thp-1細(xì)胞grp78/bip、gadd153、gadd34、gadd45α和bax基因的表達(dá),并下調(diào)bcl-2和mcl-1基因的表達(dá),同時(shí)增強(qiáng)caspase-3的活性。結(jié)論:rgd修飾的重組腺病毒載體ad.rgd-il-24對(duì)某些種類的髓系白血病細(xì)胞具有較高的基因轉(zhuǎn)導(dǎo)能力,內(nèi)源性il-24的過(guò)表達(dá)可明顯抑制白血病細(xì)胞生長(zhǎng),并一定程度地誘導(dǎo)分化。ad.rgd-il-24能通過(guò)調(diào)節(jié)凋亡相關(guān)蛋白的表達(dá),誘導(dǎo)thp-1細(xì)胞凋亡。第四部分il-24基因表達(dá)對(duì)髓系白血病細(xì)胞的免疫調(diào)變作用目的:觀察il-24基因表達(dá)對(duì)髓系白血病細(xì)胞免疫原性的調(diào)變作用。方法:為觀察il-24對(duì)髓系白血病細(xì)胞的免疫調(diào)變作用,fcm檢測(cè)il-24對(duì)白血病細(xì)胞表面cd80、cd86、hla-abc、hla-dr、mica/b、cd137、cd137l、cd200等免疫分子表達(dá)的影響,elisa檢測(cè)ad.rgd-il-24對(duì)白血病細(xì)胞分泌vegf、tnf-α、il-6和il-8細(xì)胞因子的影響,細(xì)胞毒試驗(yàn)檢測(cè)白血病細(xì)胞感染重組病毒后對(duì)cik細(xì)胞毒敏感性的變化。體內(nèi)成瘤試驗(yàn)觀察轉(zhuǎn)基因白血病細(xì)胞在裸鼠體內(nèi)的生長(zhǎng)和成瘤情況,免疫組織化學(xué)法檢測(cè)vegf、cd31、cd34、collageniv、cd147、mt1-mmp、mmp-2和mmp-9等因子的表達(dá)。結(jié)果:髓系白血病細(xì)胞低表達(dá)上述分子,而IL-24可影響部分免疫分子的表達(dá),并對(duì)白血病細(xì)胞分泌某些與免疫相關(guān)的細(xì)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(xì)胞對(duì)免疫殺傷細(xì)胞的敏感性,并抑制裸鼠白血病細(xì)胞移植瘤的生長(zhǎng)。分子機(jī)制檢測(cè)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(dá),同時(shí)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(dá)。結(jié)論:IL-24對(duì)髓系白血病細(xì)胞的免疫原性具有調(diào)變作用,能增加白血病細(xì)胞對(duì)CIK細(xì)胞體外殺傷作用的敏感性,還能通過(guò)抑制腫瘤血管生成和降低其侵襲性,抑制裸鼠白血病細(xì)胞移植瘤的生長(zhǎng)。
[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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.7
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本文編號(hào)：2146733