白细胞介素-23对黑色素瘤血管生成拟态形成影响的研究

发布时间：2018-07-01 19:18

本文选题：IL-23 + 黑色素瘤　；参考：《天津医科大学》2017年硕士论文

【摘要】：【研究目的】双向分化恶性肿瘤是指可以同时向间充质样组织和上皮样组织分化的恶性肿瘤,而上皮样分化和间充质样分化不同之处在于分化后的肿瘤细胞的形态以及组织排列两个方面。恶性黑色素瘤就是一种常见的双向分化恶性肿瘤[1]。血管生成拟态(vasculogenic mimicry,VM)是与传统内皮依赖性血管不同的肿瘤血供形式之一,其构成不依赖于内皮细胞,而是在特定的微环境条件下肿瘤细胞自身通过塑形以及与细胞外基质相互作用而形成的血管壁类似结构,是可为肿瘤输送血液的管道,VM与肿瘤的生长、浸润和转移有着密切的关系[2,3]。由炎症细胞和炎症因子参与构成的肿瘤微环境,对肿瘤的增殖、生存和转移都有着重要影响,是肿瘤形成不可缺少的一个方面[4-10]。而白细胞介素-23(IL-23)作为肿瘤微环境中的一个重要炎性细胞因子,通常是结合细胞表面膜受体复合物,激活下游STAT3、STAT4和NF-κβ等信号通路,从而发挥其生物学功能[11,12]。“肿瘤可能起源于慢性炎症”的假说Virchow早在1863年就已提出[13],这引起了后续一系列的有关炎症和肿瘤之间关系的研究,但IL-23在黑色素瘤双向分化及血管生成拟态形成过程中所起的作用尚未见报道。本研究旨在检测IL-23在黑色素瘤中的表达并探讨IL-23在黑色素瘤双向分化及血管生成拟态(VM)形成过程中的作用,进而研究其对黑色素瘤细胞迁移侵袭能力的影响。【研究方法】1.根据IL-23在黑色素瘤细胞系A375,A875,MUM-2B,MUM-2C中的表达水平,将IL-23表达质粒转染至黑色素瘤细胞系A375,MUM-2B,MUM-2C中,使高表达的A375,MUM-2B中IL-23外源性降表达,低表达的MUM-2C中诱导IL-23外源性过表达。利用Western blot及细胞免疫荧光实验检测IL-23的转染效果。2.在倒置显微镜下观察IL-23转染后A375,MUM-2B,MUM-2C的细胞形态学改变。3.通过MTT增殖实验检测IL-23转染后A375,MUM-2B,MUM-2C细胞增殖能力的变化情况。4.利用细胞划痕实验检测IL-23转染后A375,MUM-2B,MUM-2C细胞迁移运动能力的变化情况。5.通过Transwell侵袭实验检测IL-23转染后A375,MUM-2B,MUM-2C细胞侵袭能力的变化情况。6.利用三维培养实验检测IL-23转染后A375,MUM-2B,MUM-2C细胞成管能力的变化情况。7.利用Western blot及免疫荧光实验检测IL-23转染后A375,MUM-2B,MUM-2C细胞中相关蛋白(NF-κβ、N-cadherin、Vimentin、Snail、MMP-2和VE-cadherin)表达变化情况。8.利用明胶酶谱实验检测IL-23转染后A375,MUM-2B细胞中MMP-2和MMP-9表达活性的变化情况。9.将转染的降表达A375和过表达MUM-2C黑色素瘤细胞接种至裸鼠体内,观察肿瘤成瘤及生长情况。10.包埋制成组织切片,运用Endomucin/PAS双染和免疫组化检测移植瘤组织内VM和相关蛋白(Vimentin、Snail和VE-cadherin)表达情况,分析其表达差异。【研究结果】1.Western blot及细胞免疫荧光实验结果显示经慢病毒质粒转染并药筛得到稳定的IL-23外源性降表达的A375和MUM-2B细胞,IL-23外源性过表达的MUM-2C细胞。2.在倒置显微镜下观察IL-23转染后A375,MUM-2B,MUM-2C的细胞形态发生明显改变。IL-23降表达的A375和MUM-2B细胞变大变圆,伪足减少,而IL-23过表达的MUM-2C细胞变小,呈梭型,伪足增多。3.MTT增殖实验显示IL-23降表达的A375和MUM-2B细胞增殖能力显著降低,而IL-23过表达的MUM-2C细胞增殖能力升高。4.细胞划痕实验结果显示IL-23降表达的A375和MUM-2B细胞迁移运动能力显著降低,而IL-23过表达的MUM-2C细胞迁移运动能力明显提高。5.Transwell侵袭实验显示IL-23降表达的A375和MUM-2B细胞侵袭能力显著降低,而IL-23过表达的MUM-2C细胞侵袭能力明显提高。6.三维培养实验结果显示IL-23降表达的A375和MUM-2B细胞形成管状结构的能力都显著降低,而IL-23过表达的MUM-2C细胞形成管状结构的能力明显提高。7.Western blot及免疫荧光实验结果显示IL-23降表达的A375和MUM-2B细胞间充质表型蛋白N-cad和Vimentin表达降低,VM相关蛋白MMP-2和VE-cadherin表达降低,相关调节蛋白NF-κβ、Snail表达也显著降低,而IL-23过表达的MUM-2C细胞间充质表型蛋白N-cad和Vimentin表达升高,VM相关蛋白MMP-2和VE-cadherin表达升高,相关调节蛋白NF-κβ、Snail表达也显著升高。8.明胶酶谱实验结果显示IL-23降表达的A375和MUM-2B细胞中MMP-2和MMP-9表达活性都显著降低。9.接种IL-23降表达A375和过表达MUM-2C黑色素瘤细胞的裸鼠移植瘤成瘤率及大小均明显小于对照组移植瘤。10.Endomucin/PAS双染和免疫组化结果显示接种IL-23降表达A375移植瘤组织内VM和相关蛋白(Vimentin、Snail和VE-cadherin)表达降低,接种IL-23过表达MUM-2C移植瘤组织内未形成VM而相关蛋白(Vimentin、Snail和VE-cadherin)表达均升高。【研究结论】1.IL-23可促进黑色素瘤细胞向间充质表型分化,促进黑色素瘤的增殖、迁移和侵袭,并促进黑色素瘤血管生成拟态(VM)的形成。2.IL-23可能是通过NF-κβ调节Snail的表达进而调节N-cadherin和Vimentin的表达促进黑色素瘤细胞向间充质表型分化。3.IL-23通过调节间充质表型蛋白N-cadherin和Vimentin促进黑色素瘤细胞向间充质表型分化改变细胞形态使其更易于迁移和侵袭,调节MMP-2和MMP-9表达活性提供空间结构基础,并同时调节MMP-2和VE-cadherin的表达,进而进一步促进血管生成拟态(VM)的形成。
[Abstract]:[Objective] bi-directional differentiation of malignant tumors is a malignant tumor that can differentiate into mesenchymal like tissue and epithelioid tissue at the same time. The differentiation of epithelioid and mesenchymal like differentiation lies in two aspects of the morphology and tissue arrangement of the differentiated tumor cells. Malignant melanoma is a common bidirectional differentiation malignant tumor. The tumor [1]. vasculogenic mimicry (VM) is one of the form of blood supply different from the traditional endothelium dependent blood vessel, and its composition is not dependent on the endothelial cells, but in the specific microenvironment, the tumor cell itself is formed by the shape of the tumor cells themselves and the interaction of the extracellular matrix. VM has a close relationship with the growth, infiltration and metastasis of tumor, which is closely related to the tumor microenvironment composed of inflammatory cells and inflammatory factors. It has an important influence on the proliferation, survival and metastasis of the tumor. It is an indispensable aspect of tumor formation, [4-10]. and IL-23 as a tumor. An important inflammatory cytokine in microenvironment, usually combined with cell surface membrane receptor complexes, activates the downstream STAT3, STAT4, and NF- kappa beta signaling pathways, thus giving play to the hypothesis that the biological function [11,12]. "tumor may originate in chronic inflammation", Virchow has been proposed in 1863, which has caused a series of follow-up. The study of the relationship between inflammation and tumor, but the role of IL-23 in the bi-directional differentiation of melanoma and angiogenesis has not yet been reported. The purpose of this study was to detect the expression of IL-23 in melanoma and to explore the role of IL-23 in the formation of melanoma bi-directional differentiation and angiogenesis (VM). The effect on the migration and invasion of melanoma cells. [method] 1. according to the expression level of IL-23 in the melanoma cell line A375, A875, MUM-2B, MUM-2C, the IL-23 expression plasmid was transfected into the melanoma cell line A375, MUM-2B, and MUM-2C to induce the expression of the high expression A375, MUM-2B IL-23, and low expression. Western blot and cell immunofluorescence test were used to detect the transfection effect of IL-23.2. under inverted microscope to observe the cell morphological changes of A375, MUM-2B and MUM-2C after IL-23 transfection..3. through MTT proliferation test was used to detect the proliferation of IL-23 cells. Changes in the mobility of A375, MUM-2B, and MUM-2C cells after IL-23 transfection were detected by the scratch test..5. was used to detect the changes of A375, MUM-2B, MUM-2C cell invasiveness after IL-23 transfection by Transwell invasion experiment. RN blot and immunofluorescence test detected the changes in the expression of related proteins (NF- kappa, N-cadherin, Vimentin, Snail, MMP-2 and VE-cadherin) in A375, MUM-2B, MUM-2C cells after IL-23 transfection. MUM-2C melanoma cells were inoculated into nude mice, and the tumor growth and tumor growth were observed by.10. embedding into tissue sections. The expression of VM and related proteins (Vimentin, Snail and VE-cadherin) in the transplanted tumor tissues were detected by Endomucin/PAS double staining and immunohistochemical staining, and the difference of expression was analyzed. [results] 1.Western blot and The results of cell immunofluorescence test showed that A375 and MUM-2B cells were stabilized by IL-23 transfection and drug sieves, and the IL-23 exogenous MUM-2C cell.2. was observed under inverted microscope for A375 in IL-23 transfection, and the cell morphology of MUM-2B and MUM-2C changed obviously to A375 and refinement of.IL-23 descending expression. The cells changed greatly and the pseudo foot decreased, while the MUM-2C cells overexpressed by IL-23 became smaller and showed shuttle type. The proliferation test of.3.MTT in.3.MTT showed that the proliferation ability of A375 and MUM-2B cells decreased significantly, while the proliferation ability of MUM-2C cells expressed by IL-23 was higher than that of.4. cells in.4. cells. The A375 and MUM-2B cell migration of IL-23 decreased expression. The migration ability of the MUM-2C cells expressed by IL-23 was significantly increased and the.5.Transwell invasion experiment showed that the invasion ability of A375 and MUM-2B cells decreased significantly, while the invasion ability of MUM-2C cells expressed by IL-23 was obviously enhanced by.6. three-dimensional culture experiment, which showed A375 and MUM-2 with IL-23 reduced expression. The ability of B cells to form tubular structure decreased significantly, while the ability of IL-23 over expressed MUM-2C cells to form tubular structures significantly increased.7.Western blot and immunofluorescence test results showed that IL-23 reduced expression of A375 and MUM-2B cells decreased in N-cad and Vimentin expressions of MUM-2B cells, VM related proteins MMP-2 and expression decreased. The expression of NF- kappa beta and Snail decreased significantly, while the expression of N-cad and Vimentin in IL-23 overexpressed MUM-2C cells increased, the expression of MMP-2 and VE-cadherin in VM related proteins increased, and the expression of NF- kappa beta and Snail expression of the related regulatory proteins increased significantly. The expression of MMP-2 and MMP-9 in B cells significantly decreased the tumor formation rate and size of nude mice transplanted with IL-23 descending A375 and overexpression of MUM-2C melanoma cells. The results showed that.10.Endomucin/PAS double staining and immunohistochemical results showed that IL-23 descending expression of A375 was VM and related proteins in A375 transplanted tumor tissues (Vimenti). The expression of N, Snail and VE-cadherin decreased, and the expression of VM and related proteins (Vimentin, Snail and VE-cadherin) were not formed in the MUM-2C transplanted tumor tissues of IL-23 over expression. [Conclusion] 1.IL-23 can promote the differentiation of melanoma cells into the mesenchymal phenotype, promote the proliferation, migration and invasion of melanoma, and promote the blood vessels of melanoma. The formation of.2.IL-23 (VM) may be mediated by the expression of Snail by NF- kappa beta and then regulating the expression of N-cadherin and Vimentin to promote the differentiation of melanoma cells into mesenchymal phenotypic differentiation.3.IL-23 by regulating mesenchymal phenotypic protein N-cadherin and Vimentin to promote melanoma cells to differentiate into mesenchymal phenotype and change cell morphology. It is easier to migrate and invasiveness, to regulate the spatial structure of MMP-2 and MMP-9 expression activity, and to regulate the expression of MMP-2 and VE-cadherin, and further promote the formation of angiogenic mimicry (VM).
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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