Foxp3在鼠黑素瘤细胞中的表达及功能的初步研究

发布时间：2018-06-08 12:01

本文选题：黑色素瘤 + Foxp3　；参考：《中国人民解放军医学院》2012年硕士论文

【摘要】：恶性黑色素瘤（恶黑）是一种来源于黑色素细胞的高度恶性肿瘤，其对放化疗不敏感，且耐药率也是最明显的一种。近年来，临床应用肿瘤免疫治疗恶黑取得显著效果，通过肿瘤疫苗和DC细胞、CIK细胞等回输治疗，促进体内肿瘤抗原特异性T细胞数量增加，从而攻击并破坏肿瘤细胞。但体外实验表明，黑色素肿瘤组织中活化的肿瘤抗原特异性T细胞数量较预期数量有所减少，且不能有效破坏肿瘤细胞。由此现象，人们认识到恶黑肿瘤微环境中存在免疫耐受机制，，肿瘤细胞可通过某些机制诱导自身免疫耐受从而实现肿瘤免疫逃逸。自1995年Sakaguchi等[1]首先提出CD4~+CD25~+Treg之后，引起了免疫学界越来越多的关注。大量实验研究表明，CD4~+CD25~+Treg可通过抑制自身反应性T细胞来维持免疫耐受，同时也抑制了肿瘤抗原特异性自身反应性T细胞发挥有效的抗肿瘤应答。Foxp3作为Treg的特异性分子标志，在调控CD4~+CD25~+Treg的发育上起很着重要作用。Foxp3分子的表达曾被认为严格限制于T细胞系。近年来，在体外研究中发现Foxp3在其他人肿瘤细胞（肠癌、肺癌、乳腺癌、胶质母细胞瘤、黑色素瘤细胞等）和组织中均有不同程度的表达。目前，对Foxp3的研究多集中在人的肿瘤，而在鼠系的肿瘤中的研究较少。因此，我们推断Foxp3分子也可能表达于小鼠黑色素瘤细胞，并参与维持黑色素瘤肿瘤微环境的免疫耐受。针对以上假设进行以下实验。目的：证实小鼠B16黑色素瘤细胞是否表达Foxp3，研究其对效应T细胞的增殖和功能的抑制作用。方法：通过实时荧光定量PCR检测小鼠黑色素瘤细胞中Foxp3mRNA表达水平；通过Westernblot和流式细胞术检测小鼠黑色素瘤细胞中Foxp3蛋白的表达；通过免疫荧光检测小鼠黑色素瘤细胞中Foxp3分子的表达；检测干扰Foxp3后免疫抑制相关分子表达量的变化及对CD4~+CD25-T淋巴细胞增殖能力的影响。结果：证实在小鼠黑色素瘤细胞中存在Foxp3的mRNA转录和分子表达；免疫荧光显示Foxp3分子定位于黑色素瘤细胞的胞核及核周部位。通过Foxp3siRNA的转染，可实现对B16细胞Foxp3表达的沉默，可下调肿瘤细胞对CD4~+CD25-T淋巴细胞增殖抑制的能力，并且下调TGF-β1、TGF-β2和IL-10等细胞因子的表达，尤其是TGF-β2的表达。结论：小鼠B16细胞表达Foxp3，利用RNA干扰技术可抑制小鼠黑色素瘤细胞靶基因Foxp3的表达，并对CD4~+CD25-T淋巴细胞增殖抑制的能力减弱，同时减弱抑制性细胞因子的分泌。综上所述，肿瘤细胞所引起的CD4~+CD25~+Treg细胞产生及功能增强是肿瘤逃避免疫监视机制之一，而黑素瘤细胞Foxp3的表达可能成为黑素瘤肿瘤微环境抑制自身免疫系统的一种新机制。因此深入研究Foxp3在黑素瘤细胞中的作用机制将为黑素瘤的免疫治疗提供新靶标和新的治疗策略。
[Abstract]:Malignant melanoma (malignant melanoma) is a highly malignant tumor derived from melanocytes, which is insensitive to radiotherapy and chemotherapy, and the rate of drug resistance is the most obvious. In recent years, the clinical application of tumor immunotherapy to malignant melanoma has achieved remarkable results. Through tumor vaccine and DC cell CIK cells, the number of tumor antigen-specific T cells is increased, and the tumor cells are attacked and destroyed. But in vitro experiments showed that the number of activated tumor antigen-specific T cells in melanoma tissues was lower than expected and could not effectively destroy tumor cells. Therefore, it is recognized that there is an immune tolerance mechanism in the microenvironment of malignant melanoma, and tumor cells can induce autoimmune tolerance through some mechanisms to achieve tumor immune escape. [1] after Sakaguchi et al. [1] first proposed CD4- CD25 ~ Treg in 1995, More and more attention has been paid to immunology. A large number of experimental studies have shown that CD4 ~ CD25 ~ Treg can maintain immune tolerance by inhibiting autoreactive T cells, and also inhibit tumor antigen-specific autoreactive T cells to play an effective anti-tumor response. Foxp3 is a specific molecular marker of Treg. The expression of Foxp3 in CD4 ~ CD25 ~ Treg was thought to be strictly restricted to T cell lines. In recent years, in vitro studies have found that Foxp3 is expressed in various degrees in other human tumor cells (bowel cancer, lung cancer, breast cancer, glioblastoma, melanoma cells, etc.). At present, the study of Foxp3 is mainly focused on human tumors, but less on murine tumors. Therefore, we infer that Foxp3 may also be expressed in mouse melanoma cells and participate in maintaining the immune tolerance of melanoma tumor microenvironment. Aim: to investigate the inhibitory effect of Foxp3 on the proliferation and function of effector T cells in murine B16 melanoma cells. Methods: the expression of Foxp3 mRNA in mouse melanoma cells was detected by real-time fluorescent quantitative PCR, and the expression of Foxp3 protein in mouse melanoma cells was detected by Western blot and flow cytometry. The expression of Foxp3 in mouse melanoma cells was detected by immunofluorescence, and the expression of immunosuppressive related molecules and the proliferation of CD4 ~ CD25-T lymphocytes were detected after interfering with Foxp3. Results: the mRNA transcription and molecular expression of Foxp3 were confirmed in mouse melanoma cells, and the localization of Foxp3 molecule in the nucleus and perinuclear region of melanoma cells was demonstrated by immunofluorescence. By transfection of Foxp3 siRNA, the expression of Foxp3 in B16 cells was silenced, the ability of tumor cells to inhibit the proliferation of CD4 ~ CD25-T lymphocytes was down-regulated, and the expression of cytokines such as TGF- 尾 1, TGF- 尾 2 and IL-10 was down-regulated, especially the expression of TGF- 尾 2. Conclusion: mouse B16 cells express Foxp3. RNA interference can inhibit the expression of target gene Foxp3 in murine melanoma cells, weaken the ability of inhibiting the proliferation of CD4 ~ CD25-T lymphocytes, and weaken the secretion of inhibitory cytokines. The production and function of CD4 ~ CD25 ~ Treg cells induced by tumor cells is one of the mechanisms of tumor escape from immune surveillance. The expression of Foxp3 in melanoma cells may be a new mechanism for melanoma tumor microenvironment to inhibit the autoimmune system. Therefore, further study of the mechanism of Foxp3 in melanoma cells will provide a new target and new therapeutic strategy for immunotherapy of melanoma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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