發(fā)布時間：2018-06-08 12:01

  本文選題：黑色素瘤 + Foxp3��； 參考：《中國人民解放軍醫(yī)學院》2012年碩士論文

【摘要】：惡性黑色素瘤（惡黑）是一種來源于黑色素細胞的高度惡性腫瘤，其對放化療不敏感，且耐藥率也是最明顯的一種。近年來，臨床應用腫瘤免疫治療惡黑取得顯著效果，通過腫瘤疫苗和DC細胞、CIK細胞等回輸治療，促進體內(nèi)腫瘤抗原特異性T細胞數(shù)量增加，從而攻擊并破壞腫瘤細胞。但體外實驗表明，黑色素腫瘤組織中活化的腫瘤抗原特異性T細胞數(shù)量較預期數(shù)量有所減少，且不能有效破壞腫瘤細胞。由此現(xiàn)象，人們認識到惡黑腫瘤微環(huán)境中存在免疫耐受機制，，腫瘤細胞可通過某些機制誘導自身免疫耐受從而實現(xiàn)腫瘤免疫逃逸。 自1995年Sakaguchi等[1]首先提出CD4~+CD25~+Treg之后，引起了免疫學界越來越多的關注。大量實驗研究表明，CD4~+CD25~+Treg可通過抑制自身反應性T細胞來維持免疫耐受，同時也抑制了腫瘤抗原特異性自身反應性T細胞發(fā)揮有效的抗腫瘤應答。Foxp3作為Treg的特異性分子標志，在調(diào)控CD4~+CD25~+Treg的發(fā)育上起很著重要作用。Foxp3分子的表達曾被認為嚴格限制于T細胞系。近年來，在體外研究中發(fā)現(xiàn)Foxp3在其他人腫瘤細胞（腸癌、肺癌、乳腺癌、膠質(zhì)母細胞瘤、黑色素瘤細胞等）和組織中均有不同程度的表達。目前，對Foxp3的研究多集中在人的腫瘤，而在鼠系的腫瘤中的研究較少。因此，我們推斷Foxp3分子也可能表達于小鼠黑色素瘤細胞，并參與維持黑色素瘤腫瘤微環(huán)境的免疫耐受。 針對以上假設進行以下實驗。目的：證實小鼠B16黑色素瘤細胞是否表達Foxp3，研究其對效應T細胞的增殖和功能的抑制作用。方法：通過實時熒光定量PCR檢測小鼠黑色素瘤細胞中Foxp3mRNA表達水平；通過Westernblot和流式細胞術(shù)檢測小鼠黑色素瘤細胞中Foxp3蛋白的表達；通過免疫熒光檢測小鼠黑色素瘤細胞中Foxp3分子的表達；檢測干擾Foxp3后免疫抑制相關分子表達量的變化及對CD4~+CD25-T淋巴細胞增殖能力的影響。結(jié)果：證實在小鼠黑色素瘤細胞中存在Foxp3的mRNA轉(zhuǎn)錄和分子表達；免疫熒光顯示Foxp3分子定位于黑色素瘤細胞的胞核及核周部位。通過Foxp3siRNA的轉(zhuǎn)染，可實現(xiàn)對B16細胞Foxp3表達的沉默，可下調(diào)腫瘤細胞對CD4~+CD25-T淋巴細胞增殖抑制的能力，并且下調(diào)TGF-β1、TGF-β2和IL-10等細胞因子的表達，尤其是TGF-β2的表達。結(jié)論：小鼠B16細胞表達Foxp3，利用RNA干擾技術(shù)可抑制小鼠黑色素瘤細胞靶基因Foxp3的表達，并對CD4~+CD25-T淋巴細胞增殖抑制的能力減弱，同時減弱抑制性細胞因子的分泌。 綜上所述，腫瘤細胞所引起的CD4~+CD25~+Treg細胞產(chǎn)生及功能增強是腫瘤逃避免疫監(jiān)視機制之一，而黑素瘤細胞Foxp3的表達可能成為黑素瘤腫瘤微環(huán)境抑制自身免疫系統(tǒng)的一種新機制。因此深入研究Foxp3在黑素瘤細胞中的作用機制將為黑素瘤的免疫治療提供新靶標和新的治療策略。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.5

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本文編號：1995721