本文關(guān)鍵詞：microRNAs在HIFU增強(qiáng)小鼠抗黑色素瘤免疫中的作用及機(jī)制探討　出處：《重慶醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HIFU 黑色素瘤 抗腫瘤免疫 microRNA 共刺激分子

【摘要】：研究背景和目的高強(qiáng)度聚焦超聲(HIFU)是一種新興的治療實(shí)體腫瘤的非侵入性技術(shù)。進(jìn)一步的研究發(fā)現(xiàn),HIFU不僅能夠破壞原發(fā)腫瘤、減少遠(yuǎn)處轉(zhuǎn)移,還能增強(qiáng)機(jī)體抗腫瘤免疫。但是,其機(jī)制尚未闡明。microRNAs(miRNAs)是一類內(nèi)源性非編碼RNA,通過與目標(biāo)mRNA的3'UTR序列互補(bǔ)配對,調(diào)控靶基因的表達(dá)或翻譯,參與生物體的各種生命活動。越來越多的證據(jù)表明,miRNAs參與免疫細(xì)胞的分化、增殖及激活過程,在機(jī)體的免疫應(yīng)答與免疫耐受中均起著十分重要的作用。因此,我們假設(shè)miRNAs參與HIFU增強(qiáng)抗腫瘤免疫的作用,并基于文獻(xiàn)篩選出8個與免疫密切相關(guān)的miRNAs作為研究對象,包括miR-34,miR-106a,miR-126a, miR-134,miR-155, miR-181a,miR-221和miR-222；構(gòu)建HIFU輻照黑色素瘤的小鼠模型,在驗(yàn)證HIFU增強(qiáng)機(jī)體抗腫瘤免疫后,通過qRT-PCR,生物信息學(xué)、熒光素酶報告試驗(yàn)等多種方法篩選、鑒定HIFU處理引起的脾臟及黑色素瘤組織或細(xì)胞中差異表達(dá)的miRNAs,分別探討它們在HIFU增強(qiáng)小鼠抗黑色素瘤免疫中的作用及機(jī)制,為進(jìn)一步闡明HIFU增強(qiáng)機(jī)體抗腫瘤免疫的機(jī)制和促進(jìn)HIFU在黑色素瘤的臨床治療中的應(yīng)用提供實(shí)驗(yàn)和理論依據(jù)。實(shí)驗(yàn)方法1.構(gòu)建HIFU輻照黑色素瘤的小鼠模型及觀察HIFU增強(qiáng)機(jī)體抗腫瘤免疫的作用1)構(gòu)建C57BL/6J小鼠黑色素瘤B16的皮下移植瘤模型,隨機(jī)分HIFU組與假照組,HIFU組用9.3 MHz、4.5 W的HIFU腫瘤治療系統(tǒng)進(jìn)行治療,假照組進(jìn)行假照處理,后續(xù)處理如下：(1)取輻照后即刻的腫瘤組織,包埋、切片及HE染色,觀察輻照靶區(qū)消融效果；(2)輻照7天后,尾靜脈注射B16單細(xì)胞懸液200u1(1×106個細(xì)胞)／鼠,繼續(xù)飼養(yǎng)至自然死亡。期間,監(jiān)測瘤體體積；死后計(jì)數(shù)肺部腫瘤轉(zhuǎn)移結(jié)節(jié)及計(jì)算累積生存率；(3)輻照后14天,用qRT-PCR檢測外周血中黑色素瘤細(xì)胞標(biāo)志物MAGE和Melan-A的mRNA,ELISA檢測小鼠血清中的IFN-γ及TNF-α。2)將HIFU組與假照組小鼠的脾淋巴細(xì)胞分別與B16細(xì)胞共培養(yǎng),采用細(xì)胞毒性試驗(yàn)檢測脾淋巴細(xì)胞對B16細(xì)胞的殺傷活性,ELISA檢測共培養(yǎng)上清中的IFN-γ及TNF-α。2.HIFU引起的脾組織中差異表達(dá)的microRNAs的篩選及靶基因鑒定1) qRT-PCR檢測HIFU組與假照組的脾組織中的差異miRNAs。2)用生物信息學(xué)方法預(yù)測差異miRNAs的潛在靶基因。3) RT-PCR及Western blot驗(yàn)證脾組織中miRNAs潛在靶基因的表達(dá)。4)構(gòu)建熒光素酶報告質(zhì)粒,驗(yàn)證差異miRNAs與潛在靶基因之間的直接調(diào)控關(guān)系。5)將差異miRNAs的mimics轉(zhuǎn)染脾淋巴細(xì)胞,用RT-PCR及Western blot檢測其靶基因的變化,以進(jìn)一步確認(rèn)其對靶基因表達(dá)的調(diào)控。3.HIFU引起的黑色素瘤組織及細(xì)胞中差異表達(dá)的microRNAs的篩選、靶基因鑒定及其在HIFU增強(qiáng)抗黑色素瘤免疫中的作用及機(jī)制探討1) qRT-PCR檢測HIFU組與假照組腫瘤組織中的差異miRNAs。2)用生物信息學(xué)方法預(yù)測差異miRNAs的潛在靶基因,通過RT-PCR及Western blot驗(yàn)證腫瘤組織中靶基因的表達(dá)。3)構(gòu)建熒光素酶報告質(zhì)粒,驗(yàn)證差異miRNAs對潛在靶基因的直接調(diào)控作用。4)再將差異miRNAs的]mimics轉(zhuǎn)染B16細(xì)胞,RT-PCR及Western blot檢測其靶基因的變化,以進(jìn)一步確認(rèn)差異其對靶基因表達(dá)的調(diào)控。5)采用HIFU腫瘤治療系統(tǒng)輻照B16細(xì)胞：(1)檢測輻照后B16細(xì)胞中差異表達(dá)miRNA及靶基因的水平。(2)共培養(yǎng)輻照后的B16細(xì)胞與B16細(xì)胞預(yù)激活的脾淋巴細(xì)胞,采用細(xì)胞毒試驗(yàn)檢測脾淋巴細(xì)胞對B16細(xì)胞的殺傷活性,ELISA檢測共培養(yǎng)上清中的IFN-y及TNF-α。6)用siRNA干擾B16細(xì)胞中差異miRNA的靶基因表達(dá),再進(jìn)行HIFU輻照；輻照后的B16細(xì)胞與激活的脾淋巴細(xì)胞共培養(yǎng),細(xì)胞毒性試驗(yàn)檢測脾淋巴細(xì)胞對B16細(xì)胞的殺傷活性。實(shí)驗(yàn)結(jié)果1.HIFU能有效治療黑色素瘤,并提高機(jī)體抗黑色素瘤的免疫力。1)HIFU輻照立即引起靶區(qū)腫瘤組織發(fā)生凝固性壞死；輻照11天后,HIFU組瘤體體積開始小于假照組,且隨飼養(yǎng)時間延長,其統(tǒng)計(jì)學(xué)差異更加明顯；HIFU組肺部腫瘤轉(zhuǎn)移結(jié)節(jié)明顯少于假照組(P0.01),累積生存率明顯高于假照組(P0.01)。2)輻照14天后,HIFU組外周血中黑色素瘤細(xì)胞標(biāo)志物MAGE和Melan-A的mRNA水平明顯降低(P0.01)；免疫效應(yīng)因子IFN-y的水平增加(P0.05),TNF-α也呈增加趨勢(P0.05)。3)HIFU輻照增強(qiáng)脾淋巴細(xì)胞對B16細(xì)胞的殺傷活性(P0.05),增加脾淋巴細(xì)胞與B16細(xì)胞共培養(yǎng)上清中的IFN-γ (P0.05)及TNF-α(P0.05)的水平。2. HIFU輻照能夠下調(diào)脾臟中miR-181a的表達(dá),并因此上調(diào)免疫共刺激分子ICAM-1的表達(dá)。1)HIFU組小鼠脾組織中miR-181a的水平明顯低于對照組(P0.01)。2)用生物信息學(xué)方法預(yù)測出共刺激分子ICAM-1是miR-181a的潛在靶基因。3)HIFU組脾組織中ICAM-1的mRNA(P0.01)和蛋白質(zhì)(P0.01)均明顯高于對照組。4) miR-181a mimics顯著抑制LUC-ICAM-1-3'UTR報告質(zhì)粒中熒光素酶的活性(P0.01),并下調(diào)脾淋巴細(xì)胞中ICAM-1的mRNA(P0.05)和蛋白質(zhì)的表達(dá)(P0.01)。證明miR-181a負(fù)調(diào)控ICAM-1。3. HIFU輻照通過抑制B16黑色素瘤細(xì)胞中miR-134對CD86表達(dá)的負(fù)向調(diào)控作用,從而增強(qiáng)抗黑色素瘤的免疫效應(yīng)。1)HIFU組腫瘤組織中miR-134(P0.01)和miR-222 (P0.01)明顯低于對照組。2)用生物信息學(xué)方法預(yù)測出CD86和ICAM-1分別是miR-134和miR-222的潛在靶基因。3)HIFU組腫瘤組織中：(1)CD86的mRNA(P0.05)和蛋白質(zhì)(P0.05)高于對照組；(2) ICAM-1的mRNA (P.01)和蛋白質(zhì)(P0.01)明顯高于對照組。4) miR-134 mimics顯著抑制LUC-CD86-3'UTR報告質(zhì)粒的熒光素酶活性,并下調(diào)B16細(xì)胞中CD86的mRNA (P.05)和蛋白質(zhì)(P0.05)；但,miR-222不能抑制LUC-ICAM-1-3'UTR報告質(zhì)粒的熒光素酶活性。提示miR-134負(fù)調(diào)控CD86, miR-222對ICAM-1無調(diào)控作用。5)HIFU輻照的B16細(xì)胞中miR-134的水平降低,CD86的水平增加,且miR-134與CD86呈負(fù)相關(guān)。6)將HIFU輻照后的B16細(xì)胞與正常脾淋巴細(xì)胞(已用B16細(xì)胞激活)進(jìn)行體外共培養(yǎng)24 h和48 h后：(1)HIFU組B16細(xì)胞存活率明顯低于對照組(P0.05);(2) HIFU組共培養(yǎng)上清中IFN-γ和TNF-α高于對照組,且隨著輻照時間的增加而增加(P0.05)。7) CD86 siRNA增加B16細(xì)胞的存活率(P0.01)。提示CD86的下調(diào)可抑制脾淋巴細(xì)胞對B16細(xì)胞的殺傷活性,結(jié)論1. HIFU輻照能夠使靶區(qū)腫瘤組織發(fā)生凝固型壞死、抑制腫瘤生長和遠(yuǎn)處轉(zhuǎn)移,提高宿主生存率,增強(qiáng)機(jī)體抗腫瘤免疫。2. HIFU輻照通過抑制腫瘤組織中miR-134對共刺激分子CD86的負(fù)調(diào)控作用,從而增強(qiáng)其抗腫瘤免疫。3. HIFU輻照可以抑制脾組織中miR-181a對共刺激分子ICAM-1的負(fù)調(diào)控作用,這可能是HIFU輻照增強(qiáng)抗腫瘤免疫的機(jī)制之一。本研究為闡明HIFU輻照增強(qiáng)機(jī)體抗腫瘤免疫功能的作用機(jī)制提供了新的思路和積累了實(shí)驗(yàn)依據(jù),并有利于促進(jìn)HIFU技術(shù)在黑色素瘤及其它實(shí)體瘤的臨床治療中的應(yīng)用。
[Abstract]:Background and objective of high intensity focused ultrasound (HIFU) non invasive technique is a new treatment of solid tumors. Further study found that HIFU can not only destroy the primary tumor, reduce distant metastasis, but also enhance the anti-tumor immunity of the body. However, its mechanism has not been clarified.MicroRNAs (miRNAs) is a kind of endogenous non by encoding RNA, 3'UTR sequence and target mRNA complementary pairs, regulate target gene expression or translation, participate in various life activities of the organism. More and more evidence that miRNAs is involved in the differentiation of immune cells, proliferation and activation process, plays an important role in immune response and immune tolerance in the body so. We assume that miRNAs is involved in HIFU, enhance anti-tumor immunity, and literature and selected 8 closely related with immune miRNAs as the research object, based on miR-106a, including miR-34, miR-126a, miR- 134, miR-155, miR-181a, miR-221 and miR-222; to establish an experimental model of HIFU irradiated melanoma, enhance the anti-tumor immunity of the body in the verification of HIFU, through qRT-PCR, bioinformatics, luciferase reporter test methods such as screening, identification of HIFU induced spleen and melanoma cells in different tissues or expression of miRNAs. Of them to enhance the effect and mechanism of anti melanoma immunity in mice HIFU, to further elucidate the mechanism underlying HIFU enhanced anti-tumor immunity and promote the clinical application of HIFU in the treatment of melanoma in to provide experimental and theoretical basis. 1. experimental methods to construct HIFU irradiated melanoma mice model and observation of HIFU enhanced anti tumor immune function 1) subcutaneous transplantation tumor model of C57BL/6J mouse melanoma B16, were randomly divided into HIFU group and sham group, HIFU group with 9.3 MHz, 4.5 W HIFU tumor treatment Treatment system for treatment, the sham group were sham treatment, follow-up treatment is as follows: (1) tumor tissue, immediately after irradiation the embedding, sectioning and HE staining, observe the irradiation target ablation effect; (2) 7 days after irradiation, intravenous injection of B16 cell suspension (1 200u1 * 106 cells / rat), continue to raise to natural death. During the monitoring of tumor volume; cumulative survival rate was calculated and the count of lung cancer metastatic nodules after death; (3) 14 days after irradiation, detected by qRT-PCR in peripheral blood of melanoma cell markers MAGE and Melan-A mRNA, ELISA detection in the serum of mice IFN- gamma and TNF- alpha.2) HIFU group and sham group mice spleen lymphocytes were co cultured with B16 cells, the cytotoxicity of spleen lymphocytes to detect the cytotoxic activity of B16 cells, expression of IFN- and TNF- in the supernatant of gamma alpha.2.HIFU induced splenic tissue in microR co cultured with ELISA detection NAs screening and identification of target gene 1) qRT-PCR detected differences in HIFU group and sham group of spleen tissue in miRNAs.2) potential target gene.3 method with bioinformatics prediction difference miRNAs) expression of.4 miRNAs and Western blot RT-PCR target genes were verified in spleen tissue) to construct luciferase reporter plasmid,.5 control to validate the relationship between the differences of miRNAs and potential target genes) will be the difference of miRNAs mimics transfection of spleen lymphocytes, the change of target gene was detected by RT-PCR and Western blot, to further confirm the screening of differentially expressed melanoma tissues and cells on the regulation of.3.HIFU gene expression induced by microRNAs in the target gene and its identification effect and mechanism of anti melanoma immunity of 1 in HIFU group and HIFU qRT-PCR) enhanced detection of sham group differences in tumor tissue and miRNAs.2) method to forecast the difference of miRNAs by Bioinformatics The potential target gene expression by.3 RT-PCR and Western blot to verify the tumor target gene) to construct luciferase reporter plasmid, direct regulation of.4 miRNAs to verify the differences in potential target genes) then the difference of miRNAs]mimics was transfected into B16 cells, RT-PCR and Western blot to test the change of target gene, to further confirm the regulation of.5 the difference of the expression of target gene) with HIFU tumor treatment system of irradiated B16 cells: (1) miRNA and the target gene expression level in B16 cells after radiation detection. (2) B16 cells were co cultured with B16 cells after pre irradiation activated spleen lymphocytes, the cytotoxicity test of lymphocyte on B16 the cytotoxic activity of IFN-y and TNF- alpha.6 in the supernatant of ELISA co culture detection) gene expression differences of B16 in miRNA cells by siRNA interference, and then HIFU irradiation; B16 cells irradiated with activation Co cultured spleen cells, the killing activity of spleen lymphocyte cytotoxicity assay on B16 cells. The experimental results of 1.HIFU can be effective in the treatment of melanoma, and improve the anti melanoma immunity.1) HIFU irradiation immediately caused tumor tissue necrosis; 11 days after irradiation, HIFU group tumor volume less than the false control group, and with the feeding time, the difference is more obvious; the HIFU group of lung metastasis nodules was significantly less than the sham group (P0.01), the cumulative survival rate was significantly higher than that of sham group (P0.01).2) 14 days after irradiation, the level of mRNA melanoma cell markers MAGE and Melan-A in the peripheral blood of patients with HIFU decreased significantly (P0.01); immune effect factor increased the level of IFN-y (P0.05), alpha TNF- also showed an increasing trend (P0.05).3) HIFU irradiation enhanced the killing activity of spleen lymphocytes on B16 cells (P0.05), increased spleen lymphocytes and B1 IFN- in the supernatant of cultured 6 cells (P0.05) and TNF- alpha (P0.05) expression level of.2. HIFU irradiation could down regulate the miR-181a in spleen, and therefore increase the expression of costimulatory molecules ICAM-1 expression.1) miR-181a spleen tissue of mice in HIFU group was significantly lower than the control group (P0.01).2) by Bioinformatics Method to predict the costimulatory molecule ICAM-1 is a potential target gene of.3 miR-181a ICAM-1) spleen tissues of HIFU group mRNA (P0.01) and protein (P0.01) were significantly higher than the control group.4 miR-181a mimics LUC-ICAM-1-3'UTR) significantly inhibited the activity of Luciferase Report plasmid (P0.01), and down-regulation of ICAM-1 in spleen lymphocytes (P0.05 mRNA) expression and protein (P0.01). MiR-181a HIFU proved that the negative regulation of ICAM-1.3. irradiation to regulation by inhibiting miR-134 B16 melanoma cells on the expression of CD86, so as to enhance the immune effect of anti melanoma.1 H) MiR-134 IFU group in tumor tissue (P0.01) and miR-222 (P0.01) was significantly lower than the control group.2) were predicted by Bioinformatics Method CD86 and ICAM-1 are the potential target gene of.3 miR-134 and miR-222 HIFU) group in tumor tissue: (1) CD86 mRNA (P0.05) and protein (P0.05) was higher than the control group; (2) ICAM-1 mRNA (P.01) and protein (P0.01) was significantly higher than the control group.4 miR-134 mimics LUC-CD86-3'UTR) significantly inhibited reporter plasmid luciferase activity, and down-regulation of CD86 B16 cells in mRNA (P.05) and protein (P0.05); however, miR-222 did not inhibit LUC-ICAM-1-3'UTR reporter plasmid luciferase activity indicated that miR-134 negative. The regulation of CD86, miR-222 ICAM-1 miR-134) the role of.5 in regulation of HIFU irradiated B16 cells in the lower level, the level of CD86 increased, and miR-134 was negatively correlated with CD86.6) HIFU irradiated B16 cells and normal lymphocytes (have The activation of B16 cells) were co cultured with 24 h and 48 h (1): HIFU group B16 the cell survival rate was significantly lower than the control group (P0.05); (2) HIFU group in culture supernatant of IFN- gamma and alpha TNF- higher than the control group, and increased with the increase of irradiation time (P0.05).7) CD86 siRNA increased the survival rate of B16 cells (P0.01). It is suggested that downregulation of CD86 inhibits lymphocyte on the cytotoxic activity of B16 cells. Conclusion 1. HIFU irradiation can make tumor tissue coagulation necrosis, inhibit tumor growth and metastasis, improve the survival rate of the host, to enhance the negative regulation of miR-134 anti-tumor immune.2. HIFU irradiation by inhibiting the tumor tissue of the costimulatory molecule CD86, thereby enhancing the antitumor immunity of.3. HIFU can inhibit miR-181a irradiation and negatively regulate spleen tissues of costimulatory molecule ICAM-1, which is probably the HIFU irradiation enhanced the antitumor immunity of machine One of the mechanisms. This study provides new ideas and experimental evidence for elucidating the mechanism of HIFU irradiation enhancing the body's anti-tumor immune function, and helps to promote the application of HIFU technology in the clinical treatment of melanoma and other solid tumors.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R739.5

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5 記者 張e，

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