microRNAs在HIFU增强小鼠抗黑色素瘤免疫中的作用及机制探讨

发布时间：2018-01-06 20:05

本文关键词：microRNAs在HIFU增强小鼠抗黑色素瘤免疫中的作用及机制探讨　出处：《重庆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的高强度聚焦超声(HIFU)是一种新兴的治疗实体肿瘤的非侵入性技术。进一步的研究发现,HIFU不仅能够破坏原发肿瘤、减少远处转移,还能增强机体抗肿瘤免疫。但是,其机制尚未阐明。microRNAs(miRNAs)是一类内源性非编码RNA,通过与目标mRNA的3'UTR序列互补配对,调控靶基因的表达或翻译,参与生物体的各种生命活动。越来越多的证据表明,miRNAs参与免疫细胞的分化、增殖及激活过程,在机体的免疫应答与免疫耐受中均起着十分重要的作用。因此,我们假设miRNAs参与HIFU增强抗肿瘤免疫的作用,并基于文献筛选出8个与免疫密切相关的miRNAs作为研究对象,包括miR-34,miR-106a,miR-126a, miR-134,miR-155, miR-181a,miR-221和miR-222；构建HIFU辐照黑色素瘤的小鼠模型,在验证HIFU增强机体抗肿瘤免疫后,通过qRT-PCR,生物信息学、荧光素酶报告试验等多种方法筛选、鉴定HIFU处理引起的脾脏及黑色素瘤组织或细胞中差异表达的miRNAs,分别探讨它们在HIFU增强小鼠抗黑色素瘤免疫中的作用及机制,为进一步阐明HIFU增强机体抗肿瘤免疫的机制和促进HIFU在黑色素瘤的临床治疗中的应用提供实验和理论依据。实验方法1.构建HIFU辐照黑色素瘤的小鼠模型及观察HIFU增强机体抗肿瘤免疫的作用1)构建C57BL/6J小鼠黑色素瘤B16的皮下移植瘤模型,随机分HIFU组与假照组,HIFU组用9.3 MHz、4.5 W的HIFU肿瘤治疗系统进行治疗,假照组进行假照处理,后续处理如下：(1)取辐照后即刻的肿瘤组织,包埋、切片及HE染色,观察辐照靶区消融效果；(2)辐照7天后,尾静脉注射B16单细胞悬液200u1(1×106个细胞)／鼠,继续饲养至自然死亡。期间,监测瘤体体积；死后计数肺部肿瘤转移结节及计算累积生存率；(3)辐照后14天,用qRT-PCR检测外周血中黑色素瘤细胞标志物MAGE和Melan-A的mRNA,ELISA检测小鼠血清中的IFN-γ及TNF-α。2)将HIFU组与假照组小鼠的脾淋巴细胞分别与B16细胞共培养,采用细胞毒性试验检测脾淋巴细胞对B16细胞的杀伤活性,ELISA检测共培养上清中的IFN-γ及TNF-α。2.HIFU引起的脾组织中差异表达的microRNAs的筛选及靶基因鉴定1) qRT-PCR检测HIFU组与假照组的脾组织中的差异miRNAs。2)用生物信息学方法预测差异miRNAs的潜在靶基因。3) RT-PCR及Western blot验证脾组织中miRNAs潜在靶基因的表达。4)构建荧光素酶报告质粒,验证差异miRNAs与潜在靶基因之间的直接调控关系。5)将差异miRNAs的mimics转染脾淋巴细胞,用RT-PCR及Western blot检测其靶基因的变化,以进一步确认其对靶基因表达的调控。3.HIFU引起的黑色素瘤组织及细胞中差异表达的microRNAs的筛选、靶基因鉴定及其在HIFU增强抗黑色素瘤免疫中的作用及机制探讨1) qRT-PCR检测HIFU组与假照组肿瘤组织中的差异miRNAs。2)用生物信息学方法预测差异miRNAs的潜在靶基因,通过RT-PCR及Western blot验证肿瘤组织中靶基因的表达。3)构建荧光素酶报告质粒,验证差异miRNAs对潜在靶基因的直接调控作用。4)再将差异miRNAs的]mimics转染B16细胞,RT-PCR及Western blot检测其靶基因的变化,以进一步确认差异其对靶基因表达的调控。5)采用HIFU肿瘤治疗系统辐照B16细胞：(1)检测辐照后B16细胞中差异表达miRNA及靶基因的水平。(2)共培养辐照后的B16细胞与B16细胞预激活的脾淋巴细胞,采用细胞毒试验检测脾淋巴细胞对B16细胞的杀伤活性,ELISA检测共培养上清中的IFN-y及TNF-α。6)用siRNA干扰B16细胞中差异miRNA的靶基因表达,再进行HIFU辐照；辐照后的B16细胞与激活的脾淋巴细胞共培养,细胞毒性试验检测脾淋巴细胞对B16细胞的杀伤活性。实验结果1.HIFU能有效治疗黑色素瘤,并提高机体抗黑色素瘤的免疫力。1)HIFU辐照立即引起靶区肿瘤组织发生凝固性坏死；辐照11天后,HIFU组瘤体体积开始小于假照组,且随饲养时间延长,其统计学差异更加明显；HIFU组肺部肿瘤转移结节明显少于假照组(P0.01),累积生存率明显高于假照组(P0.01)。2)辐照14天后,HIFU组外周血中黑色素瘤细胞标志物MAGE和Melan-A的mRNA水平明显降低(P0.01)；免疫效应因子IFN-y的水平增加(P0.05),TNF-α也呈增加趋势(P0.05)。3)HIFU辐照增强脾淋巴细胞对B16细胞的杀伤活性(P0.05),增加脾淋巴细胞与B16细胞共培养上清中的IFN-γ (P0.05)及TNF-α(P0.05)的水平。2. HIFU辐照能够下调脾脏中miR-181a的表达,并因此上调免疫共刺激分子ICAM-1的表达。1)HIFU组小鼠脾组织中miR-181a的水平明显低于对照组(P0.01)。2)用生物信息学方法预测出共刺激分子ICAM-1是miR-181a的潜在靶基因。3)HIFU组脾组织中ICAM-1的mRNA(P0.01)和蛋白质(P0.01)均明显高于对照组。4) miR-181a mimics显著抑制LUC-ICAM-1-3'UTR报告质粒中荧光素酶的活性(P0.01),并下调脾淋巴细胞中ICAM-1的mRNA(P0.05)和蛋白质的表达(P0.01)。证明miR-181a负调控ICAM-1。3. HIFU辐照通过抑制B16黑色素瘤细胞中miR-134对CD86表达的负向调控作用,从而增强抗黑色素瘤的免疫效应。1)HIFU组肿瘤组织中miR-134(P0.01)和miR-222 (P0.01)明显低于对照组。2)用生物信息学方法预测出CD86和ICAM-1分别是miR-134和miR-222的潜在靶基因。3)HIFU组肿瘤组织中：(1)CD86的mRNA(P0.05)和蛋白质(P0.05)高于对照组；(2) ICAM-1的mRNA (P.01)和蛋白质(P0.01)明显高于对照组。4) miR-134 mimics显著抑制LUC-CD86-3'UTR报告质粒的荧光素酶活性,并下调B16细胞中CD86的mRNA (P.05)和蛋白质(P0.05)；但,miR-222不能抑制LUC-ICAM-1-3'UTR报告质粒的荧光素酶活性。提示miR-134负调控CD86, miR-222对ICAM-1无调控作用。5)HIFU辐照的B16细胞中miR-134的水平降低,CD86的水平增加,且miR-134与CD86呈负相关。6)将HIFU辐照后的B16细胞与正常脾淋巴细胞(已用B16细胞激活)进行体外共培养24 h和48 h后：(1)HIFU组B16细胞存活率明显低于对照组(P0.05);(2) HIFU组共培养上清中IFN-γ和TNF-α高于对照组,且随着辐照时间的增加而增加(P0.05)。7) CD86 siRNA增加B16细胞的存活率(P0.01)。提示CD86的下调可抑制脾淋巴细胞对B16细胞的杀伤活性,结论1. HIFU辐照能够使靶区肿瘤组织发生凝固型坏死、抑制肿瘤生长和远处转移,提高宿主生存率,增强机体抗肿瘤免疫。2. HIFU辐照通过抑制肿瘤组织中miR-134对共刺激分子CD86的负调控作用,从而增强其抗肿瘤免疫。3. HIFU辐照可以抑制脾组织中miR-181a对共刺激分子ICAM-1的负调控作用,这可能是HIFU辐照增强抗肿瘤免疫的机制之一。本研究为阐明HIFU辐照增强机体抗肿瘤免疫功能的作用机制提供了新的思路和积累了实验依据,并有利于促进HIFU技术在黑色素瘤及其它实体瘤的临床治疗中的应用。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.5

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