脂氧素A4通过调节性B细胞在抗肿瘤免疫中的作用研究

发布时间：2018-03-01 03:08

本文关键词： 脂氧素A4 调节性B细胞白介素10 调节性T细胞　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：脂氧素(LXA4)在炎症的调控中发挥着重要作用。LXA4同样具有抗肿瘤作用。我们实验室先前的研究发现,LXA4能通过重塑肿瘤微环境和抗肿瘤血管生成来抑制肝癌肿瘤细胞的生长,但是其免疫学机制仍不是十分清楚。调节性B细胞,是一种能分泌IL-10的B细胞,已被证明在炎症、自身免疫性疾病和肿瘤的调控中发挥重要作用。最新的研究表明人和小鼠的B细胞上都有LXA4受体的表达。本研究旨在通过研究LXA4对调节性B细胞的作用来阐明其抑制肿瘤生长的免疫学机制。方法：(1)将不同的肿瘤细胞系通过皮下接种至小鼠,随机分为两组,其中一组腹腔注射LXA4类似物BML-111(1mg/kg)进行治疗,观察并记录肿瘤生长情况。此外,用CFSE标记小鼠肝癌H22细胞,培养24h后,检测LXA4对细胞生长的影响。(2)为寻找LXA4抑制肿瘤的机制,将肝癌H22细胞接种至SCID小鼠,并分为四组：对照组、LXA4治疗组、CD3+T细胞过继转移组、CD3+T细胞过继转移+LXA4治疗组,观察并记录肿瘤生长的情况。(3)为探讨LXA4对调节性B细胞的作用,体外条件下,分离小鼠脾脏B细胞,培养液中加入或不加LXA4,并在PMA、离子霉素、LPS和莫能菌素四种因子的刺激下,5h后通过流式细胞术检测LXA4对调节性B细胞生成的影响。体内条件下,将LXA4治疗的肿瘤小鼠的脾脏取出,比较治疗组和对照组调节性B细胞的数量。为探讨LXA4对B细胞其他功能的作用,通过CFSE标记、流式细胞术和HE染色的方法检测LXA4对B细胞增殖、浆细胞分化以及生发中心结构的影响。(4)将裸鼠皮下接种H22细胞,分为两组,其中一组每两天用LXA4治疗一次,观察并绘制肿瘤生长曲线,并通过流式细胞术和ELISA法检测小鼠脾脏调节性B细胞的数量及外周血、脾脏组织内炎症因子IL-6.TNF-α和IL-1β的蛋白表达情况。(5)将纯化的CD4+T细胞标记CFSE,检测LXA4对其增殖的影响；将纯化的CD8+T细胞用PMA和离子霉素刺激,检测LXA4对其分泌IFN-γ能力的影响。(6)将H22细胞皮下接种至Balb/c小鼠,分为两组,其中一组每两天用LXA4治疗一次,第15天取脾脏、接种肿瘤侧引流淋巴结并分离肿瘤组织内浸润的淋巴细胞,通过流式细胞术检测调节性T细胞的数量和CD8+T细胞的杀伤能力。并通过免疫组织化学的方法检测肿瘤组织内浸润的T细胞的数量。(7)通过磁珠分选的方法将CD4+CD25-T细胞纯化出来后,在刺激性抗体CD3和CD28以及细胞因子TGF-β和IL-2的作用下,72h后检测LXA4对诱导生成的调节性T细胞数量的影响。另外,将纯化出来的CD4+T细胞与B细胞按照1：1的比例在CD3和LPS的作用下共培养,72h后检测LXA4能否通过B细胞影响诱导的调节性T细胞的生成。(8)为寻找LXA4影响调节性B细胞的分子机制,将LXA4治疗小鼠的脾脏细胞或纯化的B细胞提取蛋白质,并通过Western Blot的方法检测转录因子ERK和STAT3的磷酸化水平。此外,体外诱导调节性B细胞并加入ERK或STAT3的磷酸化抑制剂,5h后检测调节性B细胞生成的情况。结果：(1)LXA4类似物BML-111对多种不同的肿瘤细胞系都有抑制其在体内生长的作用。此外,LXA4对肿瘤的生长没有直接的影响。(2)与对照组相比,LXA4能够促进SCID小鼠的肿瘤生长；将CD3+T细胞过继回输至SCID小鼠体内后,增强了抗肿瘤的效应,但是LXA4同样也不影响肿瘤的生长。(3)LXA4在体外和体内都能明显抑制调节性B细胞的生成,但是LXA4不影响B细胞的增殖、生发中心的结构以及浆细胞的分化。(4)在裸鼠中,LXA4能明显抑制调节性B细胞的生成,同时降低了炎症因子IL-6、TNF-α和IL-1β的蛋白表达,但是肿瘤在裸鼠中的生长并没有受到影响。(5)LXA4不影响CD4+T细胞的增殖以及CD8+T细胞分泌IFN-γ的能力。(6)LXA4抑制了引流淋巴结和肿瘤组织内的调节性T细胞的数量,并增强了CD8+T细胞的杀伤能力,但是对脾脏中的这两种细胞没有影响；免疫组化的结果显示LXA4增强了CD3+T细胞浸润肿瘤的能力。(7)LXA4不能通过直接作用影响调节性T细胞的生成,但是可以通过B细胞来抑制这种细胞的数量。(8)LXA4能有效地抑制B细胞而不是脾脏细胞中转录因子ERK和STAT3的磷酸化水平。加入ERK或STAT3的磷酸化抑制剂后能有效地抑制调节性B细胞的生成,说明这两种转录因子的磷酸化与调节性B细胞的生成密切相关。结论：LXA4能有效地抑制多种肿瘤细胞的生长；LXA4对肿瘤细胞没有直接作用,而是通过对调节性B细胞的抑制从而增强了抗肿瘤效应；LXA4通过抑制调节性B细胞的生成从而减少了引流淋巴结和肿瘤组织内调节性T细胞的数量并增强了CD8+T细胞的杀伤能力；LXA4通过降低B细胞内转录因子ERK和STAT3的磷酸化水平来抑制调节性B细胞的生成。
[Abstract]:Objective: lipoxin (LXA4) in the regulation of inflammation plays an important role in.LXA4 also has anti-tumor effect. Previous studies have found that our laboratory, LXA4 can reshape the tumor microenvironment and anti angiogenesis to inhibit the growth of tumor cells, but the immune mechanism is not very clear regulatory B. Cell is a IL-10 secreted by B cells, has been shown to play an important role in the regulation of inflammation, autoimmune diseases and tumors. Recent studies show that human and mouse B cells have the expression of LXA4 receptor. This study aims at through the study of LXA4 on regulatory B cells to clarify the immunological mechanism of inhibiting tumor growth.
Methods: (1) the different tumor cell lines by subcutaneous inoculation into mice, were randomly divided into two groups, one group received intraperitoneal injection of LXA4 analogue BML-111 (1mg/kg) treatment, observe and record the growth of tumors. In addition, using mouse hepatoma H22 cell marker CFSE, 24h after culture, to study the effect of LXA4 on cell growth. (2) to find the mechanism of LXA4 inhibiting tumor, the hepatocellular carcinoma H22 cells inoculated to SCID mice were divided into four groups: control group, LXA4 treatment group, CD3+T cell adoptive transfer CD3+T cells adoptive transfer group, +LXA4 treatment group, observe and record the tumor growth. (3) to investigate the effect of LXA4 on regulatory B cells, in vitro, isolated from mouse spleen B cells, cultured in the presence or absence of LXA4, and in PMA, ionomycin, LPS and monensin four factors under the stimulation of 5h cells by flow cytometry to detect the LXA4 on the generation of regulatory B cells the shadow Ring. In vivo, the treatment of LXA4 tumor in mice spleen removed, the number of treatment group compared with control group of regulatory B cells. To investigate the effect of LXA4 on B cells of other functions, through the method of CFSE labeling, flow cytometry and HE staining of LXA4 on the proliferation of B cells and plasma cell differentiation effect of germinal center structure. (4) the nude mice were inoculated with H22 cells, divided into two groups, one group every two days of treatment with LXA4 at a time, to observe and draw tumor growth curve, and by mouse spleen cells was detected by flow cytometry and ELISA regulatory B cells in peripheral blood and the number of the expression of inflammatory cytokines in spleen tissue, IL-6.TNF- alpha and IL-1 beta protein. (5) the CD4+T cell marker CFSE purification, detection effect of LXA4 on cell proliferation; purified CD8+T cell stimulated by PMA and ionomycin, affect the detection of LXA4 IFN- secretion on the ability of the H22 (6). Cells were inoculated subcutaneously into Balb/c mice, divided into two groups, one group by LXA4 every two days for a fifteenth day from spleen inoculation side of tumor draining lymph node and tumor infiltrating lymphocytes isolated from tissues of regulatory T cells by flow cytometry and the number of CD8+T cell killing ability. And the invasion of T cells by immunohistochemistry in tumor tissue. (7) through the method of sorted CD4+CD25-T cells were purified, CD3 and CD28 in stimulating antibody and cytokine TGF- beta and IL-2, to study the effect of LXA4 on the number of regulatory T cells induced by 72h. In addition, the purified CD4+T cells and B cells according to the ratio of 1:1 in CD3 and LPS under the effect of co culture, 72h can detect LXA4 regulatory T cells generated by B cells induced by LXA4. (8) for effect The molecular mechanism of regulatory B cells, LXA4 treated mice spleen cells or purified B cell protein was extracted by Western method, and the detection of Blot transcription factor ERK and phosphorylation of STAT3. In addition, phosphorylation inhibitor in vitro induction of regulatory B cells with ERK or STAT3, detection of regulatory B cells the generation of 5h.
Results: (1) tumor cell line LXA4 analogue BML-111 on different have inhibited the in vivo growth. In addition, LXA4 on tumor growth has no direct effect. (2) compared with the control group, LXA4 SCID can promote tumor growth in mice; CD3+T cells adoptive infusion into SCID mice in vivo, enhanced anti-tumor effect, but LXA4 also did not affect tumor growth. (3) LXA4 can significantly inhibit the generation of regulatory B cells in vitro and in vivo, but LXA4 does not affect the proliferation of B cells in germinal center structure and plasma cell differentiation in nude mice (4). In LXA4 can obviously inhibit the generation of regulatory B cells, and reduce the inflammatory factor IL-6, expression of TNF- alpha and IL-1 beta protein, but the tumor growth in nude mice was not affected. (5) the ability of LXA4 does not affect the proliferation of CD4+T cells and CD8+T cells to secrete IFN-. ( 6) LXA4 inhibited the drainage quantity of regulatory T cells in tumor tissue and lymph nodes, and enhanced CD8+T cell killing ability, but has no effect on these two kinds of cells in the spleen; immunohistochemistry results showed that LXA4 enhanced the ability of CD3+T cells to tumor invasion. (7) do not produce the LXA4 regulation T cells by direct effect, but the number can be through B cell to inhibit this cell. (8) LXA4 can effectively inhibit B cells and transcription factor ERK and STAT3 cells in the spleen phosphorylation. Phosphorylation inhibitor with ERK or STAT3 can effectively inhibit the generation of regulatory B cells, indicating that these two transcription factor phosphorylation is closely related with the generation of regulatory B cells.
Conclusion: LXA4 can effectively inhibit tumor cell growth; LXA4 has no direct effect on tumor cells, but through inhibition of regulatory B cells to enhance the anti-tumor effect; LXA4 by inhibiting the generation of regulatory B cells in order to reduce the drainage of lymph node in tumor tissue and the number of regulatory T cells and enhanced the CD8+T cell killing ability; LXA4 by reducing the transcription factor ERK and STAT3 in B cells to inhibit the phosphorylation of generation of regulatory B cells.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.2

【相似文献】