调节性T细胞在胶质母细胞瘤中的预后作用及分化机制研究

发布时间：2018-08-08 14:04

【摘要】：[目的]胶质母细胞瘤(GBM)是中枢神经系统常见的恶性肿瘤,目前综合治疗效果不佳,免疫逃逸可能在其恶性进展中发挥了重要作用。调节性T细胞(Treg)可以诱导机体的免疫抑制,在胶质瘤组织中也有浸润,已有研究试图探究Treg对GBM病人的预后作用,但是结论不一,此外,Treg在胶质瘤中的分化机制亦罕有报道。本课题希望首先明确Treg在GBM中的聚集情况,然后深入分析这类免疫抑制细胞对临床转归的预后价值,最后揭示naive CD4+T细胞向Treg分化的可能机制。[方法]在第一部分中,首先采用Western Blot检测了不同级别胶质瘤组织、脑外伤组织中Foxp3蛋白的含量,然后对62例GBM石蜡切片进行了Foxp3、CD8、 p53、MGMT、Ki-67免疫组化染色,镜下计数各指标的表达情况,并对Foxp3和CD8实施了线性相关分析,根据临床病理资料对病人分组,比较不同组间Foxp3CD8等指标的差异。在第二部分中,以Foxp3的中位表达密度为分界点将上述62例病人等分为两组,结合预后资料,针对无进展生存期(PFS)和总生存期(0S)进行了Kaplan-Meier生存分析,采用同样的方法对CD8、Foxp3+/CD8+的预后作用进行了分析,此外,还根据p53、MGMT、Ki-67等病理指标对病人分组,进一步研究了Foxp3、 CD8、Foxp3+/CD8+在各个亚组内的预后价值,最后,纳入所有临床、病理、免疫指标,进行了单因素和多因素分析。在第三部分中,首先,收集了不同级别胶质瘤病人的外周血、脑脊液、肿瘤组织匀浆液,采用FlowCytomix及ELISA的方法检测其中IL-6、IL-10的含量并做比较,再以不同浓度的IL-6、IL-10分别孵育从健康人外周血提取的naiveCD4+T细胞72小时,用流式细胞术检测CD4+细胞中CD25+Foxp3+田胞的比例；其次,从新鲜胶质母细胞瘤标本中分选得到胶质瘤干细胞(GSC)及CD133-细胞,再分别用GSC及上清、CD133-细胞及上清孵育naive CD4+T细胞,检测Treg的比例；最后,通过慢病毒转染健康人巨噬细胞而获得B7-H4阳性巨噬细胞,再分别用这类细胞及未转染B7-H4的对照细胞孵育naive CD4+T细胞,同上检测Treg的比例。[结果]胶质瘤级别越高,肿瘤组织中Foxp3蛋白含量越高,而正常脑组织中几乎无Foxp3表达。Foxp3主要位于细胞核,Foxp3+淋巴细胞在肿瘤标本中呈散在分布,平均密度为8.6个/mm2,CD8+淋巴细胞平均密度为49.6个/mm2,显著高于前者(P0.001),而且这两者之间呈正相关(rs=0.375、P=0.003)。此外,Foxp3表达密度和临床病理特征无关。Foxp3高表达组的中位PFS为9个月,显著低于Foxp3低表达组的中位PFS12个月(P=0.001),两组的中位0S分别为16个月和20个月,Foxp3高表达组也显著短于Foxp3低表达组(P=0.003)。CD8高、低表达的两组间的PFS和OS均无显著性差异。高Foxp3+/CD8+组的中位PFS为9个月,而低Foxp3+/CD8+组为12个月,两者间的差异有统计学意义(P=0.031),两者间的OS的差异尚不够显著。不同病理分型下Foxp3的预后意义也不同,比如,在Ki-67低表达组,Foxp3能够提示不良预后,而在高表达组,却无阳性发现。针对PFS和OS的多因素分析均显示,Foxp3是GBM病人的一个独立预后因素。IL-6在不同级别胶质瘤病人的体液中无明显差异,但在高级别胶质瘤的组织匀浆液中却显著富集,1 ng/ml IL-6处理后的CD4+细胞中Treg的比例就显著高于未经处理的细胞(P 0.05),5 ng/ml IL-6处理后的细胞中Treg的比例显著高于1ng/ml IL-6处理后的细胞(P0.01),而5 ng/ml和10 ng/ml IL-6处理的两组间无显著差异。IL-10在不同级别胶质瘤病人的体液中无明显差异,在高级别胶质瘤的组织匀浆液中呈增多趋势,但无统计学意义,用不同浓度IL-10处理的naive CD4+T细胞中Treg比例无明显差异。GSC及其上清处理组的Treg比例显著高于其余各组,而这两组之间无显著性差异,CD133-肿瘤细胞及其上清处理组与对照组间均无显著性差异。经LV-B7-H4转染的巨噬细胞处理的CD4+T细胞中,Treg比例显著高于由正常培养基、正常巨噬细胞或LV-GFP转染的巨噬细胞处理的其他各组。[结论]Treg在胶质瘤中的浸润程度随肿瘤级别升高而上调,GBM肿瘤组织中Treg和CD8+细胞的数目呈正相关。Treg是GBM病人的一个独立预后因素,肿瘤微环境中Treg越多,病人生存期越短。IL-6、GSC、B7-H4阳性巨噬细胞均可以促进naive CD4+T细胞向Treg的分化。
[Abstract]:[Objective] glioblastoma (GBM) is a common malignant tumor in the central nervous system. The effect of integrated treatment is not good at present. Immune escape may play an important role in its malignant progress. Regulatory T cells (Treg) can induce immune suppression in the body and also have infiltration in glioma tissue. Research has been made to explore Treg to GBM patients. In addition, the differentiation mechanism of Treg in glioma is rarely reported. In this subject, we hope to clarify the aggregation of Treg in GBM, and then analyze the prognostic value of this kind of immunosuppressive cells for clinical outcome, and finally reveal the possible mechanism of naive CD4+T cell to Treg differentiation. [method] in the first part First, Western Blot was used to detect the content of Foxp3 protein in different grades of glioma tissue and brain trauma. Then 62 cases of GBM paraffin section were treated with Foxp3, CD8, p53, MGMT, Ki-67 immunohistochemical staining, and the expression of each index was counted under the microscope. The linear correlation analysis was carried out on Foxp3 and CD8, and the disease was based on the clinicopathological data. In the second part, the 62 patients were divided into two groups with the median expression density of Foxp3 in the second part, and the Kaplan-Meier survival analysis was carried out for the progression free survival (PFS) and the total survival time (0S), and the same method was used for CD8, Foxp3+/CD8+. The post effect was analyzed. In addition, the patients were grouped according to p53, MGMT, Ki-67 and other pathological indexes, and the prognostic value of Foxp3, CD8, Foxp3+/CD8+ in each subgroup was further studied. Finally, all clinical, pathological and immune indexes were included in the analysis of single factor and multiple factors. In the third part, first, the different grade gliomas were collected. The patients' peripheral blood, cerebrospinal fluid and tumor tissue homogenate were used to detect the content of IL-6 and IL-10 by FlowCytomix and ELISA, and then the naiveCD4+T cells extracted from the peripheral blood of healthy people were incubated with IL-6 and IL-10 at different concentrations for 72 hours, and the proportion of CD25+Foxp3+ in CD4+ cells was detected by flow cytometry; secondly, the proportion of CD25+Foxp3+ in the CD4+ cells was detected; secondly, the proportion of CD25+Foxp3+ in the CD4+ cells was detected by flow cytometry. From the fresh glioblastoma specimens, the glioma stem cells (GSC) and CD133- cells were obtained, and the naive CD4+T cells were incubated with GSC and supernatant, CD133- cell and supernatant respectively, and the proportion of Treg was detected. Finally, B7-H4 positive macrophages were obtained by transfection of the healthy human macrophage by lentivirus, and then the cells and the untransfected B were respectively used. The control cells of 7-H4 incubated the naive CD4+T cells, and the ratio of Treg was detected. [results] the higher the level of the glioma, the higher the Foxp3 protein content in the tumor tissue, while the Foxp3 expression of.Foxp3 in the normal brain was mainly located in the nucleus, and the Foxp3+ lymphocytes were scattered in the tumor specimens, with an average density of 8.6 /mm2 and CD8+ lymph thin. The average density of the cell was 49.6 /mm2, significantly higher than the former (P0.001), and there was a positive correlation between the two (rs=0.375, P=0.003). In addition, the Foxp3 expression density and the clinicopathological features were not related to the middle PFS of the high expression group of.Foxp3, which was significantly lower than the median PFS12 month (P=0.001) of the low expression group of Foxp3, and the median 0S of the two groups was 16 months and 20, respectively. The Foxp3 high expression group was also significantly shorter than the Foxp3 low expression group (P=0.003).CD8, and there was no significant difference in PFS and OS between the two groups of low expression. The median PFS of the high Foxp3+/CD8+ group was 9 months, and the low Foxp3+/CD8+ group was 12 months. The difference between the two groups was statistically significant (P= 0.031). The difference between the two groups was not significant. The prognostic significance of Foxp3 is different, for example, in the Ki-67 low expression group, Foxp3 can indicate poor prognosis, but in the high expression group, there is no positive discovery. The multivariate analysis of PFS and OS shows that Foxp3 is an independent prognostic factor of GBM patients,.IL-6 is not significantly different in the body fluids of patients with different levels of glioma, but in the high level gel The proportion of Treg in the CD4+ cells after 1 ng/ml IL-6 treatment was significantly higher than that of untreated cells (P 0.05). The proportion of Treg in the cells treated with 5 ng/ml IL-6 was significantly higher than that of the 1ng/ml IL-6 treated cells (P0.01), but there was no significant difference between the 5 ng/ and 10 groups. There was no significant difference in the body fluid of the patients with glioma at different levels, but there was an increasing trend in the tissue homogenate of high grade glioma, but there was no statistical significance. There was no significant difference in the proportion of Treg in the naive CD4+T cells treated with different concentrations of IL-10. The Treg ratio of.GSC and the supernatant treatment group was significantly higher than that of the other groups, but there was no significant difference between the two groups. There was no significant difference between the CD133- tumor cell and its supernatant treatment group and the control group. In the CD4+T cells treated by LV-B7-H4 transfected macrophages, the proportion of Treg was significantly higher than that of other groups treated by normal medium, normal macrophage or LV-GFP transfected macrophages. [conclusion]Treg in glioma infiltration degree. The number of Treg and CD8+ cells in GBM tumor tissue is positively correlated with the number of CD8+ cells..Treg is an independent prognostic factor of GBM patients. The more Treg in the tumor microenvironment, the shorter the patient's survival time, the.IL-6, GSC, and B7-H4 positive macrophages can promote naive CD4+T cell to differentiate to Treg.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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