TAMs在原发肿瘤及骨转移瘤中表型的观察研究

发布时间：2018-07-03 15:49

本文选题：肿瘤相关巨噬细胞 + 乳腺癌　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景与目的巨噬细胞作为广泛存在于组织、器官中的一类免疫细胞,在机体非特异性免疫反.应中发挥主要作用,具有清除病原体和组织碎片的作用,担负着抵御外来侵袭第一道防线的任务。研究表明,其功能受微环境的制约与调控[1-3]。炎性因子、细菌产物可导致巨噬细胞分化成为M1型的巨噬细胞;M1型的巨噬细胞表现为高表达白细胞介素12(interleukin 12,IL-12),低表达IL-10,参与促炎反应;IL-4、IL-10、IL-13和糖皮质激素等可促进巨噬细胞向M2型的巨噬细胞进行分化;M2型巨噬细胞高表达IL-10但低表达IL-12,在促进免疫耐受中起重要作用[4-6]。肿瘤相关巨噬细胞(tumor associated macrophages,TAMs)是一种浸润入肿瘤组织内存在的巨噬细胞,来源具有多样性,可由外周募集的单核细胞分化而来[7]。文献报道TAMs可与肿瘤细胞发生相互作用,相互影响,TAMs可分泌多种细胞因子,进而调节干细胞微环境,广泛参与肿瘤进程,发挥促瘤血管生成、促瘤细胞侵袭及转移、促瘤免疫耐受等作用[7-9]。在乳腺癌、前列腺癌、胰腺癌等临床病人组织样本中,巨噬细胞标志性分子CD68的表达水平与不良预后正相关[10-12]。乳腺癌作为女性最常见的恶性肿瘤之一,约有8%的患者发生了骨转移,其中高度恶性患者骨转移率甚至可高达69%[13]。乳腺癌脊柱转移患者约占所有骨转移患者的三分之二,大多数乳腺癌脊柱转移是呈溶骨性改变的,可使脊椎骨质量下降,患者发生压缩性骨折概率较高[14-17],容易发生神经损伤甚至截瘫等严重并发症;加之此类患者预期寿命较长,伴随剧烈癌性疼痛,患者承受严重的身心痛苦。有研究报道,乳腺癌中肿瘤相关白细胞的浸润水平很高,且巨噬细胞占其中的多半[18]。TAMs与乳腺癌发生、发展存在密切联系,其浸润程度与肿瘤转移呈正相关,是乳腺癌独立的预后因素[19]。乳腺癌中最常见的是浸润性导管癌,但其病理学机制仍尚未研究清楚。乳腺癌患者原发灶和骨转移灶中肿瘤相关巨噬细胞的差异国内外尚未见相关报道。分析TAMs在乳腺癌发生、发展、转移等过程中的表型变化,或可为肿瘤诊治提供思路和借鉴。鉴于肿瘤进程经历发生、发展多个过程,那么,肿瘤微环境的变化是否介导TAMs表型的转换,从而调控TAMs的功能就成为一个十分重要的问题[20]。本研究利用MMTV-PyMT基因型的小鼠乳腺癌自发模型,模拟乳腺癌发生、发展过程,通过分离、比较不同病程中TAMs的M1型巨噬细胞及M2型巨噬细胞标志性分子的表达,进而分析TAMs的表型变化。筛选临床乳腺癌原发肿瘤及乳腺癌骨转移瘤病灶样本进行组织学染色观察,对其中的肿瘤相关巨噬细胞进行计数,统计学计算分析肿瘤相关巨噬细胞数目及M2型肿瘤相关巨噬细胞所占阳性比例有无差异,分析M2型肿瘤相关巨噬细胞所占阳性比例与发生淋巴结转移、骨转移的相关性。1.实验方法1.1小鼠自发乳腺癌模型中肿瘤相关巨噬细胞的表型检测与分析筛选出MMTV-PyMT基因型的小鼠后,按试剂盒说明书的方法提取小鼠基因组并对该小鼠自发乳腺癌模型进行基因型的鉴定。通过分析不同周龄小鼠肿瘤体积的普遍规律,对肿瘤的进程进行早期与晚期的区分(16周且肿瘤总体积小于100 mm3的肿瘤分为早期组;20周且肿瘤总体积大于800 mm3的肿瘤分为晚期组)。进而分离肿瘤细胞及肿瘤相关巨噬细胞。按照试剂盒说明书将分选好的肿瘤相关巨噬细胞经RNA提取及反转录。获得早期及晚期肿瘤相关巨噬细胞的cDNA,实时定量PCR测定M1型巨噬细胞标志性分子及M2型巨噬细胞标志性分子种类。1.2临床原发肿瘤及骨转移瘤病灶中TAMs的观察研究查询解放军总医院2006年1月至2016年12月诊断为“乳腺癌并发骨转移”的临床病例资料,选取原发病灶及骨转移病灶蜡块均留存于解放军总医院病理科的病例为研究对象共6例样本,另选取单纯乳腺癌病人中同样病理类型且无转移灶的乳腺癌6例样本作为对照组。所选蜡块均做连续切片6张(每张切片厚度约5μm),选取紧邻的两张切片依次进免疫组化染色,余切片分别行HE染色和甲苯胺蓝染色,镜下观察染色结果,记录并分析相关数据,根据镜下观察对其中的肿瘤相关巨噬细胞进行计数,统计学计算分析肿瘤相关巨噬细胞数目及M2型肿瘤相关巨噬细胞所占阳性比例有无差异,分析M2型肿瘤相关巨噬细胞所占阳性比例与发生淋巴结转移、骨转移的相关性。2.实验结果2.1.1待鉴定小鼠基因组进行PCR,筛选出与阳性对照出现相同条带的小鼠即为MMTV-PyMT小鼠,可用于后续实验。2.1.2根据出生日期相差1周内的10只MMTV-PyMT雌性小鼠进行肿瘤体积的跟踪监测,通过求和得到单只小鼠肿瘤的总体积,将总体积小于100 mm3的小鼠定为处于肿瘤早期,而总体积大于800 mm3的小鼠定为处于肿瘤晚期。2.1.3通过CD45的表达圈选肿瘤组织浸润CD45+白细胞,然后通过MHCⅡ及CD11b的表达,圈选出MHCⅡ+CD11b+的TAMs亚群(图4)。其中,白细胞占肿瘤组织细胞2.07%,而TAMs占白细胞中的31.3%。2.1.4利用M1型的标志性分子以及M2型的标志性分子的引物检测早期及晚期TAMs的表型后,结果证实,在肿瘤的进程中,肿瘤相关巨噬细胞发生了由M1型到M2型的表型转换。2.2.1根据文献报道,可用CD68抗体来标记巨噬细胞,CD163抗体标记M2型肿瘤相关巨噬细胞。在乳腺癌并发骨转移的患者中,其原发灶中CD68阳性细胞数比单纯乳腺癌患者要高,差异有统计学意义(P0.01),亦比其对应骨转移灶中高,差异有统计学意义(P0.01);而单纯乳癌患者病灶中CD68阳性细胞数比乳腺癌骨转移病灶高,差异有统计学意义(P0.01)。乳腺癌骨转移灶中CD163阳性细胞/CD68阳性细胞比值较其原发灶高,差异具有统计学意义(p0.05);合并骨转移的乳腺癌原发灶中CD163阳性细胞/CD68阳性细胞比值较单纯乳腺癌患者高,差异具有统计学意义(p0.01);乳腺癌骨转移灶中CD163阳性细胞/CD68阳性细胞比值较单纯乳腺癌患者高,差异具有统计学意义(p0.01)。2.2.2乳腺癌患者是否发生淋巴结转移及骨转移与肿瘤组织中CD163阳性细胞/CD68阳性细胞比值正相关。结论1.小鼠自发乳腺癌模型中,TAMs在肿瘤早期到晚期的进程中发生了由M1型到M2型的表型转换。2.本研究中可见乳腺癌骨转移灶中M2型TAMs的阳性率较其原发灶高;乳腺癌合并骨转移的患者其原发灶的M2型TAMs的阳性率要高于未发生骨转移的乳腺癌患者的原发灶。CD68阳性及CD163阳性细胞在乳腺癌骨转移灶中均低于其原发灶相应数目,但乳腺癌骨转移灶中CD163阳性细胞数/CD68阳性细胞数比值(即可认为M2型巨噬细胞)要高于原发灶。此外,乳腺癌患者是否发生淋巴结转移或骨转移与肿瘤组织中M2型TAMs的阳性率呈正相关。由此推测M2型巨噬细胞阳性比例增多可能是促进恶性肿瘤进展、甚至发生骨转移的原因之一。
[Abstract]:Background and objective macrophages, as a kind of immune cells that exist widely in tissues and organs, play a major role in the non specific immunity of the body, play a role in eliminating pathogens and tissue debris, and undertake the task of resisting the first line of defense against foreign invasion. 1-3]. inflammatory factors, bacterial products can cause macrophages to differentiate into M1 type macrophages; M1 type macrophages show high expression of interleukin 12 (interleukin 12, IL-12), low expression of IL-10, and participate in proinflammatory reaction; IL-4, IL-10, IL-13, and glucocorticoid can promote macrophages to differentiate into M2 type macrophages; M2 type giant The high expression of IL-10 but low expression of IL-12, which plays an important role in promoting immune tolerance, [4-6]. tumor related macrophages (tumor associated macrophages, TAMs) is a kind of macrophage infiltrated into the tumor tissue. The source is diverse and can be differentiated by peripheral collection of mononuclear cells. [7]. literature reports that TAMs can be associated with tumor. Cells interact and interact with each other. TAMs secretes a variety of cytokines, and then regulates the microenvironment of stem cells, widely participates in tumor progression, plays the role of tumor promoting angiogenesis, tumor promoting cell invasion and metastasis, and tumor immunololerance, and [7-9]. in breast cancer, prostate cancer, pancreatic cancer and other clinical patient tissue samples, macrophage markers The expression level of molecular CD68 is positively associated with poor prognosis. [10-12]. breast cancer is one of the most common malignant tumors in women. Bone metastases occur in about 8% of the patients, of which the rate of bone metastasis in highly malignant patients is even up to 2/3 of all patients with 69%[13]. breast cancer, most of the breast cancer Metastasis is dissolving bone, which can reduce the quality of vertebra bone, the probability of compression fracture of the patient is higher [14-17], and it is easy to have serious complications such as nerve injury and paraplegia. In addition, the patients have long life expectancy and severe cancer pain, and the patients suffer severe physical and mental pain. The level of leukocyte infiltration is very high, and macrophages account for the majority of [18].TAMs and breast cancer. There is a close relationship between the development and development of breast cancer. The degree of infiltration is positively correlated with tumor metastasis. It is an independent prognostic factor of breast cancer. The most common of [19]. breast cancer is invasive ductal carcinoma, but its pathological mechanism is still not well studied. The difference of tumor related macrophages in primary and bone metastases has not been reported at home and abroad. Analysis of the phenotypic changes of TAMs in the occurrence, development and metastasis of breast cancer may provide some ideas and references for the diagnosis and treatment of tumor. The conversion of TAMs phenotypes to regulate the function of TAMs becomes a very important problem. [20]., a mouse model of breast cancer using MMTV-PyMT genotypes, is used to simulate the occurrence and development of breast cancer. Through the separation, the expression of the M1 type macrophage cell and the expression of the M2 type macrophage markers in TAMs is compared in different course of disease, and then the expression of the M2 type macrophage marker molecules is compared. The phenotypic changes of TAMs were analyzed. The samples of primary breast cancer and breast cancer bone metastases were screened by histological staining, and the tumor related macrophages were counted. The number of macrophages related to tumor related macrophages and the positive proportion of M2 tumor related macrophages were statistically analyzed, and M2 type was analyzed. The correlation between the positive proportion of tumor related macrophages and the occurrence of lymph node metastasis and bone metastasis.1. experimental method 1.1 mouse model of spontaneous breast cancer was detected and analyzed by the phenotype of tumor related macrophages. After screening the MMTV-PyMT genotypes in mice, the mouse genome was extracted according to the prescription of the kit and the self milk of the mouse was obtained. The adenocarcinoma model was used to identify the genotypes. By analyzing the general rules of tumor volume at different weeks of age, the early and late stages of the tumor were differentiated (16 weeks and the total volume of tumor less than 100 mm3 was divided into early group; 20 weeks and the total volume of tumor larger than 800 mm3 was divided into late group), and then the tumor cells and swelling were separated. Tumor related macrophages. RNA extraction and reverse transcription of tumor related macrophages were selected according to the kit instructions. CDNA of early and late tumor related macrophages was obtained. Real-time quantitative PCR determination of M1 type macrophage marker molecules, M2 macrophage markers,.1.2 clinical primary tumor and bone metastases The observation and study of TAMs in the focus of the General Hospital of the PLA from January 2006 to December 2016 diagnosed as the clinical case data of "breast cancer complicated with bone metastases". The primary focus and the bone metastases were left in the pathology department of the General Hospital of the PLA for the study of 6 samples, and the same disease in simple breast cancer patients was selected as the same disease. 6 cases of breast cancer with no metastatic foci were used as control group. The selected paraffin blocks were divided into 6 pieces of continuous slice (the thickness of each slice was about 5 m). Two sections of the adjacent sections were selected for immunohistochemical staining in turn. The remaining slices were stained with HE and toluidine blue respectively. The staining results were observed under the microscope, and the related data were recorded and analyzed under the microscope. The tumor related macrophages were counted, the number of macrophages and the positive proportion of M2 type tumor related macrophages were statistically analyzed. The correlation between the positive proportion of M2 type tumor related macrophages and the occurrence of lymph node metastasis and the correlation between bone transfer and.2. test were to be identified in mice. The genome was PCR, and the mice with the same band as the positive control were selected as MMTV-PyMT mice. It could be used in follow-up experiment.2.1.2 to track the tumor volume according to 10 female mice of 1 weeks different from the birth date. The total volume of the tumor in a single mouse was obtained by finding the total volume of the mice with a total volume of less than 100 mm3. In the early stage of the tumor, the mice with the total volume of more than 800 mm3 were located in the tumor tissue infiltrating CD45+ white cells in the tumor tissue of the advanced.2.1.3 through CD45, and then the TAMs subgroup of MHC II +CD11b+ was selected by the expression of MHC II and CD11b (Figure 4). Among them, leukocytes accounted for 2.07% of the tumor tissue cells, while TAMs accounted for 31.3%.2.1 in white cells. .4 uses the primers of M1 type markers and M2 type markers to detect the phenotype of early and late TAMs. The results show that in the process of cancer, the tumor related macrophages have developed from M1 to M2 phenotype conversion.2.2.1 according to the literature, using CD68 antibody to mark macrophages and CD163 antibody marker M2 type tumor. In the patients with breast cancer complicated with bone metastases, the number of CD68 positive cells in the primary foci was higher than that of the simple breast cancer patients (P0.01), and the difference was statistically significant (P0.01), but the number of CD68 positive cells in the breast cancer patients was more than that of the breast cancer bone metastases. The difference was statistically significant (P0.01). The ratio of /CD68 positive cells to CD163 positive cells in bone metastasis of breast cancer was higher than that of its primary cell, and the difference was statistically significant (P0.05). The ratio of /CD68 positive cells to CD163 positive cells in the primary foci of breast cancer with bone metastases was higher than that of those with pure breast cancer (P0.01). The ratio of /CD68 positive cells in CD163 positive cells in adenocarcinoma bone metastases is higher than that in simple breast cancer patients. The difference is statistically significant (P0.01) whether.2.2.2 breast cancer patients have lymph node metastasis and bone metastasis and the /CD68 positive cell ratio of CD163 positive cells in tumor tissues. Conclusion TAMs is in the 1. mouse model of spontaneous breast cancer. In the early and late stages of the tumor, the phenotypic conversion of type M1 to type M2 occurred in the.2. study. In this study, the positive rate of M2 type TAMs in the bone metastases of breast cancer was higher than that of the original one; the positive rate of M2 type TAMs in the primary foci of breast cancer patients with bone metastasis was higher than that of the primary.CD68 positive and C in the patients with breast cancer without bone metastases. The number of D163 positive cells in the bone metastases of breast cancer is lower than that of the primary foci, but the ratio of the number of CD163 positive cells to the positive cells of /CD68 in the bone metastases of breast cancer is higher than that of the primary macrophage. In addition, whether the lymph node metastasis or the bone metastasis of the breast cancer patients and the positive M2 TAMs in the tumor tissue are positive. It is presumed that the increase of M2 macrophage positive ratio may be one of the reasons for promoting malignant tumor progression and even bone metastasis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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