本文選題：腫瘤相關(guān)巨噬細(xì)胞 + 乳腺癌��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景與目的巨噬細(xì)胞作為廣泛存在于組織、器官中的一類免疫細(xì)胞,在機體非特異性免疫反.應(yīng)中發(fā)揮主要作用,具有清除病原體和組織碎片的作用,擔(dān)負(fù)著抵御外來侵襲第一道防線的任務(wù)。研究表明,其功能受微環(huán)境的制約與調(diào)控[1-3]。炎性因子、細(xì)菌產(chǎn)物可導(dǎo)致巨噬細(xì)胞分化成為M1型的巨噬細(xì)胞;M1型的巨噬細(xì)胞表現(xiàn)為高表達白細(xì)胞介素12(interleukin 12,IL-12),低表達IL-10,參與促炎反應(yīng);IL-4、IL-10、IL-13和糖皮質(zhì)激素等可促進巨噬細(xì)胞向M2型的巨噬細(xì)胞進行分化;M2型巨噬細(xì)胞高表達IL-10但低表達IL-12,在促進免疫耐受中起重要作用[4-6]。腫瘤相關(guān)巨噬細(xì)胞(tumor associated macrophages,TAMs)是一種浸潤入腫瘤組織內(nèi)存在的巨噬細(xì)胞,來源具有多樣性,可由外周募集的單核細(xì)胞分化而來[7]。文獻報道TAMs可與腫瘤細(xì)胞發(fā)生相互作用,相互影響,TAMs可分泌多種細(xì)胞因子,進而調(diào)節(jié)干細(xì)胞微環(huán)境,廣泛參與腫瘤進程,發(fā)揮促瘤血管生成、促瘤細(xì)胞侵襲及轉(zhuǎn)移、促瘤免疫耐受等作用[7-9]。在乳腺癌、前列腺癌、胰腺癌等臨床病人組織樣本中,巨噬細(xì)胞標(biāo)志性分子CD68的表達水平與不良預(yù)后正相關(guān)[10-12]。乳腺癌作為女性最常見的惡性腫瘤之一,約有8%的患者發(fā)生了骨轉(zhuǎn)移,其中高度惡性患者骨轉(zhuǎn)移率甚至可高達69%[13]。乳腺癌脊柱轉(zhuǎn)移患者約占所有骨轉(zhuǎn)移患者的三分之二,大多數(shù)乳腺癌脊柱轉(zhuǎn)移是呈溶骨性改變的,可使脊椎骨質(zhì)量下降,患者發(fā)生壓縮性骨折概率較高[14-17],容易發(fā)生神經(jīng)損傷甚至截癱等嚴(yán)重并發(fā)癥;加之此類患者預(yù)期壽命較長,伴隨劇烈癌性疼痛,患者承受嚴(yán)重的身心痛苦。有研究報道,乳腺癌中腫瘤相關(guān)白細(xì)胞的浸潤水平很高,且巨噬細(xì)胞占其中的多半[18]。TAMs與乳腺癌發(fā)生、發(fā)展存在密切聯(lián)系,其浸潤程度與腫瘤轉(zhuǎn)移呈正相關(guān),是乳腺癌獨立的預(yù)后因素[19]。乳腺癌中最常見的是浸潤性導(dǎo)管癌,但其病理學(xué)機制仍尚未研究清楚。乳腺癌患者原發(fā)灶和骨轉(zhuǎn)移灶中腫瘤相關(guān)巨噬細(xì)胞的差異國內(nèi)外尚未見相關(guān)報道。分析TAMs在乳腺癌發(fā)生、發(fā)展、轉(zhuǎn)移等過程中的表型變化,或可為腫瘤診治提供思路和借鑒。鑒于腫瘤進程經(jīng)歷發(fā)生、發(fā)展多個過程,那么,腫瘤微環(huán)境的變化是否介導(dǎo)TAMs表型的轉(zhuǎn)換,從而調(diào)控TAMs的功能就成為一個十分重要的問題[20]。本研究利用MMTV-PyMT基因型的小鼠乳腺癌自發(fā)模型,模擬乳腺癌發(fā)生、發(fā)展過程,通過分離、比較不同病程中TAMs的M1型巨噬細(xì)胞及M2型巨噬細(xì)胞標(biāo)志性分子的表達,進而分析TAMs的表型變化。篩選臨床乳腺癌原發(fā)腫瘤及乳腺癌骨轉(zhuǎn)移瘤病灶樣本進行組織學(xué)染色觀察,對其中的腫瘤相關(guān)巨噬細(xì)胞進行計數(shù),統(tǒng)計學(xué)計算分析腫瘤相關(guān)巨噬細(xì)胞數(shù)目及M2型腫瘤相關(guān)巨噬細(xì)胞所占陽性比例有無差異,分析M2型腫瘤相關(guān)巨噬細(xì)胞所占陽性比例與發(fā)生淋巴結(jié)轉(zhuǎn)移、骨轉(zhuǎn)移的相關(guān)性。1.實驗方法1.1小鼠自發(fā)乳腺癌模型中腫瘤相關(guān)巨噬細(xì)胞的表型檢測與分析篩選出MMTV-PyMT基因型的小鼠后,按試劑盒說明書的方法提取小鼠基因組并對該小鼠自發(fā)乳腺癌模型進行基因型的鑒定。通過分析不同周齡小鼠腫瘤體積的普遍規(guī)律,對腫瘤的進程進行早期與晚期的區(qū)分(16周且腫瘤總體積小于100 mm3的腫瘤分為早期組;20周且腫瘤總體積大于800 mm3的腫瘤分為晚期組)。進而分離腫瘤細(xì)胞及腫瘤相關(guān)巨噬細(xì)胞。按照試劑盒說明書將分選好的腫瘤相關(guān)巨噬細(xì)胞經(jīng)RNA提取及反轉(zhuǎn)錄。獲得早期及晚期腫瘤相關(guān)巨噬細(xì)胞的cDNA,實時定量PCR測定M1型巨噬細(xì)胞標(biāo)志性分子及M2型巨噬細(xì)胞標(biāo)志性分子種類。1.2臨床原發(fā)腫瘤及骨轉(zhuǎn)移瘤病灶中TAMs的觀察研究查詢解放軍總醫(yī)院2006年1月至2016年12月診斷為“乳腺癌并發(fā)骨轉(zhuǎn)移”的臨床病例資料,選取原發(fā)病灶及骨轉(zhuǎn)移病灶蠟塊均留存于解放軍總醫(yī)院病理科的病例為研究對象共6例樣本,另選取單純?nèi)橄侔┎∪酥型瑯硬±眍愋颓覠o轉(zhuǎn)移灶的乳腺癌6例樣本作為對照組。所選蠟塊均做連續(xù)切片6張(每張切片厚度約5μm),選取緊鄰的兩張切片依次進免疫組化染色,余切片分別行HE染色和甲苯胺藍染色,鏡下觀察染色結(jié)果,記錄并分析相關(guān)數(shù)據(jù),根據(jù)鏡下觀察對其中的腫瘤相關(guān)巨噬細(xì)胞進行計數(shù),統(tǒng)計學(xué)計算分析腫瘤相關(guān)巨噬細(xì)胞數(shù)目及M2型腫瘤相關(guān)巨噬細(xì)胞所占陽性比例有無差異,分析M2型腫瘤相關(guān)巨噬細(xì)胞所占陽性比例與發(fā)生淋巴結(jié)轉(zhuǎn)移、骨轉(zhuǎn)移的相關(guān)性。2.實驗結(jié)果2.1.1待鑒定小鼠基因組進行PCR,篩選出與陽性對照出現(xiàn)相同條帶的小鼠即為MMTV-PyMT小鼠,可用于后續(xù)實驗。2.1.2根據(jù)出生日期相差1周內(nèi)的10只MMTV-PyMT雌性小鼠進行腫瘤體積的跟蹤監(jiān)測,通過求和得到單只小鼠腫瘤的總體積,將總體積小于100 mm3的小鼠定為處于腫瘤早期,而總體積大于800 mm3的小鼠定為處于腫瘤晚期。2.1.3通過CD45的表達圈選腫瘤組織浸潤CD45+白細(xì)胞,然后通過MHCⅡ及CD11b的表達,圈選出MHCⅡ+CD11b+的TAMs亞群(圖4)。其中,白細(xì)胞占腫瘤組織細(xì)胞2.07%,而TAMs占白細(xì)胞中的31.3%。2.1.4利用M1型的標(biāo)志性分子以及M2型的標(biāo)志性分子的引物檢測早期及晚期TAMs的表型后,結(jié)果證實,在腫瘤的進程中,腫瘤相關(guān)巨噬細(xì)胞發(fā)生了由M1型到M2型的表型轉(zhuǎn)換。2.2.1根據(jù)文獻報道,可用CD68抗體來標(biāo)記巨噬細(xì)胞,CD163抗體標(biāo)記M2型腫瘤相關(guān)巨噬細(xì)胞。在乳腺癌并發(fā)骨轉(zhuǎn)移的患者中,其原發(fā)灶中CD68陽性細(xì)胞數(shù)比單純?nèi)橄侔┗颊咭?差異有統(tǒng)計學(xué)意義(P0.01),亦比其對應(yīng)骨轉(zhuǎn)移灶中高,差異有統(tǒng)計學(xué)意義(P0.01);而單純?nèi)榘┗颊卟≡钪蠧D68陽性細(xì)胞數(shù)比乳腺癌骨轉(zhuǎn)移病灶高,差異有統(tǒng)計學(xué)意義(P0.01)。乳腺癌骨轉(zhuǎn)移灶中CD163陽性細(xì)胞/CD68陽性細(xì)胞比值較其原發(fā)灶高,差異具有統(tǒng)計學(xué)意義(p0.05);合并骨轉(zhuǎn)移的乳腺癌原發(fā)灶中CD163陽性細(xì)胞/CD68陽性細(xì)胞比值較單純?nèi)橄侔┗颊吒?差異具有統(tǒng)計學(xué)意義(p0.01);乳腺癌骨轉(zhuǎn)移灶中CD163陽性細(xì)胞/CD68陽性細(xì)胞比值較單純?nèi)橄侔┗颊吒?差異具有統(tǒng)計學(xué)意義(p0.01)。2.2.2乳腺癌患者是否發(fā)生淋巴結(jié)轉(zhuǎn)移及骨轉(zhuǎn)移與腫瘤組織中CD163陽性細(xì)胞/CD68陽性細(xì)胞比值正相關(guān)。結(jié)論1.小鼠自發(fā)乳腺癌模型中,TAMs在腫瘤早期到晚期的進程中發(fā)生了由M1型到M2型的表型轉(zhuǎn)換。2.本研究中可見乳腺癌骨轉(zhuǎn)移灶中M2型TAMs的陽性率較其原發(fā)灶高;乳腺癌合并骨轉(zhuǎn)移的患者其原發(fā)灶的M2型TAMs的陽性率要高于未發(fā)生骨轉(zhuǎn)移的乳腺癌患者的原發(fā)灶。CD68陽性及CD163陽性細(xì)胞在乳腺癌骨轉(zhuǎn)移灶中均低于其原發(fā)灶相應(yīng)數(shù)目,但乳腺癌骨轉(zhuǎn)移灶中CD163陽性細(xì)胞數(shù)/CD68陽性細(xì)胞數(shù)比值(即可認(rèn)為M2型巨噬細(xì)胞)要高于原發(fā)灶。此外,乳腺癌患者是否發(fā)生淋巴結(jié)轉(zhuǎn)移或骨轉(zhuǎn)移與腫瘤組織中M2型TAMs的陽性率呈正相關(guān)。由此推測M2型巨噬細(xì)胞陽性比例增多可能是促進惡性腫瘤進展、甚至發(fā)生骨轉(zhuǎn)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738

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8 徐海濱;胡祖健;胡望華;史紅仙;何俊玲;黃建;;乳腺癌骨轉(zhuǎn)移綜合治療初探[J];中華乳腺病雜志(電子版);2007年01期

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10 周圍;周恩相;王澤強;郭瓊;;乳腺癌骨轉(zhuǎn)移57例回顧性分析[J];實用醫(yī)藥雜志;2007年05期

相關(guān)會議論文 前10條

1 魏志真;董峰;;乳腺癌骨轉(zhuǎn)移的臨床特點分析[A];中華醫(yī)學(xué)會放射醫(yī)學(xué)與防護學(xué)分會第三次全中國青年學(xué)術(shù)交流會論文摘要匯編[C];2001年

2 魏志真;董峰;;乳腺癌骨轉(zhuǎn)移的臨床特點分析[A];中華醫(yī)學(xué)會放射醫(yī)學(xué)與防護學(xué)分會第三次全國中青年學(xué)術(shù)交流會論文匯編[C];2001年

3 徐海濱;胡祖健;胡望華;史紅仙;何俊玲;黃建;;乳腺癌骨轉(zhuǎn)移綜合治療初探[A];2005年浙江省外科學(xué)術(shù)會議論文匯編[C];2005年

4 蔡鋼;陳佳藝;朱驥;俞曉立;章真;郭小毛;胡超蘇;;乳腺癌骨轉(zhuǎn)移的放射治療和預(yù)后因素[A];2007第六屆全國放射腫瘤學(xué)學(xué)術(shù)年會論文集[C];2007年

5 蔡鋼;陳佳藝;朱驥;俞曉立;章真;郭小毛;胡超蘇;;乳腺癌骨轉(zhuǎn)移的放射治療和預(yù)后因素[A];2007第六屆全國放射腫瘤學(xué)學(xué)術(shù)年會論文集[C];2007年

6 劉兆U，

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