肿瘤相关巨噬细胞在子宫内膜癌雌激素敏感性中的作用及机制研究

发布时间：2018-08-29 07:35

【摘要】：肿瘤相关巨噬细胞(TAMs)在调控Ⅰ型子宫内膜癌雌激素敏感性中的作用及机制研究子宫内膜癌(Endometrial carcinoma, EC)是女性最常见的生殖系统恶性肿瘤之一,其中70%-80%为Ⅰ型子宫内膜样腺癌,普遍认为其发生与长期无孕激素拮抗的雌激素刺激相关。我们的前期研究发现,Ⅰ型子宫内膜癌及其癌前病变患者血清雌激素水平并不高于健康同龄妇女,提示可能存在其他机制参与Ⅰ型内膜癌的发生发展。对其他实体肿瘤的研究发现,炎症刺激在肿瘤的发生发展中起重要作用。大量流行病学研究发现,Ⅰ型子宫内膜癌与胰岛素抵抗密切相关,而慢性炎症是后者的重要临床表现之一。也有研究显示,作为慢性炎症的具体表现形式,在子宫内膜癌病灶存在巨噬细胞浸润,后者与子宫内膜癌的不良预后密切相关。提示巨噬细胞浸润可能在Ⅰ型内膜癌的发生发展中起重要作用。但其作用和机制还有待进一步研究。人体内的单核/巨噬细胞可被诱导活化为不同表型：M1型和M2型,目前,M2型巨噬细胞被认为是在多数肿瘤中浸润的巨噬细胞,也被称为肿瘤相关巨噬细胞(Tumor-associated macrophages, TAMs)。以TAMs浸润为主的炎症反应与多种肿瘤的预后不良有关。研究发现,巨噬细胞浸润可调控肿瘤细胞激素受体的表达。在前列腺癌,巨噬细胞浸润可上调雄激素受体表达,促进雄激素对前列腺的致癌作用。在乳腺癌,肥胖造成的慢性炎症状态可激活乳腺癌局部的雌激素受体。因此,我们推测子宫内膜病灶局部TAMs浸润可能通过调控雌激素受体,增加内膜局部对雌激素的敏感性,促进癌症的发生和发展。本研究拟通过分析巨噬细胞浸润与子宫内膜癌和癌前病变的相关性,以及巨噬细胞对子宫内膜癌雌激素受体的调控及其对雌激素的敏感性,验证上述假设,并进一步分析可能的机制所在。具体分为四部分：第一部分：Ⅰ型子宫内膜癌及内膜增生性病变患者血清雌激素水平分析；第二部分：巨噬细胞浸润与Ⅰ型子宫内膜癌及内膜增生性病变的相关性分析；第三部分：TAMs通过ER α调控Ⅰ型子宫内膜癌细胞雌激素敏感性；第四部分：TAMs通过IL-17调控Ⅰ型子宫内膜癌细胞ER α表达。第一部分：Ⅰ型子宫内膜癌及癌前病变患者血清雌激素水平分析目的：检测血清雌激素水平与Ⅰ型子宫内膜癌发生发展的相关性。方法：1.收集2011年9月至2013年12月,子宫内膜正常及发生增生性病变患者的一般情况和血清雌二醇(Estradiol,E2)水平资料,包括对照组39例,增生紊乱69例,单纯性增生过长168例,复杂性增生过长67例,不典型增生33例,Ⅰ型子宫内膜癌25例,共收集病例401例。所有血液学检测均在本院检验科执行。统计分析血清雌激素水平,采用Graph pad prism 5.0软件进行one-way ANOVA分析,以P=0.05为显著性统计学差异标准。2.收集2011年9月至2013年12月,子宫内膜正常及内膜癌患者血清,包括对照组34例,Ⅰ型子宫内膜癌34例,共收集病例68例。相同年龄正常及内膜癌患者配对比较。ELISA检测血清雌激素水平。采用Graph pad prism 5.0进行paired t test分析,以P=0.05为显著性统计学差异标准。结果：1.临床资料显示,雌二醇水平在各组分布分别为：95.00±68.81ng/L、151.8±163.2 ng/L、156.4±178.8 ng/L、138.4±330.0 ng/L、125.5±156.7 ng/L、36.52 ±45.35 ng/L,结果不具有统计学差异(P0.05)。以40岁为界,将患者分为25-40岁和40-55岁两个年龄段,25-40岁年龄段中,雌二醇水平在各组分布分别为：107.1 ±70.73 ng/L、140.3±165.2 ng/L、122.1±129.6 ng/L、76.59±59.86 ng/L和120.1±179.2 ng/L；40～55岁年龄段中,雌二醇水平在各组分布分别为：102.2±63.36 ng/L、 161.1±154.5 ng/L、164.0±20.6 ng/L、148.1±152.6 ng/L、148.3±122.5 ng/L和76.86±68.13 ng/L,同一年龄段的各组间雌二醇水平分布不具有统计学差异(P0.05)。2.Elisa结果显示,雌二醇水平在对照组与Ⅰ型子宫内膜癌组按同一年龄配对比较：P=0.8338,无统计学意义。结论：Ⅰ型子宫内膜癌及癌前病变患者血清雌激素水平并不高于内膜正常妇女。提示可能存在其他机制参与内膜癌的发生发展。第二部分：巨噬细胞浸润与Ⅰ型子宫内膜癌及内膜增生性病变的相关性分析目的：检测巨噬细胞在Ⅰ型子宫内膜癌及癌前病变中的浸润情况,分析二者间的相关性。方法：收集正常及增生性病变子宫内膜组织共140例：包括增殖期、分泌期、单纯型增生、复杂型增生子宫内膜各20例,不典型增生子宫内膜、Ⅰ型子宫内膜癌各30例,CD68免疫组化染色,计数各期别CD68(+)细胞数,评价CD68(+)巨噬细胞在子宫内膜中的浸润情况。采用Graph pad prism 5.0进行one-way ANOVA分析,以P=0.05为显著性统计学差异标准。结果：1.CD68表达于巨噬细胞的胞质和胞膜。2.各组中CD68+细胞数目分别是(每400倍视野)：增殖期：6.641±5.463个：分泌期：13.82±10.36个；单纯型增生：20.45±10.17个；复杂型增生：35.70±14.73个；不典型增生期：41.98±15.74个；Ⅰ型子宫内膜癌：57.80±25.72个,与Ⅰ型子宫内膜癌相比均有P0.001,CD68(+)细胞数与子宫内膜增生性病变进展呈正相关。3.随子宫内膜增生性病变进展,巨噬细胞浸润部位由血管周围、间质向腺上皮转变。4.随病变进展,巨噬细胞形态逐渐成熟。结论：CD68(+)巨噬细胞在子宫内膜浸润数目、部位及形态可能与Ⅰ子宫内膜癌发生发展有关。第三部分：TAMs通过ERα调控Ⅰ型子宫内膜癌细胞雌激素敏感性目的：研究TAMs在促进Ⅰ型子宫内膜癌细胞增殖及内膜癌细胞对雌二醇敏感性中的作用,并进一步分析可能的机制。方法：采用两种雌激素受体阳性的子宫内膜癌细胞株Ishikawa和HEC-1-A作为研究对象；培养单核细胞系THP-1,并诱导为M2型巨噬细胞,即TAMs;子宫内膜癌细胞与M2型THP-1共培养,或经M2型THP-1的条件培养基培养后,CCK-8检测共培养前后内膜癌细胞增殖情况,以及对生理剂量(10-9M)E2的敏感性改变,并将此变化与过表达ERα或应用ER α抑制剂ICI后的趋势相比较,采用Graph pad prism 5.0进行one-way ANOVA分析,以P=0.05为显著性统计学差异标准；Real-time PCR和Western Blot检测内膜癌细胞与M2型THP-1共培养后雌激素受体α (ERα)的表达变化,并采用细胞增殖相关蛋白CyclinDl验证细胞增殖情况。结果：1.人M2型THP-1巨噬细胞诱导活化成功,形态改变明显,表型鉴定CD68、CD163、CD204和CD206显著升高。2.M2型THP-1与Ⅰ型子宫内膜癌细胞Ishikawa或HEC-1-A共培养后,内膜癌细胞增殖均被促进；生理剂量的E2对两株细胞的促增殖作用亦被上调；该现象可被雌激素受体抑制剂ICI抑制。3.M2型THP-1的条件培养基(CM)可时问依赖性促进Ishikawa和HEC-1-A细胞增殖,上调生理剂量雌激素对内膜癌细胞的促增殖作用,其促增殖作用同样可被ICI抑制。4.M2型THP-1的条件培养基(CM)可时间依赖性上调雌激素受体α表达,而对雌激素受体β无明显作用；CM可时间依赖性上调cyclinDl表达；5.M2型THP-1对雌激素促增殖作用的上调作用与内膜癌细胞过表达ER α后细胞增殖增强情况类似。结论：TAMs可促进子宫内膜癌细胞Ishikawa和HEC-1-A增殖及对雌激素的敏感性,这种作用可能是通过上调内膜癌细胞雌激素受体α实现的。第四部分：TAMs通过IL-17调控Ⅰ型子宫内膜癌细胞ERα表达目的：筛选TAMs中可能在促进内膜癌细胞增殖中起关键作用的炎症因子,研究其在促Ⅰ型子宫内膜癌细胞增殖及雌激素敏感性方面的作用,并探索可能的机制。方法：M2型THP-1与子宫内膜癌细胞HEC-1-A经Transwell共培养后,提取M2型THP-1细胞的RNA行Real-time PCR筛选稳定作用的炎症因子；采用CCK-8验证筛选的炎症因子对HEC-1-A的促增殖及促雌激素敏感性作用,采用Graph pad prism 5.0进行one-way ANOVA分析,以P=0.05为显著性统计学差异标准；Real-time PCR和Western blot验证该炎症因子对HEC-1-A的ER α表达调控；Western blot验证该炎症因子和E2共同作用于HEC-1-A细胞后,对调控细胞增殖的p-AKT信号通路的活化作用。结果：1.经筛选,与HEC-1-A共培养后M2型THP-1细胞炎症因子IL-10和IL-17表达明显增加；2.IL-10和IL-17均可上调E2对HEC-1-A细胞的促增殖作用,且IL-17作用更强；3IL-10、IL-17或E2处理后,均可上调HEC-1-A的ER a表达,且IL-17作用更强,进一步筛选出IL-17作为研究对象；4.IL-17与E2共同处理HEC-1-A细胞后,其p-AKT信号通路活化显著增强,此作用与HEC-1-A细胞转染过表达ERα后加用E2相似。结论：TAMs通过IL-17上调子宫内膜癌细胞ER a的表达,从而促进内膜癌细胞对雌激素敏感性增加,增强雌激素对内膜癌细胞的促增殖作用。上述结果提示：1.血清雌激素水平与Ⅰ型子宫内膜增生性病变的进展无显著相关性,雌激素对内膜的促癌作用可能存在其他机制；2.巨噬细胞浸润与工型子宫内膜增生性病变进展呈正相关,慢性炎症可能在Ⅰ型子宫内膜癌发生发展中发挥重要作用；3.肿瘤相关巨噬细胞通过上调内膜癌细胞雌激素受体α表达增强雌激素对Ⅰ型子宫内膜癌细胞的促增殖作用,慢性炎症对雌激素敏感性的调控可能是Ⅰ型子宫内膜癌发生发展的重要原因；4.肿瘤相关巨噬细胞通过分泌炎症因子,如白介素-17,上调内膜癌细胞雌激素受体α表达,从而提高内膜细胞对雌激素敏感性。可能是内膜癌发生发展的重要机制之一本课题研究表明：无孕激素拮抗的高雌激素水平可能不是雌激素促进Ⅰ型子宫内膜癌发生的唯一方式,慢性炎症调控的内膜雌激素敏感性增加可能为内膜癌发生的机制提供补充。靶向性抗炎治疗可能为预防Ⅰ型子宫内膜癌及癌前病变的发生提供新的思路。
[Abstract]:The role and mechanism of tumor-associated macrophages (TAMs) in regulating estrogen sensitivity in type I endometrial carcinoma Endometrial carcinoma (EC) is one of the most common female reproductive malignancies, 70% - 80% of which are type I endometrioid adenocarcinoma. It is generally believed that the occurrence of TAMs is associated with estrogen without progesterone antagonism for a long time. Our previous study found that serum estrogen levels in patients with type I endometrial carcinoma and its precancerous lesions were not higher than those in healthy women of the same age, suggesting that there may be other mechanisms involved in the development of type I endometrial carcinoma. A large number of epidemiological studies have found that type I endometrial carcinoma is closely related to insulin resistance, and chronic inflammation is one of the important clinical manifestations of the latter. It is suggested that macrophage infiltration may play an important role in the genesis and development of type I endometrial carcinoma, but its role and mechanism need to be further studied.Monocytes/macrophages in human body can be induced to activate into different phenotypes: M1 and M2. At present, M2 macrophages are considered as infiltrating macrophages in most tumors, also known as macrophages. Tumor-associated macrophages (TAMs). Inflammatory reactions predominantly with TAMs infiltration are associated with poor prognosis in a variety of tumors. Macrophage infiltration can regulate the expression of hormone receptors in tumor cells. In prostate cancer, macrophage infiltration can up-regulate the expression of androgen receptors and promote the effect of androgen on prostate. In breast cancer, chronic inflammation caused by obesity can activate estrogen receptor in breast cancer. Therefore, we speculate that local TAMs infiltration in endometrial lesions may promote the occurrence and development of cancer by regulating estrogen receptor and increasing local sensitivity to estrogen. The relationship between cell infiltration and endometrial carcinoma and precancerous lesions, and the regulation of macrophages on estrogen receptor and their sensitivity to estrogen in endometrial carcinoma were studied to verify the hypothesis and further analyze the possible mechanism. Analysis of serum estrogen level; Part II: Correlation between macrophage infiltration and type I endometrial carcinoma and endometrial hyperplasia; Part III: TAMs regulate estrogen sensitivity of type I endometrial carcinoma cells through ERalpha; Part IV: TAMs regulate ERalpha expression of type I endometrial carcinoma cells through IL-17. Objective: To detect the correlation between serum estrogen levels and the occurrence and development of type I endometrial carcinoma. Methods: 1. To collect the general information and serum estradiol (E2) of patients with normal and proliferative endometrium from September 2011 to December 2013. Data of serum estrogen levels were collected in 401 cases, including 39 cases of control group, 69 cases of hyperplasia disorder, 168 cases of simple hyperplasia, 67 cases of complex hyperplasia, 33 cases of atypical hyperplasia, 25 cases of type I endometrial carcinoma. E-way ANOVA analysis, with P = 0.05 as the significant statistical difference standard. 2. Collected from September 2011 to December 2013, normal endometrial and endometrial cancer patients serum, including 34 cases of control group, 34 cases of type I endometrial cancer, a total of 68 cases. The same age of normal and endometrial cancer patients matched comparison. ELISA detection of serum estrogen levels. Results: 1. The clinical data showed that the distribution of estradiol levels in each group were 95.00 (+ 68.81 ng/L), 151.8 (+ 163.2 ng/L), 156.4 (+ 178.8 ng/L), 138.4 (+ 330.0 ng/L), 125.5 (+ 156.7 ng/L) and 36.52 (+ 45.35 ng/L), respectively. The results were not statistically significant (P 0.05). 05). Patients were divided into 25-40 years old and 40-55 years old. The distribution of estradiol in each group was 107.1 (+ 70.73 ng/L), 140.3 (+ 165.2 ng/L), 122.1 (+ 129.6 ng/L), 76.59 (+ 59.86 ng/L) and 120.1 (+ 179.2 ng/L) in 25-40 years old, respectively. 63.36 ng/L, 161.1+154.5 ng/L, 164.0+20.6 ng/L, 148.1+152.6 ng/L, 148.3+122.5 ng/L and 76.86+68.13 ng/L. There was no significant difference in the distribution of estradiol levels among the same age groups (P 0.05). 2. Elisa results showed that estradiol levels in the control group and the type I endometrial cancer group were matched by the same age: P = 0.8338, no significant difference. Conclusion: The serum estrogen level of patients with type I endometrial carcinoma and precancerous lesions is not higher than that of normal endometrial women, suggesting that there may be other mechanisms involved in the occurrence and development of endometrial carcinoma. Methods: 140 normal and proliferative endometrial tissues were collected, including 20 cases of proliferative endometrium, 20 cases of simple hyperplasia, 20 cases of complex hyperplasia, 30 cases of atypical hyperplasia and 30 cases of type I endometrial carcinoma. The number of CD68 (+) cells in each stage was counted and the infiltration of CD68 (+) macrophages in endometrium was evaluated. Graph pad prism 5.0 was used for one-way ANOVA analysis. P = 0.05 was used as the standard of significant statistical difference. Results: 1. CD68 was expressed in the cytoplasm and membrane of macrophages. 2. The number of CD68 (+) cells in each group was separately analyzed. Proliferative phase: 6.641 + 5.463: Secretory phase: 13.82 + 10.36; Simple hyperplasia: 20.45 + 10.17; Complex hyperplasia: 35.70 + 14.73; Atypical hyperplasia: 41.98 + 15.74; Type I endometrial carcinoma: 57.80 + 25.72; Compared with type I endometrial carcinoma, there were all P 0.001, CD68 (+) cells and sons. With the development of endometrial hyperplasia, the infiltration sites of macrophages changed from perivascular to mesenchymal to glandular epithelial. 4. With the progress of endometrial hyperplasia, the morphology of macrophages gradually matured. Conclusion: The number, location and morphology of CD68 (+) macrophages infiltration in endometrium may be related to the occurrence of endometrial cancer. Part III: TAMs regulate estrogen sensitivity of type I endometrial carcinoma cells through ERalpha. Objective: To study the role of TAMs in promoting the proliferation of type I endometrial carcinoma cells and the sensitivity of endometrial carcinoma cells to estradiol, and to further analyze the possible mechanisms. Membrane cancer cell lines Ishikawa and HEC-1-A were used as research objects; monocyte THP-1 was cultured and induced into M2 macrophages, namely TAMs; endometrial cancer cells were co-cultured with M2 THP-1 or cultured on M2 THP-1 conditioned medium. CCK-8 was used to detect the proliferation of endometrial cancer cells before and after co-culture, and the sensitivity to physiological dose (10-9M) E2. The perceptual changes were compared with those after overexpression of ER-alpha or ICI. One-way ANOVA analysis was performed with Graph pad prism 5.0 and P=0.05 as the significant statistical difference standard. The expression of ER-alpha in endometrial carcinoma cells co-cultured with M2 THP-1 was detected by Real-time PCR and Western Blot. Results: 1. Human M 2 THP-1 macrophages were successfully induced and activated, and the morphological changes were obvious. The phenotypes of CD68, CD163, CD204 and CD206 were significantly increased. 2. M 2 THP-1 co-cultured with Ishikawa or HEC-1-A, the proliferation of endometrial cancer cells was promoted. Physiological dose of E2 also up-regulated the proliferation of the two cell lines; this phenomenon can be inhibited by estrogen receptor inhibitor ICI. 3. M2 THP-1 conditioned medium (CM) can promote the proliferation of Ishikawa and HEC-1-A cells in a time-dependent manner, and up-regulate the proliferation of endometrial cancer cells induced by physiological dose of estrogen, the same as its proliferation-promoting effect. Conditioned medium (CM) inhibited by ICI could up-regulate the expression of estrogen receptor alpha in a time-dependent manner, but had no significant effect on estrogen receptor beta; CM could up-regulate the expression of cyclin Dl in a time-dependent manner; 5. Conclusion: TAMs can promote the proliferation and estrogen sensitivity of endometrial carcinoma cells Ishikawa and HEC-1-A. This effect may be achieved by up-regulating estrogen receptor alpha in endometrial carcinoma cells. Part IV: TAMs regulate the expression of ERalpha in type I endometrial carcinoma cells through IL-17. Objective: To screen TAMs may promote the proliferation of endometrial carcinoma cells. Methods: M2 THP-1 and HEC-1-A cells were co-cultured with Transwell, and RNA of M2 THP-1 cells was extracted to screen stable inflammatory factors by Real-time PCR. Graph pad prism 5.0 was used for one-way ANOVA analysis, and P = 0.05 was used as the significant statistical difference standard; Real-time PCR and Western blot were used to verify the regulation of the inflammatory factors on the expression of ERalpha in HEC-1-A; Western blot was used to verify the inflammation. Results: 1. After co-culture with HEC-1-A, the expression of inflammatory factors IL-10 and IL-17 in M 2 THP-1 cells was significantly increased; 2. Both IL-10 and IL-17 could up-regulate the proliferation of HEC-1-A cells, and the effect of IL-17 was stronger. After treatment with IL-17 or E2, the expression of HEC-1-A was up-regulated, and the effect of IL-17 was stronger. IL-17 was further screened out as the research object. 4. After treatment with IL-17 and E2, the activation of p-AKT signaling pathway in HEC-1-A cells was significantly enhanced, which was similar to that of HEC-1-A cells transfected with ER-a and then treated with E2. These results suggest that: 1. There is no significant correlation between serum estrogen levels and the progression of type I endometrial hyperplasia, and estrogen may have other mechanisms to promote the proliferation of endometrial cancer cells. 2. Macrophage infiltration is positively correlated with the development of type I endometrial hyperplasia. Chronic inflammation may play an important role in the development of type I endometrial carcinoma. 3. Tumor-associated macrophages enhance the proliferation of type I endometrial carcinoma cells by up-regulating the expression of estrogen receptor alpha. The regulation of sex inflammation on estrogen sensitivity may be an important reason for the occurrence and development of type I endometrial carcinoma. This study suggests that high estrogen levels without progesterone antagonism may not be the only way estrogen promotes the development of type I endometrial carcinoma. Increased estrogen sensitivity in the endometrium regulated by chronic inflammation may complement the mechanism of endometrial carcinoma. Targeted anti-inflammatory therapy may prevent type I endometrial carcinoma and New ideas are provided for the occurrence of precancerous lesions.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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